Neurotoxicology and Teratology 64 (2017) 1–7

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Neurotoxicology and Teratology

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Full length article

In vivo assessment of hair cell damage and developmental toxicity caused by MARK
gestational caffeine exposure using zebrafish (Danio rerio) models
Yoon Chan Raha,1, Myung Hoon Yoob,1, June Choia,⁎, Saemi Parka, Hae-Chul Parkc,
Kyoung Ho Oha, Seung Hoon Leea, Soon-Young Kwona
a
Department of Otorhinolaryngology-Head and Neck Surgery, Korea University Ansan Hospital, Korea University, College of Medicine, Seoul, Republic of Korea
b
Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
c
Laboratory of Neurodevelopmental Genetics, Graduate School of Medicine, Korea University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the present study was to evaluate hair cell damage and associated developmental toxicity caused by
Caffeine gestational caffeine exposure.
Embryotoxicity We exposed embryos to various caffeine concentrations (25 μM, 125 μM, 250 μM, and 500 μM) and evaluated
Teratogenicity developmental toxicity of the embryos at 72 and 120 h and hair cell damage at 120 h after fertilization. The
Hair cell
average number of total hair cells within four neuromasts exposed to various concentrations of caffeine was
Zebrafish
compared with that of the control group. To seek the underlying mechanisms, TUNEL and DASPEI assay were
carried out to evaluate hair cell apoptosis and mitochondrial damage, respectively. Morphologic abnormality,
mortality, hatching rate, and heart rate were also evaluated.
Caffeine induced significant hair cell damage compared with control group (p < 0.01, control;
35.64 ± 10.48 cells, 500 μM caffeine; 23.32 ± 12.14 cells, n = 25–30). Significant increase in the hair cell
apoptosis was confirmed in a dose-dependent manner (p < 0.01, TUNEL assay) and the mitochondrial damage
in high caffeine concentrations (250, 500 μM) (p < 0.01, DASPEI assay).Morphologic abnormalities were sig-
nificantly increased in high caffeine concentrations (250 or 500 μM) for body shape, notochord, and heart at
both 3-, and 5-dpf. The control group exhibited 3.3% mortality which increased up to 11.6% at 500 μM caffeine.
Rapid hatching was present at 48 h (control; 46.6%, 500 μM caffeine; 100%).
In conclusion, gestational caffeine exposure caused significant hair cell damage and developmental toxicities
in zebrafish at early developmental stages.

1. Introduction reduction of mitochondrial membrane potential (Saiki et al.,

The embryogenesis is highly susceptible to toxicant exposure;
however, many chemicals and foods currently available for commercial
use lack safety information (Beker van Woudenberg et al., 2014;
Selderslaghs et al., 2009). Caffeine is a white crystalline xanthine al-
kaloid that acts as a stimulant drug for the central nervous system
(Abdelkader et al., 2013; Rana et al., 2010; Saiki et al., 2011). It is
typically used to relieve headache and pain as one of the most generally
consumed psychoactive compounds in the world (Abdelkader et al.,
2013; Rana et al., 2010). Many beverages also contain caffeine with
around 87% of the population consumes an average of 193 mg of caf-
feine per day (Abdelkader et al., 2013).Recently, it was determined that
caffeine induces cell death in human osteoblasts (Lu et al., 2008)and a
2011),which is primarily attributed to apoptosis (Lu et al., 2008; Saiki
et al., 2011). However, the developmental toxicity of caffeine has not
yet been fully examined.
The zebrafish (Danio rerio) have several advantages for the evalua-
tion of developmental toxicity with well characterized developmental
information, high fecundity, rapid embryonic development, and simple
recognition of various anomalies from the transparency of the embryo
(Chen et al., 2014; Embry et al., 2010; Fang et al., 2015; Nagel, 2002;
Lantz-McPeak et al., 2015; Berghmans et al., 2008; Ku et al., 2015). The
zebrafish has also been used as a highly efficient tool for evaluating hair
cell changes with a series of mechanosensory neuromasts which con-
tains bundle of hair cells structurally and functionally similar to human
inner ear hair cells (Bever and Fekete, 2002).

⁎
Corresponding author at: Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, 123, Jeokgeum-ro (Gojan-dong), Danwon-gu, Ansan-si,
Gyeonggi-do 425-707, Republic of Korea.
E-mail address: mednlaw@korea.ac.kr (J. Choi).
1
Yoon Chan Rah and Myung Hoon Yoo contributed equally to this work as first authors.

http://dx.doi.org/10.1016/j.ntt.2017.08.003
Received 18 August 2016; Received in revised form 17 May 2017; Accepted 24 August 2017
Available online 25 August 2017
0892-0362/ © 2017 Published by Elsevier Inc.
Y.C. Rah et al.
This study was aimed to evaluate the developmental hair cell
changes with its underlying mechanisms and the various developmental
toxicities caused by the gestational caffeine exposure. By these, we'd
like to see the possibility of potential congenital hearing loss caused by
gestational caffeine exposure.
2. Materials and methods
2.1. Zebrafish housing and chemicals
Adult zebrafish were maintained in aquaria with a continuous re-
circulating system under a light (14 h):dark (10 h) photoperiod. Adult
zebrafish were fed two times a day with newly hatched Artemia brine
shrimp (San Francisco Bay Brand Inc., Newark, CA, USA). Male and
female pairs were separated with a barrier in a seeding box containing a
mesh bottom to prevent the seeded embryos from being devoured. The
boxes were incubated overnight in a 28.5 ± 1 °C zebrafish facility at
the Korea University Ansan Hospital. The next day, the barrier was
removed at the beginning of the light period and the adult zebrafish
started seeding eggs (Abdelkader et al., 2013).After fertilization, wild-
type AB zebrafish and transgenic (Tg) zebrafish (brn3c:EGFP) modified
to express GFP in neuromasts were maintained in embryo medium
(15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 0.15 mM
KH2PO4, 0.05 mM NH2PO4, and 0.7 mM NaHCO3) (Westerfield, 2000)
and staged according to days post-fertilization (dpf) (Kimmel et al.,
1995). Caffeine, 1,3,7-trimethylxanthine (ReagentPlus®, powder), was
purchased from Sigma-Aldrich (CAS No. 58-08-2). The caffeine solution
was prepared at concentrations of 25 μM, 125 μM, 250 μM, and 500 μM
by dissolving caffeine powder in distilled water. This study protocol
was approved by the Korea University Institutional Animal Care and
Use Committee (approval no. KUIACUC-2015-173). All experiments
were performed in accordance with the guidelines of the Animal Care
Ethics Committee of Korea University Medical Center and the National
Institutes of Health guidelines.
2.2. Developmental toxicity evaluation of the embryos to caffeine exposure
For toxicity studies, healthy embryos were transferred to a petri
dish. Different concentrations of caffeine (25 μM, 125 μM, 250 μM, and
500 μM) were added to the dishes and incubated at 28.5 ± 1°Cfor
72 h. Hatching rate was expressed as the percentage of the number of
embryos that hatched compared with the control at 48 hpf (n = 56–74
embryos for each concentrations).Heart rate at 48 hpf was recorded
using a stopwatch under direct microscopic observation for 60 s
(n = 60 embryos for each concentration). The mortality of the embryos
was expressed as the percentage of the total number of dead embryos
compared with the total embryos at 48 hpf (n = 52–57 embryos for
each concentrations). Morphologic changes were assessed based on the
general morphologic scoring system (0.5: not evident structures, 1:
multiple abnormalities, 2: two or more abnormalities, 3: only 1 ab-
normality, 5: entirely normal) suggested by Panzica-Kelly et al. for body
shape, somites, notochord, tail, dorsal fins, pectoral fins, and heart
(n = 37–40 for each concentrations) (Panzica-Kelly et al., 2010).
2.3. Hair cell toxicity evaluation caused by gestational caffeine exposure
The wild type AB zebrafish embryos were exposed to caffeine for
48 h (from 72 hpf to 120 hpf) at each of the following concentrations:
25 μM, 125 μM, 250 μM, and 500 μM. At 120 hpf, the embryos were
then washed with embryo medium three times and anesthetized using
tricaine (3-aminobenzoic acid 0.4 g/ethyl ester; 100 mL; pH 7, adjusted
using Tris buffer) for 5 min as described in other publications (Chang
et al., 2013; Choi et al., 2014; Ou et al., 2009; Park et al., 2014; Song
et al., 2014).The embryos were then mounted on a depression slide in
methylcellulose. The number of hair cells present within the 4 neuro-
masts (supraorbital [SO1 and SO2], otic [O1] and occipital [OC1]) on
2
Neurotoxicology and Teratology 64 (2017) 1–7
one side of each fish was determined (Ou et al., 2007; Owens et al.,
2007; Song et al., 2014). The average number of total hair cells within
the 4 neuromasts of each zebrafish under all experimental and control
conditions (n = 25–30 for each concentrations) was determined using a
fluorescence microscope (LSM5 PASCAL; Carl Zeiss, Germany).
In addition, hair cell apoptosis of Tg (brn3c:EGFP) embryos was
investigated by performing terminal deoxynucleotidyl transferase
[TdT]-mediated dUTP-biotin nick end labeling (TUNEL) staining using
an in situ cell detection kit (Roche Molecular Biochemicals, Mannheim,
Germany), in accordance with the manufacturers protocol at 120 hpf.
We previously reported the use of this protocol (Choi et al., 2013; Hong
et al., 2013).Hair cell apoptosis within the four neuromasts (SO1, SO2,
O1, and OC1) were assessed under a fluorescence microscope (LSM5
PASCAL; Carl Zeiss, Germany) and scored by averaging the TUNEL-
positive cells across those neuromasts (n = 27–33 embryos for each
concentrations).
To evaluate the mitochondrial damage by caffeine exposure, the 2-
(4-[dimethylamino] styryl)-N-ethylpyridinium iodide (DASPEI;
Invitrogen, Carlsbad, CA, USA) was used to stain mitochondria within
the hair cells of wild (AB) type zebrafish embryos at 120 hpf.
Considering that the DASPEI stains the mitochondrial inside the cyto-
plasm, the results were quantified by measuring the relative fluores-
cence-positive area inside the hair cells and averaged across four neu-
romasts by using image J software (National Institutes of Health,
Besthesda, MD, USA) (n = 29–31 embryos for each concentrations).
2.4. Statistical analysis
All data are presented as the mean ± standard deviation if ap-
plicable. For statistical comparisons, chi-squared tests were used for
variables with nominal values such as hatching rate. Fisher's exact test
was used for cases with observed value < 5 cases instead of chi-squared
test. One-way analysis of variance (ANOVA) was used for multi-group
comparisons. Post hoc analysis was completed using the Tukey's hon-
estly significant difference test. P < 0.05 was considered to be statis-
tically significant. Statistical analysis was performed with IBM SPSS
20.0 for Windows (IBM, Armonk, NY, USA).
3. Results
3.1. Developmental toxicity caused by gestational caffeine exposure
Morphologic abnormality was increased in high caffeine con-
centrations (Fig. 1A). In the analysis of 3 dpf larva, significantly low
morphologic scores were confirmed in high caffeine concentrations
(500 μM for somites, body shape, and dorsal fin; 250 and 500 μM for
notochord and heart) (Fig. 1B). For 5 dpf larva, significantly low mor-
phologic scores were also confirmed in high caffeine concentrations
(500 μM for body shape, tail, and heart; 250 and 500 μM for notochord)
(Fig. 1C). Morphologic scoring was carried out by scoring system sug-
gested by Panzica-Kelly et al. (2010).
Hatching rate was increased as the caffeine concentration increases.
A total of 46.6% of embryos hatched in the normal control and 100%
hatched in 500 μM caffeine at 48 hpf (p < 0.01, n = 60, chi-squared
test, Fig. 2). Mortality was increased in a dose-dependent manner. In
the normal control, morality was 3.3%. Caffeine exposure at 500 μM
increased mortality to 11.7% at 48 hpf; however, this difference was
not statistically significant (p = 0.565, Fisher's exact test, Fig. 2). The
heart rate of caffeine-exposed embryos showed a slight decrease at
higher caffeine concentrations and reached an average of
118.62 ± 16.17 heart beats per min at 500 μM caffeine from that of
126.07 ± 19.74 heart beats per min in the normal control; however,
the difference was not statistically significant (p = 0.073, one way
analysis of variance, Fig. 2).
Y.C. Rah et al. Neurotoxicology and Teratology 64 (2017) 1–7

Fig. 1. Morphologic abnormality was increased as the caffeine concentration increases (A).Significantly low morphologic score was confirmed with high dosecaffeine administration than
the normal control groupfor somite (p < 0.01§, 500 μM*), body shape (p < 0.01§, 500 μM*), notochord (p < 0.01§, 250 μM*, 500 μM*), dorsal fin (p = 0.01§, 500 μM†), heart
(p < 0.01§, 250 μM*, 500 μM*) at 3 dpf. For 5 dpf larva, low morphologic score was also confirmed for body shape (p < 0.01§, 500 μM*), notochord (p < 0.01§, 250 μM*, 500 μM*),
tail (p = 0.033§, 500 μM‡), heart (p < 0.01§, 500 μM#) (C).·The morphologic scoring was carried out by using the scoring system suggested by Panzica-Kelly et al.
*Significant differences between all other caffeine concentrations and control group in multiple pairwise tests.
†Significant differences between 500 μM and control group; 500 μM and 125 μM in multiple pairwise tests.
‡Significant difference between 500 μM and control group in multiple pairwise test.
#Significant difference between 500 μM and 250 μM in multiple pairwise test.
§
Significant differences among groups in one way analysis of variance tests.
All Post-hoc analysis by Turkey's honestly significant difference test.

Fig. 2. Physiologic changes after gestational caffeine ex-

posure. Hatching rate increased at high caffeine con-
centrations. A total of 46.6% of embryos hatched in the
normal control, 67.3% in 25 μM caffeine, 73.2% in 125 μM
caffeine, 82.1% in 250 μM caffeine, and 100% in 500 μM
caffeine-exposed group (p < 0.01, chi-squared test).
Mortality was 3.3% in the normal control, 8.3% in 25 μM
caffeine, 6.7% in 125 μM caffeine, 6.7% in 250 μM caffeine,
and 11.7% in the 500 μM caffeine-exposured group.
However, these differences were not statistically significant
(p = 0.565, Fisher's exact test). Heart rate of caffeine-ex-
posed embryos showed a slight decrease at higher caffeine
concentrations that reached an average of
118.62 ± 16.17 heart beats/min at 500 μM caffeine from
that of 126.07 ± 19.74 heart beats/min in the normal control (124.02 ± 12.99 in 25 μM caffeine, 122.94 ± 16.83 in 125 μM caffeine, and 122.81 ± 19.34 in 250 μM caffeine).
However, the increase was not statistically significant (p = 0.073, ANOVA, n = 60). All data were evaluated at 48 hpf.

3
Y.C. Rah et al.
3.2. Hair cell damage caused by gestational caffeine exposure
The relationship between caffeine concentration and hair cell da-
mage was evaluated in four neuromasts (SO1, SO2, O1, and OC1) of the
wild type AB zebrafish embryos. Caffeine exposure for 48 h (from
72 hpf to 120 hpf) significantly reduced the average number of total
hair cells in all caffeine exposure groups compared with the normal
control (35.64 ± 10.48 cells, n = 30, p < 0.01, one way analysis of
variance, Fig. 3A, B). However, there was no significant difference
among caffeine-exposed groups (25 μM caffeine: 24.72 ± 7.32 cells,
125 μM caffeine: 24.28 ± 8.70 cells, 250 μM caffeine: 25.24 ± 9.97
cells, and 500 μM caffeine: 23.32 ± 12.14 cells, Fig. 3B).
4
Neurotoxicology and Teratology 64 (2017) 1–7
Fig. 3. Hair cells in the four neuromasts (SO1, SO2, O1, and OC1)
decreased in caffeine-exposed groups under a fluorescent micro-
scopic examination (× 40, wild type AB zebrafish, at 120 hpf) (A).
Caffeine exposure for 48 h (from 72 hpf to 120 hpf) significantly
decreased the number of total hair cells of the 4 neuromasts within
caffeine exposure groups compared with that of the normal control
(B) However, there was no significant difference among caffeine-
exposed groups.
*p = 0.01; **p = 0.02 (Turkey's honestly significant difference
test).
3.3. Hair cell apoptosis and mitochondrial damages caused by gestational
caffeine exposure
The number of TUNEL-positive hair cells was increased as the caf-
feine concentration increases showing significantly different distribu-
tions among groups (p < 0.01, one way analysis of variance).Normal
control group exhibited negative TUNEL reaction, consequently
showing significant differences from all caffeine-exposed groups. The
TUNEL-positive hair cells were also increased as the caffeine con-
centration increases with significant differences between each caffeine-
exposed groups. Those results indicate that the gestational caffeine
exposure caused the hair cell apoptosis in a significant dose-dependent
manner. Details are shown in Fig. 4.
Y.C. Rah et al. Neurotoxicology and Teratology 64 (2017) 1–7

Fig. 4. Caffeine-induced apoptosis was confirmed by TUNEL assay.

Apoptotic cells appeared as bright red dots in the background of
natural green fluorescence of hair cells. The TUNEL-positive cells
were increased as the caffeine concentration increases (A, O1
neuromast) Caffeine-exposed groups had significantly more TUNEL-
positive cells than normal control which showed negative TUNEL
reaction (p < 0.01 for all comparisons, Tukey's honestly sig-
nificant difference test) and showed increasing number of TUNEL-
positive cells as the increase of the caffeine concentration in a dose
dependent manner (p < 0.01, One way analysis of variance).All
images were captured using Tg (brn3c:EGFP) zebrafish at 120 hpf.
Scale bars = 10 μm (× 10), 200 μm (× 40).
*Significantly different from normal control group.
†Significantly different from 25 μM caffeine exposed group.
‡Significantly different from 125 μM caffeine exposed group.
#Significantly different from 250 μM caffeine exposed group.
All Post-hoc analysis by Turkey's honestly significant difference test.
(For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

Fig. 5. Mitochondrial viability assessed by DASPEI assay. The mi-

tochondria in live cells were stained as bright green fluorescence by
DASPEI assay and decreased as the caffeine concentration increases
(A). The relative DASPEI-positive area was significantly different
among groups (p < 0.01, One way analysis of variance). Caffeine-
exposed group of high concentration (250 and 500 μM) showed
significantly smaller DASPEI-positive green fluorescence-occupied
area than normal control group (B).
*Significantly different from normal control group.
†Significantly different from 25 μM caffeine exposed group.
‡Significantly different from 125 μM caffeine exposed group.
All Post-hoc analysis by Turkey's honestly significant difference test.
(For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

5
Y.C. Rah et al.
The DASPEI assay which stains the mitochondria in the live cells
showed significant decrease of the DASPEI-positive green fluorescence-
occupied area in the 250 and 500 μM ethanol-exposed groups compared
with either of normal control, 25, and 125 μM ethanol exposed groups
(p < 0.01, one way analysis of variance). Those results imply that the
gestational caffeine exposure could cause mitochondrial damage that
could lead the cells to fall into apoptosis as confirmed in the TUNEL
assay in high ethanol concentrations (250 and 500 μM). Details are
shown in Fig. 5.
4. Discussion
According to our data, gestational caffeine exposure caused devel-
opmental hair cell loss with associated various developmental anoma-
lies. Regarding the underlying mechanisms of hair cell loss, it was
suggested that caffeine-induced oxidative stress might trigger apoptosis
via a mitochondria-dependent pathway (Abdelkader et al., 2013).Some
in vitro studies reported that caffeine caused a reduction of mitochon-
drial membrane potential and induced apoptosis in a dose-dependent
manner, which was further decreased by the inhibition of autophagy
with 3-methyladenine or Atg7 siRNA knockdown in various cell lines
(Saiki et al., 2011). Caffeine also triggered apoptosis by stimulating
mitochondria-dependent cell death pathways in osteoblasts (Lu et al.,
2008). In the current study, caffeine exposure for 48 h (from 72 to
120 hpf) significantly decreased the number of hair cells and increased
apoptosis as presented in the TUNEL assay, which was postulated to be
the cause of hearing loss. Mitochondrial damage was also confirmed by
the significant decrease of DASPEI-positive green fluorescence as caf-
feine concentration increases. Considering that the hair cell is the ul-
timate sensory apparatus for hearing, the developmental hair cell loss
inevitably results in congenital hearing loss (Ahroon et al., 1993).In
another animal model assessing the effect of caffeine on hearing, it was
indicated that caffeine attenuated hearing recovery after acoustic
trauma in guinea pigs with reduced remaining spiral ganglion cell
densities (Mujica-Mota et al., 2014).Another animal study using guinea
pigs also confirmed that a daily dose of caffeine could impair the re-
covery of hearing loss caused by acoustic overstimulation with inner
ear hair cell damages (Zawawi et al., 2016).Considering these experi-
mental data, more sophisticated clinical investigations necessary re-
garding the potential adverse effect of gestational caffeine exposure on
neonatal hearing and inner ear hair cells.
In the present study, embryonic mortality was increased in a dose-
dependent manner with caffeine administration at 48 hpf. These results
correspond well with those of earlier studies that reported a decreased
survival of zebrafish embryos after caffeine exposure (Pruvot et al.,
2012)and clinically, significantly increased pregnancy loss by 19%with
150 mg/day or by 8% with 2 cups/day in a meta-analysis (Li et al.,
2015).Increased hatching was confirmed by caffeine exposure at 48 hpf
in the present study, which is consistent with that of an earlier study
that showed higher maternal caffeine intake leads to a greater risk of
preterm birth in humans (Okubo et al., 2015). However, in another
study with zebrafish animal model, significantly decreased hatching
rate was confirmed by caffeine administration at 60- and 72-hpf
(Abdelkader et al., 2013).
Our data showed a slight decrease in heart rates with caffeine ad-
ministration without statistical significance. Previous studies reported a
dose-dependent decrease of heart rate by caffeine administration with
48- and 72-hpf zebrafish embryos (Pruvot et al., 2012; Rana et al.,
2010). However, the influence of gestational caffeine intake on fetal
heart rate seems to be controversial. In some studies, maternal caffeine
intake increased fetal reactivity including fetal heart rate acceleration
(Buscicchio et al., 2012) andcaffeine-induced heart rate increase was
also reported in studies using zebrafish (Luca et al., 2014; Abdelkader
et al., 2013). For the underlying mechanisms of heart rate alteration,
the antagonizing effect of caffeine against adenosine receptors (espe-
cially A1 receptor) was reported (Rana et al., 2010). The A1 adenosine
6
Neurotoxicology and Teratology 64 (2017) 1–7
receptor activation also inhibits cardiac cell proliferation and can lead
to cardiac hypoplasia (Zhao and Rivkees, 2001). Caffeine could also
affect cardiomyocyte formation and function by altering the expression
pattern of gap junctions (Ahir and Pratten, 2016). In another study, it
was postulated that caffeine affected heart rate by inhibition of ether-a-
go-go potassium channels (Rana et al., 2010).
In the present study, morphologic abnormality was increased in a
dose-dependent manner. These results correspond well with those of
earlier studies that reported gestational caffeine induced morphological
defects in zebrafish embryos (Lantz-McPeak et al., 2015; Pruvot et al.,
2012). However, the caffeine concentrations of the present study that
induced developmental toxicity was much lower than that of Lantz-
McPeak et al., which suggested that concentrations of caffeine over
500 μM could cause developmental toxicity (Lantz-McPeak et al.,
2015). Lethal concentration 50 (LC50) of caffeine ranges from 50 to
500 mg/kg, which is equivalent to a range of 260–2500 μM (Dews,
1982; Lantz-McPeak et al., 2015).In another study, developmental de-
fects occurred at a concentration of 286 μM caffeine in 48–72 hpf
zebrafish and the LC50 was estimated at 5000 μM in 48-hpf zebrafish
(Pruvot et al., 2012). It was also reported that acute treatment with
1 mM concentration of caffeine induced a typical kinking effect in the
trunk and tail region (Rana et al., 2010).
Typically, developmental toxicity was evaluated by continuous ex-
posure of embryos to the test chemicals starting at the earliest possible
zebrafish developmental stage (i.e., 2–3 hpf) because major develop-
mental events including conversion-extension, segmentation, otolith
organ formation, heart beat and blood circulation, occur during the first
48 h of development (Asharani et al., 2008; Ellis et al., 2014; Jang et al.,
2014; Massarsky et al., 2013; Nagel, 2002; Pruvot et al., 2012; Zhang
et al., 2015).In hair cell development, primary neuromasts emerge on
the lateral lines of the zebrafish at 2 days post-fertilization (dpf) and are
present on all major lateral lines including O1 (on otic lateral line), OC1
(on occipital lateral line), SO1 and SO2 (on supraorbital lateral line)
neuromast by 4 dpf (Pujol-Marti and Lopez-Schier, 2013; Raible and
Kruse, 2000). Hair cells are functional a few hours after deposition
enabling successful physiological recordings of evoked potential by
5 dpf (Brack and Ramcharitar, 2012; Kindt et al., 2012; Lopez-Schier
et al., 2004)Thus, experimental measurements for developmental hair
cell changes of the lateral lines has been carried out at 5 dpf (Kindt
et al., 2012; Nicolson et al., 1998; Lopez-Schier et al., 2004) as in the
present study.
In conclusion, the results of the present study confirmed that caf-
feine exposure in early developmental stages potentially causes hair cell
damage and increases morphologic anomalies and mortality in zebra-
fish. To our knowledge, this is the first study to investigate into the
detailed developmental hair cell damage related with gestational caf-
feine exposure in zebrafish. Further studies with mammals could pro-
vide more practical evidences for the embryotoxicity of caffeine in
human development.
Transparency document
The http://dx.doi.org/10.1016/j.ntt.2017.08.003 associated with
this article can be found, in online version.
Acknowledgments
This research was supported by a Korea University Grant and a
Grant-in-Aid from the Department Foundation of Otorhinolaryngology-
Head and Neck Surgery, College of Medicine, Korea University and Soo
ENT clinic.
Conflict of interest
The authors have no conflicts of interest to declare.
Y.C. Rah et al.
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