General and Comparative Endocrinology 327 (2022) 114095

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General and Comparative Endocrinology

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

Research paper

Water hardness alleviates the stress response caused by waterborne zinc in

goldfish Carassius auratus
Zhongze Li a, 1, Jin Ah Song b, 1, Min Ju Kim c, Cheol Young Choi a, c, *
a
Division of Marine BioScience, Korea Maritime & Ocean University, Busan 49112, Republic of Korea
b
Marine Bio-Resources Research Unit, Korea Institute of Ocean Science and Technology, Busan 49111, Republic of Korea
c
Department of Convergence Study on the Ocean Science and Technology, Korea Maritime and Ocean University, Busan 49112, Repiblic of Korea

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

Keywords: In this study, the combined effect of waterborne Zn and water hardness on the stress response in the goldfish
Zinc toxicity Carassius auratus was investigated. Goldfish were exposed to Zn concentrations of 0.5, 1.0, and 3.0 mg/L and
Water hardness water hardness of 90, 270, and 450 mg/L CaCO3 for 1, 3, 7, and 14 d. After exposure, it was determined that
Hypothalamus–pituitary–interrenal axis
higher the Zn concentration, the more obvious the stress response. However, the stress response reduced with
Na+/K+-ATPase
increasing water hardness. An increase in the Zn concentration caused stress responses in fish according to the
Goldfish
increase in the mRNA expressions of corticotropin-releasing hormone and adrenocorticotropic hormone and
cortisol level in the hypothalamus–pituitary–interrenal axis. The expression of these factors was the highest on
day 7 and decreased on day 14. Furthermore, to evaluate the stress change in the liver tissue, we analyzed
alanine aminotransferase, aspartate aminotransferase, and heat shock protein 70 concentrations to determine the
damage caused by Zn and the change in water hardness. Immunohistochemistry staining for Na+/K+-ATPase in
the gills showed that the gill activity was inhibited by Zn, and an increase in water hardness could improve Na+/
K+-ATPase. In conclusion, we found that increasing water hardness is a successful method to reduce the stress
response in goldfish caused by Zn.

1. Introduction immunity (Carruth et al., 2002; Bernier et al., 2009).

When fish are exposed to a stressful environment, heat shock pro­
Various wastes, including metals such as Zn, are generated as a result teins (HSPs) are expressed along with changes in the HPI axis. HSP is
of industrialization, and have a negative impact on aquatic ecosystems expressed to prevent destruction of protein structure due to heat shock;
when they are released into the environment (Javed and Usmani, 2019). however, even when exposed to metals, HSP expression is induced to
According to Li et al. (2019), the concentration of Zn has increased reduce stress and protect the cells (Xie, 2017; Savassi et al., 2020).
exponentially in the global combined river and lake water from the Alanine aminotransferase (ALT) and aspartate aminotransferase (AST),
1970 s (0.23 ± 0.47 mg/L) to the 2010 s (0.48 ± 1.38 mg/L). When fish which are expressed in the liver tissue, are enzymes that play important
are exposed to metals, their enzyme activity decreases and cancer and roles in amino acid metabolism. ALT and AST are also widely used as an
birth defects occur (Zhang et al., 2019). In fish, the hypothalamus- index for stress due to metal exposure because of it sensitivity to metals
pituitary-interrenal (HPI) axis defends against stress induced by (Almeida et al., 2001; Atli et al., 2015).
metals, which act as a stress factor (Hontela, 2005). In the HPI axis, Among metals, Zn is known to promote the activation of mineral
corticotropin-releasing hormone (CRH) released from the hypothalamus enzymes in the body (Anandkumar et al., 2020). Although Zn is an
stimulates the pituitary gland, which releases the adrenocorticotropic essential trace element for the survival of living organisms, high Zn
hormone (ACTH). Finally, the interrenal system releases cortisol (Bonga, concentrations can adversely affect an organism’s stress response,
1997; Mommsen et al., 1999). Cortisol, a stress indicator, is released into metabolism, and biochemical effects (Atli and Canli 2007; Gioda et al.,
the blood and changes into blood glucose through gluconeogenesis to 2007).
control various physiological phenomena such as metabolism and In particular, metal ions (including Zn2+) act on fish gills and have

* Corresponding author at: Division of Marine BioScience, Korea Maritime & Ocean University, Busan 49112, Republic of Korea.
E-mail address: [email protected] (C.Y. Choi).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ygcen.2022.114095
Received 7 March 2022; Received in revised form 24 May 2022; Accepted 8 July 2022
Available online 11 July 2022
0016-6480/© 2022 Elsevier Inc. All rights reserved.
Z. Li et al. General and Comparative Endocrinology 327 (2022) 114095

adverse effects such as inhibiting Na+/K+-ATPase activity (Bianchini Table 1

and Wood, 2003; Bianchini et al., 2005). Na+/K+-ATPase is located in Primers used for qPCR amplification.
the epithelial cells of the gills and regulates the body ion balance in fish Genes (Accession no.) Primer DNA sequences
to maintain cell volume, osmotic pressure, and membrane permeability
CRH (XM_026201362.1) Forward 5ʹ-CCC GAG ACA TCC CAG TAT T-3ʹ
in a normal state (Atli and Canli, 2013). Reverse 5ʹ-GTA ATT GCC ATC CAA GCG A-3ʹ
However, the toxicity of metals (including Zn) depends on the pH, ACTH (XM_026285142.1) Forward 5ʹ-TAT TCC ACG CTC TAC GCT A-3ʹ
temperature, and hardness of the aquatic environment, which causes a Reverse 5ʹ-AGA AGA TTT TCA CCG TAG CA-3ʹ
difference in the stress response in fish (Barron and Albeke, 2000; Qu HSP70 (AB092839.2) Forward 5ʹ-TGC TAT TGG GAT TGA CCT GG-3ʹ
Reverse 5ʹ-GGC AGG AAC TGT GAT AAC TG-3ʹ
et al., 2014; Kabir et al., 2020). Among the various environmental fac­ β-actin (LC382464)) Forward 5ʹ-TTC CCT TGC TCC TTC CAC CA-3ʹ
tors that change in water environments, water hardness, in particular, Reverse 5ʹ-TGG AGC CAC CAA TCC AGA CA-3′
which refers to the total amount of divalent ions (Ca2+ and Mg2+) in
water, affects fish toxicity due to metal exposure (Pilehvar et al., 2020).
As a result of measuring the lethal concentrations of Cd and Cu affecting mg/L of Zn in hard water (Zn 1.0 + H); 3.0 mg/L of Zn in hard water (Zn
50 % of the test population (LC50) in Fathead minnow Pimephales 3.0 + H); 0 mg/L of Zn in very hard water (Zn 0 + V); 0.5 mg/L of Zn in
promelas in a study by Sorensen (1991), it was found that the LC50 value very hard water (Zn 0.5 + V); 1.0 mg/L of Zn in very hard water (Zn 1.0
(i.e., toxicity decreased) increased as the water hardness increased. + V); and 3.0 mg/L of Zn in very hard water (Zn 3.0 + V). Four fish from
Other studies have also found that metals are more toxic in soft water each replicate treatment group were randomly sampled at each time
than hard water (Daramola and Oladimeji, 1989; Kiyani et al., 2013). period. During the exposure time, no death occurred in the fish and
However, in the case of studies related to metals and water hardness hardness, measured every day, did not change.
conducted to date, limited studies have observed the molecular physi­ Goldfish were anesthetized using 100 mg/L clove oil (C8392; Sigma)
ology of stress changes in fish due to exposure to various metal con­ (Perdikaris et al., 2010). Blood was collected rapidly from the caudal
centrations and water hardness (Ebrahimpour et al., 2010; Karthikeyan vein using a 1-mL disposable syringe coated with heparin. Plasma
and Mani, 2014). samples were separated by centrifugation (4 ◦ C at 12, 000 × g for 12
Therefore, our study was carried out to determine whether the stress min). Liver and brain samples were stored at − 80 ◦ C, and portions of the
caused by Zn exposure in goldfish reduces depending on the water liver and gill samples were stored at 4 ◦ C in 4 % paraformaldehyde (PFA)
hardness environment, and to what extent. We selected goldfish as the for further analysis.
experimental model, as this species may be exposed to Zn2+ due to
pollution of freshwater environments. After exposing goldfish to various
2.3. Total RNA extraction and cDNA synthesis
Zn concentrations and water hardness environments, we compared
changes in HPI axis-related hormones in the body and HSP70 mRNA
Total RNA was extracted from the brain and liver using TRI Re­
expression in the liver tissue by in-situ hybridization. In addition, by
agent® (TR188, Molecular Research Center, Cincinnati, OH, USA) ac­
comparing the Na+/K+-ATPase expression status in the gills, the effect of
cording to the manufacturer’s instructions. The purity (A260/A280) of
water hardness on the reduction in toxic stress in goldfish generated by
all RNA samples was between 1.8 and 2.0. According to the manufac­
Zn exposure was confirmed.
turer’s protocol of M-MLV reverse transcriptase (RT0015, Takara,
Tokyo, Japan), total RNA (2 μg) was reverse-transcribed to comple­
2. Materials and methods
mentary DNA (cDNA) using an oligo-(dT)15 anchor. All the synthesized
cDNAs were diluted by 1:50 and stored at − 20 ◦ C prior to analysis.
2.1. Experimental fishes

Immature goldfish Carassius auratus (n = 384, body length 6.8 ± 0.8 2.4. Real-time polymerase chain reaction of CRH and ACTH mRNA
cm; mass 10.4 ± 2.1 g) were purchased randomly from Choryang
Aquarium, Busan, Korea and were acclimated for one week in a 300-L The relative expression of CRH, ACTH, and β-actin was measured
freshwater tank in the laboratory. Fish were fed twice and 50 % of the using real-time quantitative polymerase chain reaction (qPCR). qPCR
water was changed daily. The water temperature was maintained at 19.5 primers were designed according to known sequences in the NCBI
± 1 ◦ C, pH was 7.5 ± 0.2, and aeration was provided. Feeding was database (Table 1). qPCR amplification was performed using Bio-Rad
discontinued 24 h before the experiment. The animal study protocol was iCycler iQ multicolor real-time PCR detection system (Bio-Rad, Contra
approved by the Institutional Animal Care and Use Committee of Korea Costa County, CA, USA), and the qPCR was performed in a 25 μL solution
Maritime and Ocean University (Approved protocol no # KMOU IACUC containing 0.5 μL of cDNA, 0.25 μM of each primer, 0.2 mM dNTP, SYBR
202202). Green, and Taq polymerase in buffer (pH 9.0 of 10 mM Tris-HCl, 50 mM
KCl, 1.4 mM MgCl2, and 20 nM fluorescein). The PCR process was set as
2.2. Zinc and water hardness treatments and sampling follows: one cycle of denaturation at 95 ◦ C for 5 min, 35 cycles of
denaturation at 95 ◦ C for 20 s, annealing at 55 ◦ C for 20 s. The ampli­
Prior to the experimentation, no zinc was detected in the water. fication efficiencies were found to be as follows: b-actin = 99.1 %, CRH
Goldfish were exposed to Zn concentrations of 0, 0.5, 1, and 3 mg/L = 98.4 %, and ACTH = 97.9 %. All data are expressed as changes with
(using ZnSO4, 83265; Sigma, St. Louis, MO, USA). The Zn concentration respect to the corresponding β-actin-calculated cycle threshold (ΔCt)
was set according to previous studies (Huo, 1996; Qu et al., 2014). Water levels. The calibrated ΔCt values (ΔΔCt) for samples and the internal
hardness was measured by the addition of CaCO3 (239216; Sigma) to control (β-actin) were calculated using the equation ΔΔCt = 2− (ΔCt
soft (S, 90 mg/L), hard (H, 270 mg/L), and very hard (V, 450 mg/L) sample− ΔCt internal control)
.
water. The tap water CaCO3 concentration was detected at 90 mg/L in
Busan, Korea, using a water hardness meter (PWH-303, Lutron, China).
Exposure times were 0, 1, 3, 7, and 14 d. 2.5. Plasma cortisol level
We named the experimental groups according to the water hardness
and Zn concentration as follows: 0 mg/L of Zn in soft water (Zn 0 + S); Cortisol level in the plasma was measured using an enzyme-linked
0.5 mg/L of Zn in soft water (Zn 0.5 + S); 1.0 mg/L of Zn in soft water immunosorbent assay kit (MBS165888, Mybiosource, San Diego, CA,
(Zn 1.0 + S); 3.0 mg/L of Zn in soft water (Zn 3.0 + S); 0 mg/L of Zn in USA) following the manufacturer’s protocol, and absorbance was
hard water (Zn 0 + H); 0.5 mg/L of Zn in hard water (Zn 0.5 + H); 1.0 measured at 450 nm.

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Z. Li et al. General and Comparative Endocrinology 327 (2022) 114095

Fig. 1. Changes in the mRNA expression of (A) corticotropin-releasing hormone CRH and (B) adrenocorticotropic hormone (ACTH) measured for 14 d. Different
letters indicate significant differences among goldfish exposed to the same Zn concentration and water hardness for a different exposure time (P < 0.05). Different
numbers indicate significant differences among goldfish exposed to different Zn concentrations and water hardness for the same duration (P < 0.05). Values are
presented as the means ± SD.

2.6. HSP70 mRNA in-situ hybridization capture images.

The HSP70 sequence for the in-situ hybridization probe was designed 2.7. Plasma parameter analysis
as shown in Table 1, amplified using PCR, and ligated into a pGEM-T
easy vector (A137A, Promega, Madison, WI, USA). Furthermore, the Plasma samples were separated by centrifugation and then plasma
antisense was confirmed by sequencing, and plasmid DNA was amplified glucose (Code 1050, Fujifilm, Tokyo, Japan), ALT (Code 3250, Fujifilm),
using PCR with the antisense and T7 primers (5ʹ-TAA TAC GAC TCA CTA and AST (Code 3150, Fujifilm) levels were measured using the dry
TAG GG-3ʹ). Digoxigenin (DIG)-labelled probes were created using a DIG multiplayer analytic slide method in a biochemistry autoanalyzer (Fuji
RNA Labeling Mix (11277073910, Merck, Darmstadt, Germany), and Dri-Chem 4000; Fujifilm).
PCR products using the anti-sense primer and T7 RNA polymerase were
used as the anti-sense labelling probes. 2.8. Immunohistochemistry (IHC) staining of Na+/K+-ATPase in liver
The liver tissues of the control (Zn 0 + S), Zn 3.0 + S, Zn 3.0 + H, and
Zn 3.0 + V groups exposed for 14 d were fixed using 4 % PFA for at least Na+/K+-ATPase expression in the liver was detected immunocyto­
18 h, and stored in 30 % sucrose to prevent freezing during the exper­ chemically based on the method described by Takata et al. (2000), with
iment. Hybridization buffer (50 mL deionized formamide, 25 mL of 20 modifications. First, the livers were fixed in 4 % PFA, dehydrated in
× saline sodium citrate (SSC), 1 mL 0.1 % Tween-20, 920 μL 1 M citric ethanol, and embedded in paraffin. In brief, paraffin-embedded sections
acid, and DEPC water up to a total volume of 100 mL) was used to hy­ were deparaffinized in xylene, rehydrated in ethanol, and then incu­
bridize the liver tissue, yeast total RNA (50 μL with 950 μL of hybridi­ bated overnight at 4 ◦ C with primary mouse anti-Na+/K+-ATPase anti­
zation buffer), and the tissue was hybridized overnight at 65 ◦ C. Tissues bodies (dilution 1:200; TA309580, Origene, Rockville, MD, USA) as well
were washed with a mixture of hybridization buffer containing 2 × SSC as with secondary antibodies afterwards (HRP-conjugated anti-mouse
and 0.2 × SSC with PBST (phosphate buffered saline + tween20). The immunoglobulin; dilution 1:1000). Antibody binding was visualized
tissues were then treated with 10 % calf serum and antibody (anti-DIG- using 3,3-diaminobenzidine as the detection system. Slides were coun­
AP) overnight at 4 ◦ C in darkness. terstained and mounted with Canada balsam for observation under a
Then, the sections were washed with PBST and treated with alkaline light microscope (DM 100; Leica, Wetzlar, Germany), and images were
Tris buffer and labeling mix. Finally, all sections were mounted using captured using a digital camera (DS-Fi1c, Nikon, Tokyo, Japan).
Aquamount (Aqua Polymount, Warrington, PA, USA) and covered with
a slip. A stereomicroscope (Nikon Eclipse Ci, Tokyo, Japan) was used to

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Z. Li et al. General and Comparative Endocrinology 327 (2022) 114095

Fig. 2. Changes in the cortisol level measured for 14 d. Different letters indicate significant differences among goldfish exposed to the same Zn concentration and
water hardness for different exposure time (P < 0.05). Different numbers indicate significant differences among goldfish exposed to different Zn concentrations and
water hardness for the same duration (P < 0.05). Values are presented as the means ± SD.

Fig. 3. In-situ hybridization of the control and Zn 3.0 treatments on day 14. (A) Zn 0 + S, (B) Zn 3.0 + S, (C) Zn 3.0 + H, (D) Zn 3.0 + V. The dark area (black arrow)
indicate HSP70 mRNA expression in the liver (scale bars = 200 μm). HE: hepatocytes.

2.9. Statistical analysis 3. Results

All experiments in the study were performed at least quadruplicate, 3.1. Changes in CRH and ACTH mRNA expression
and the data are expressed as the mean ± standard deviation (SD).
Statistical significance (P < 0.05) was assessed using SPSS version 25.0 The expressions of CRH and ACTH mRNA in the Zn exposure groups
(IMB SPSS, Armonk, NY, USA) between the different Zn concentrations, were significantly higher than those in the no Zn groups. There was no
water hardness, and different times of the sample in the data using a significant difference between the no Zn groups with changes in water
two-way ANOVA followed by Tukey’s post hoc test. hardness (Fig. 1). As the Zn concentration increased, the expressions of

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Z. Li et al. General and Comparative Endocrinology 327 (2022) 114095

Fig. 4. Changes in the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels measured for 14 d. Different letters indicate significant dif­
ferences among goldfish exposed to the same Zn concentration and water hardness for different exposure time (P < 0.05). Different numbers indicate significant
differences among goldfish exposed to different Zn concentrations and water hardness for the same duration (P < 0.05). Values are presented as the means ± SD.

CRH and ACTH mRNA increased, and peaked on day 7. Zn 3.0 + S on ng/mL, P > 0.05) groups. Among the treatments with the same Zn
day 7 exhibited the highest expressions of CRH (1.94 ± 0.17, P < 0.05) concentrations, all soft water groups showed the highest cortisol level
and ACTH (2.34 ± 0.19, P < 0.05) among all groups. ACTH expression values, whereas the very hard water groups showed the lowest values. In
was decreased in the very hard water group on day 14 compared to that the Zn 3.0 + S group, the cortisol level increased more rapidly than in
on day 7 and was lower than that on day 3. Expression of CRH on day 14 other groups; it was almost three times higher in the Zn 3.0 + S group on
had decreased compared to that on day 3. day 7 (15.07 ± 0.98 ng/mL, P < 0.05) than on day 0 (4.42 ± 0.38 ng/
mL, P < 0.05).
3.2. Changes in plasma cortisol level
3.3. HSP70 mRNA expression in the liver using in-situ hybridization
Cortisol level was similar to that of CRH and ACTH (Fig. 2). There
was no significant difference between Zn 0.5 and Zn 1.0, except in the Zn On day 14, HSP70 mRNA expression in the liver was detected by in-
0.5 + H (9.50 ± 0.84 ng/mL, P > 0.05) and Zn 1.0 + H (11.20 ± 0.94 situ hybridization (Fig. 3). In Fig. 3, the dark area (black arrow) indicates

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Z. Li et al. General and Comparative Endocrinology 327 (2022) 114095

Fig. 5. Immunohistochemistry (IHC) staining of the control (Cont.) and Zn 3.0 treatments on day 14: (A) Zn 0 + S; (B) Zn 3.0 + S; (C) Zn 3.0 + H; (D) Zn 3.0 + V. The
brown area (black arrows) indicates the Na+/K+-ATPase expression in the gills (scale bars = 200 μm). GA: gill arch; GL: secondary gill lamellae.

HSP70 mRNA expression in the cytoplasm and membranes of the reduced by increases in water hardness.
hepatocytes. The HPI axis is the center of the stress response in fish; CRH is
released from the hypothalamus, which acts on the pituitary gland to
3.4. Changes in glucose, ALT, and AST levels release ACTH, and cortisol is released from the interrenal (Bonga, 1997).
Li et al. (2014) reported that during exposure of the Chinese rare
Changes in glucose (Fig. 4A), ALT (Fig. 4B), and AST (Fig. 4C) levels minnow Gobiocypris rarus to Cd concentrations of 0.5 and 2.5 mg/L, the
increased steadily between the start of the experiment and day 7, as higher the Cd concentration, the higher the expression of CRH. Suprapto
shown in Fig. 4. After day 7, the levels began to decrease until day 14. et al. (2019) also reported that, as a result of exposing Java barb Bar­
The glucose, ALT, and AST levels in the Zn 3.0 groups were higher than bonymus gononotus to Pb concentrations of 0.66–2.65 ppm, the cortisol
those in other groups at the same time. With the increase in water level in the body increased as the Pb concentration increased. This study
hardness, the glucose, ALT, and AST levels decreased (glucose: on day 7 confirmed the stress induced by metals through changes in the con­
Zn 3.0 + S 438 ± 28 mmol/L and Zn 3.0 + V 245 ± 16 mmol/L; ALT: on centrations of HPI axis-related stress hormones, with results similar to
day 7 Zn 3.0 + S 58.7 ± 3.97 U/L and Zn 3.0 + V 34.3 ± 1.67 U/L; AST: those of the present study. Furthermore, Qu et al. (2014) compared the
on day 7 Zn 3.0 + S 786.0 ± 39.6 U/L and Zn 3.0 + V 481.0 ± 19.4 U/L, antioxidant enzyme activity after exposing goldfish to Zn concentrations
P < 0.05). of 0.1 and 1 mg/L for 3, 12, and 30 d, and reported that the antioxidant
enzyme activity in all the experimental groups was lower than that in the
control group; on day 30, there was no significant difference in any
3.5. IHC staining of Na+/K+-ATPase in the gills
group. In our study, CRH, ACTH expression, and cortisol level were the
highest on day 7 after Zn exposure and decreased again on day 14.
On day 14, IHC staining showed Na+/K+-ATPase expression in the
Karthikeyan et al. (2007) exposed the edible fish Cirrhinus mrigala to a Ni
epithelial cells of the gill secondary lamellae (GL) and gill arch (GA) of
concentration of 3.91 ppm in soft and hard water environments, and
goldfish (Fig. 5). In Fig. 5, the brown area (black arrows) indicates the
showed that the Ni accumulated in the gills, liver, and kidneys was
Na+/K+-ATPase in the gill. The control group (Fig. 5A) showed higher
significantly reduced in hard water environments compared to soft
Na+/K+-ATPase expression than the Zn-exposed group. The Zn 3.0 + S
water. Alsop and Wood (2011) also reported that the concentration of
group showed the lowest Na+/K+-ATPase expression in the epithelial
Zn2+ dissolved in water decreased as the water hardness increased.
cells of the gills. All groups, except Zn 3.0 + S, showed Na+/K+-ATPase
In our study, CRH and ACTH mRNA expression and cortisol level
expression in the GA.
increased with an increasing Zn concentration. However, it was
confirmed that the stress indices of the HPI axis decreased as the water
4. Discussion
hardness increased, which is suggested to have been because Zn accu­
mulation in the fish body decreased as the water hardness increased. The
In this study, the HPI axis-related stress index and stress index
stress induced by Zn was also significantly reduced.
expressed in the liver tissue and gills were compared after exposing
Unlike CRH and ACTH, which are stress indicators expressed along
goldfish, a freshwater fish, to various Zn concentrations and water
the HPI axis, HSP70 is mainly expressed in the cytoplasm and cell
hardness. We determined whether the stress caused by Zn exposure was

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membranes of hepatocytes when fish are exposed to stressful environ­ Acknowledgements

ments (Rendell et al., 2006; Rajeshkumar et al., 2013). Shen et al. (2004)
and Harsij et al. (2021) reported that when goldfish were exposed to This research was supported by Korea Institute of Marine Science &
metals such as Zn and Cu, HSP70 was expressed in the liver tissue to Technology Promotion (Grant numbers 20220559 & 20170392).
reduce stress. In the results of in-situ hybridization conducted in our
study, it was confirmed that HSP70 mRNA expression was significant in References
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