Ecotoxicology and Environmental Safety 249 (2023) 114440

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

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

The involvement of oxidative stress mediated endoplasmic reticulum

pathway in apoptosis of Golden Pompano (Trachinotus blochii) liver under
PS-MPs stress
Fu Cheng Yao, Yue Gu, Tian Jiang, Peng Fei Wang, Fei Biao Song, Zhi Zhou, Jun Long Sun *,
Jian Luo
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan Aquaculture Breeding Engineering Research Center, Hainan Academician Team
Innovation Center, Hainan University, Haikou 570228, China

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

Keywords: Globally, microplastics (MPs) are highly prevalent, especially in coastal areas. Unfortunately, golden pompano as
Polystyrene microplastics a major marine fish in China is typically raised in floating marine cages near coasts, facing these MPs sources.
Oxidative stress However, toxicological studies on Golden Pompano which farm in coastal areas and face actual microplastic
Endoplasmic reticulum stress
exposure are rare. Therefore, golden pompano were exposed to 10.0 μg/L, 100.0 μg/L, and 1000.0 μg/L poly­
Apoptosis
styrene MPs (PS-MPs) for 14 days to study the potential impact of the microplastics on the Golden Pompano. Fish
Metabolism
show slowed growth after 14 days of exposure. Histopathology shows irregular shaped nuclei and nuclear and
cytoplasmic vacuolation traits in liver. Oxidative stress-related enzyme activity and gene expression data show
that oxidative damage occurs in the high-concentrations (100.0 μg/L and 1000.0 μg/L) of PS-MPs exposures. Up-
regulation of Grp78, Xbp-1, Eif-2α and chop gene expression indicates the occurrence of endoplasmic reticulum
stress, and the western blot results also confirmed this. Severe oxidative stress also caused ERS, which ultimately
increased BAX/Bcl-2 ratios and induces apoptosis. Furthermore, up-regulated anaerobic respiration, altered lipid
metabolism, and immune disturbance were exhibited during PS-MPs stress. Therefore, oxidative stress appeared
to be the main toxicity issue caused by MPs, while ERS-mediated apoptosis, metabolic alterations, and immune
responses were induced by this stress. Notably, endoplasmic reticulum stress and apoptosis are a self-protective
mechanism, which may be an intermediate link in the toxicity of microplastics. This study highlights the role of
endoplasmic reticulum stress in MPs toxicology and assesses the adverse effects of microplastics on Golden
Pompano.

1. Introduction
With increased industrial development, serious issues with ecolog­
ical destruction and environmental pollution have emerged. Persistent
organic pollutants, endocrine disruptors and microplastics (MPs) are
serious global pollutants. Since the 1950′ s, plastics production has risen
dramatically, while in 2018, global production exceeded 359 million
metric tons (PlasticsEurope, 2019). However, plastic recycling rates are
very low, with 94% of plastics non-recycled and left in the environment
(Rangasamy et al., 2022). The ocean is a sink for microplastics. Previous
studies speculated that at least 1.5 million tons of MPs are released into
marine ecosystems each year and the widespread use of plastics,
improper plastic waste disposal, and erratic oceanic MPs movements
now mean that MPs are found in every corner of the world (C. Wang
et al., 2021a,2021b). MPs pollution is associated with human activities
and enters freshwater systems via domestic and industrial wastewater
(Yu et al., 2020). Plastics waste in freshwater systems then enters marine
ecosystems through runoff and is dispersed across oceans via currents
(Laskar and Kumar, 2019). In near-coastal areas, where freshwater and
marine systems intersect, high MPs concentrations have been docu­
mented (Su et al., 2020). Tang et al. reported that MPs concentrations
were 14.9 particles/L in seawater along the eastern coast of Hainan
Province (Tang et al., 2021). Similarly, MPs released into the environ­
ment accumulate in organisms, with several plastic pollution studies

* Corresponding author.
E-mail addresses: daaaahua@gmail.com (F.C. Yao), y5187244@163.com (Y. Gu), jiangtian28@126.com (T. Jiang), 1965978587@qq.com (P.F. Wang),
songfb1014@126.com (F.B. Song), zhouzhi@hainanu.edu.cn (Z. Zhou), 1988sunjunlong@sina.com (J.L. Sun), luojian@hainanu.edu.cn (J. Luo).

https://doi.org/10.1016/j.ecoenv.2022.114440
Received 14 September 2022; Received in revised form 9 December 2022; Accepted 13 December 2022
Available online 14 December 2022
0147-6513/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
F.C. Yao et al.
having focused on marine ecosystems. While it is clear that MPs induce
toxicity in marine animals, mechanistic toxicological studies are limited
(Courtene-Jones et al., 2022).
Growing evidence now suggests that MPs ingestion or tissue trans­
location cause digestive tissue damage, Oxidative stress, altered immune
responses, and metabolic disorders (Hale et al., 2020; Kim et al., 2021).
Qiao reported that MPs-induced ROS was associated with altered lipid
metabolism in zebrafish (Qiao et al., 2019). PS-MPs also induced
myocardial apoptosis via oxidative stress (Li et al., 2020). Oxidative
stress is reported in numerous microplastic toxicology articles, which
may be the main toxicity of microplastics (Kim et al., 2021) and is
closely related to physiological responses such as metabolism, apoptosis
and immunity. However, little is known about the intermediate pro­
cesses of these reactions mediated by oxidative stress in microplastic
toxicology studies. Umamaheswari et al., in their comprehensive study,
showed that zebrafish apoptosis during MPs stress was caused by
ROS-mediated alterations to p53 signaling (Umamaheswari et al.,
2021b). Protein handling, modification and folding in the endoplasmic
reticulum (ER) are tightly regulated processes that determine cell
function, fate and survival (Chen and Cubillos-Ruiz, 2021). Oxidative
stress interferes with ER protein folding, activates unfolded protein re­
sponses (UPR), and induce endoplasmic reticulum stress (ERS) (Ma
et al., 2022). Unfortunately, if the resolution of ER stress and restoration
of homeostasis are unsuccessful, UPR induces cell suicide to ensure
normal tissue function (Lemmer et al., 2021). In addition, studies have
shown that endoplasmic reticulum stress can cause metabolic disorders
(Wang et al., 2022) and immune responses (Rashid et al., 2021). Thus,
endoplasmic reticulum stress appears to play a pivotal role in oxidative
stress, apoptosis, metabolic disturbances, immune responses, and tissue
damage responses to harsh environments. However, ERS is limited re­
ported in marine life about microplastic toxicology studies.
Trachinotus blochii (Golden Pompano) are recommended for sus­
tainable marine aquaculture practices by FAO (Sumithra et al., 2022)
consumed in China, USA, Canada, Europe, Southeast Asia, and the
Middle East. MPs have higher concentrations in coastal areas (Hale
et al., 2020). Unfortunately, golden pompano is typically raised in
floating marine cages near coasts, facing these MPs sources. It is
necessary to assess the impact of microplastics on Golden Pompano.
From previous reports, the highest MPs levels in ocean circulation was
approximately 100.0 ug/L3 in oceanic gyres (Limonta et al., 2021).
PS-MPs are one of the most common MPs types, therefore they were
selected for study in this research. Here, we identified apoptosis, ERS,
oxidative stress, immunity, and metabolism indicators in golden pom­
pano. It is hypothesized that oxidative stress is the main toxic response
of microplastics, then endoplasmic reticulum stress and apoptosis which
are adaptive and defense mechanisms occur. This study may help further
elucidate potential MPs toxicological mechanism in marine animals.
2. Materials and methods
2.1. Fish
Five-hundred golden pompano (individual average weight = 125.6 g
± 10.3 g) were obtained from Ling Shui City, Hainan Province, China,
and randomly assigned to three 3000.0 L tanks with a circulating water
filtration system for one week. During the holding period, a 3–5% fish
body weight diet was supplied daily. Marine water from nearby sea
areas is used after sedimentation and sand filtration. Water quality pa­
rameters: temperature (28.0–30.0 ℃), ammonia-nitrogen and nitrite (<
0.02 mg / L), dissolved oxygen (7.0 ± 0.5 mg/L), pH (7.5 ± 0.5), salinity
(30.0–35.0‰), and a natural photoperiod.
2.2. PS-MPs
Globular 5.0 µm PS-MPs were purchased from Tianjin Baseline
Chromtech Research Center, Tianjin, China (Product No.7–3–0500).
2
Ecotoxicology and Environmental Safety 249 (2023) 114440
According to the data provided by the manufacturer, the dimensional
distribution of PS-MPs is as follows: 90% < 5.8 µm; 50% < 5.1 µm; 10%
< 4.7 µm. Commercial PS-MPs were dissolved in deionized water (2.5%
w/v) and the PS-MPs beads density was 1.06 g/cm3. To disperse MPs,
they were ultrasonically treated for 20 min, added to 1.0 L seawater,
ultrasonically treated for 20 min, and finally added to a culture barrel to
reach predetermined concentrations. PS-MPs were rapidly added with
sufficient air to aquaculture buckets so they were evenly dispersed.
During the fish breeding process, different experimental groups used
different breeding tools, such as nets and siphons to avoid cross-
contamination. To maintain PS-MPs concentrations, the seawater was
changed every two days in aquaculture buckets, and the same PS-MPs
concentration was added as before. Control fish experienced the same
conditions but minus PS-MPs.
2.3. Challenge studies
After the holding period, 120 healthy golden pompanos were
randomly assigned to four aquaculture buckets (600 L) and weighed.
Then the fish was fasted for a day. Four MPs concentration groups of 0
μg/L, 10.0 μg/L, 100.0 μg/L, and 1000.0 μg/L (the corresponding par­
ticle densities are 3.5 *1010 particles/L, 3.5 *1011 particles /L, and 3.5
*1012 particles/L, termed C, MP10, MP100, and MP1000, respectively)
were assigned (Fig. S1). During the experiment, the tanks were contin­
uously aerated to maintain the dispersion of the particles in water.
Sufficient feed was provided until the fish are not chasing food, weighed
and recorded at 8:30 am and 5 pm every day. After 2 h feeding, excreta
were withdrawn by siphon. The water in the breeding tank was
refreshed every two days, and the corresponding microplastics are
added. Water quality indicators such as ammonia nitrogen and dissolved
oxygen concentrations were maintained during temporary culture and
experimental periods.
2.4. Sample collection
After 14 days, all fish were weighed and recorded. Three fish were
randomly collected from each group and humanely anesthetized using
MS-222. The liver was removed, washed in normal saline, and divided
into two sections; one part was stored in 4.0% Paraformaldehyde for 24
h and then transferred to 95% alcohol for sectioning (Liu et al., 2022)
and the other part was frozen in liquid nitrogen and stored at − 80 ℃ for
biochemical and gene expression analyses. To prevent
cross-contamination, sampling was performed from low to high MPs
concentrations, and gloves, dissection tools, and other materials were
changed for each fish sample.
2.5. DNA damage and histopathology
Alcohol -stored liver samples were sectioned at 3 µm to assess DNA
damage and histomorphology (n = 3). Hematoxylin and eosin (H&E)
staining included, dewaxing, staining with H&E, and dehydrating. Liver
slices were observed under a microscope and photographed (Olympus
Corporation, Tokyo, Japan) for analysis. Three visual fields were
randomly collected from each section, and the number of normal cells
per filed is quantified to assess tissue damage. To assess DNA damage,
the DAB (SA-HRP) TdT-mediated dUTP-biotin nick end labeling
(TUNEL) cell apoptosis detection kit was purchased from Wuhan Serv­
icebio Technology Co., Ltd. (Wuhan, Hubei, China). Manufacturer in­
structions included; sample deparaffinization and rehydration, antigen
retrieval, blocking endogenous peroxidase, equilibrating samples,
TUNEL reactions, diaminobenzidine (DAB) development, and hema­
toxylin staining. Positive apoptosis cells had brown-yellow nuclei. In the
TUNEL assay, we randomly selected field slices, captured images, and
counted total brown-yellow cells in each field. The degree of apoptosis
was reflected by the number of positive cells/unit area.
F.C. Yao et al.
2.6. Biochemical analyses
Livers from different groups were biochemically analyzed. Total
superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), CAT,
and malondialdehyde (MDA) levels were used to assess oxidative stress.
AKP and ACP activities and LZM levels were used to assess immune
responses. Lactic acid (LD), LDH, ATP, non-esterified fatty acid (NEFA)
and triglyceride (TG) levels were used to estimate metabolic output. All
commercial kits were purchased from Jiancheng Bioeng. Inst. (Nanjing,
China). Liver tissue was diluted 10-fold in normal saline, ground with
steel balls at low temperatures, centrifuged at 2500 g at 4 ◦ C for 10 min,
and the supernatant was extracted to analyze biochemical parameters.
MDA activity was measured by the thibabituric acid method. T-sod
measured by nitrite method. GSH-PX activity was measured by its rate of
promoting the reaction of GSH and hydrogen peroxide. CAT activity is
measured by the ammonium molybdate method, and one unit of activity
represents the amount of 11 μmol of hydrogen peroxide decomposed per
milligram of hemoglobin per second. Lactate activity is measured by the
lactate dehydrogenase colorimetric method. LDH activity was tested by
its catalyzed reaction from LD to pyruvate. ATP content was determined
by the phosphomolybdic acid colorimetric method. All detection was
performed strictly according to manufacturer’s instructions. Total pro­
tein was determined using Coomassie brilliant blue method. All exper­
iments were performed in triplicate biological replicates.
2.7. RNA extraction and gene expression analysis
Total RNA was extracted using TRIzol (Invitrogen, USA), chloroform,
and isopropanol, washed in alcohol, and dissolved in RNase-free water
(Thermo Scientific, USA). RNA quality and integrity were measured by
UV spectrophotometry (NanoDrop 2000, Thermo, Wilmington, DE,
USA) and electrophoresis, respectively. Total RNA with 260/280 and
260/230 optical density ratios > 1.8 were used for cDNA synthesis.
Glutathione peroxidase (Gpx), Cat Sod1, Sod2, and Sod3 gene
expression levels were used to assess antioxidant levels. Eukaryotic
translation initiation factor 2 A (Eif-2α), B-cell lymphoma 2 (Bcl2), Bcl-2-
associated X protein (Bax), C/EBP homologous protein (Chop), X-box
binding protein 1 (Xbp-1) and glucose regulated protein 78 (Grp78)
expression levels were used to examine apoptosis reactions. Toll-like
receptor 7 (Tlr7) gene, Toll-like receptor 8 (Tlr8) gene, Toll-like recep­
tor 9 (Tlr9) gene and Interleukin 1 beta (Il-1β) gene levels were used to
examine immune reactions. Gene primers were designed according to
the NCBI and transcriptome sequencing data (GSE188265). Primer in­
formation is shown Table S1. A 10 μl final volume system (500 nM
primers, 500 ng cDNA template and 6 μl 1x SYBR Green PCR master mix
(Takara Biomedical Technology Co., Ltd., Beijing, China)) was amplified
using a real-time PCR instrument (LightCycle 96, Roche Molecular
Systems, Inc., China), incorporating denaturation 95 ◦ C for 20 s, then
95 ◦ C for 3 s, TM for 30 s, and 72 ◦ C for 60 s for 40 cycles. β-actin was
used as an internal reference and the 2− ΔΔCt method was used to
calculate gene expression levels. Three biological replicates and three
manipulation replicates were performed.
2.8. Western blot
Liver tissue was rinsed with pre-cooled PBS buffer, then homoge­
nized in lysis buffer, centrifuged at 13,000 g at 4 ◦ C for 5 min, and su­
pernatants assayed to western blot. Total protein concentration in the
supernatant was determined by the bicinchoninic acid method, sepa­
rated with 12% sodium dodecyl sulfate-polyacrylamide gel electropho­
resis. Target protein was electrotransferred to polyvinylidene fluoride
membranes with corresponding primary antibodies (Table S2) at 4 ◦ C
overnight. β-actin served as a reference protein. After excess primary
antibody was removed by Tris-buffered saline with 0.1% (v/v) Tween 20
(TBST), the membranes were incubated for 1 h room temperature in­
cubation with the horseradish peroxidase conjugated secondary
3
Ecotoxicology and Environmental Safety 249 (2023) 114440
antibodies. After washing, freshly prepared enhanced chem­
iluminescence mixture was added development. Chemiluminescent
detection of western blots was performed using an Amersham Imager
600 System (GE Healthcare Bio-Sciences, Pittsburgh, PA, United States).
2.9. Statistical analysis
SPSS software (version 18.0, IBM Corp, Armonk, NY, USA) was used
to analyze data. Origin 2019 software was used to draft histograms. Data
were presented as the mean ± standard error. Differences between
controls and experimental groups were analyzed using F-test and inde­
pendent sample t-tests. A p value < 0.05 was considered significantly
different between two groups. “* ” indicates a statistically significant
differences compared with the C group (p < 0.05). All numbers have one
significant digit.
3. Results
3.1. Food intake and weight gain
After 14 days of exposure to microplastics, the total food intake of
groups C, MP10, MP100, and MP1000 were 461.4 g, 495.2 g, 419.2 g,
and 520.3 g, respectively. According to the order of groups C, MP10,
MP100, and MP1000, the corresponding individual average weight gain
were 13.5 ± 2.2, 2.2 ± 2.6, 1.6 ± 2.2 and − 5.2 ± 3.5. Statistical
analysis showed that the body weight gain of the experimental group
was significantly decreased in a concentration-dependent manner
compared with the C group (p < 0.05). No fish dead during the
husbandry.
3.2. Histopathological and apoptosis
Histological changes in golden pompano liver at different concen­
tration exposed to PS-MPs are shown in Fig. 1. After 14 days treatment,
irregular shaped nuclei and nuclear and cytoplasmic vacuolation traits
were identified in MP100 and MP1000 groups (Fig. 1c, d). However,
normal nuclei were regular and oval-shaped, with filled cytoplasm in
control and MP10 groups (Fig. 1a, b). Therefore, clear differences were
observed between low- and medium-high MPs concentration groups. It
is obvious that tissue damage occurred in golden pompano liver under
100 μg/L, 1000 μg/L PS-MPs pressure after 14 days.
Results of the TUNEL Assay under PS-MPs exposure are shown in
Fig. 2. More brown liver cells which showed morphologic changes of
condensed and irregular nuclei were identified in MPs-treated groups
and indicated liver apoptosis when compared with group C. After 14
days, apoptotic cells in group C, MP10, MP100, and MP1000 were 36.0
± 7.0, 73.0 ± 18.0, 110.0 ± 7.0, and 96.0 ± 10.0, respectively (Fig. 2e).
Apoptotic cells were significantly increased in MP100 and MP1000
groups.
3.3. The oxidative and anti-oxidative balance is disrupted
Oxidative and anti-oxidative indicators under the stress of different
concentrations of microplastics are shown in Fig. 3. MDA levels were
significantly increased in MPs groups (p < 0.05) (Fig. 3a). Correspond­
ingly, antioxidant levels also changed. An increase in T-SOD activity was
observed in MP100 and MP1000 groups (p < 0.05) (Fig. 3b). GSH-PX
levels did not change in the MP10 group after 14 days (p > 0.05), but
significantly increased in MP100 and MP1000 groups (p < 0.05)
(Fig. 3c). CAT activity increased significantly in MP10 and MP100
groups (p < 0.05) (Fig. 3d). In conclusion, the medium and high con­
centrations of microplastics obviously lead to the increase of oxidation
and antioxidant indicators in golden pompano liver.
Differences in the oxidative and anti-oxidative genes expression
under different concentrations of PS- MPs are shown in Fig. 4. As the
gene of the SOD family, Sod1 and sod2 expression increased significantly
F.C. Yao et al. Ecotoxicology and Environmental Safety 249 (2023) 114440

Fig. 1. Photomicrographs of the liver of the Trachinotus blochii. Note: Representative pictures acquired by electron microscopy. (a) Control group; (b) MP10 group;
(c) MP100 group; (d) MP1000 group. (e) Quantification of normal cells in each field of view. The red arrows indicate the cytoplasmic vacuolation (CV); The black
arrows indicate nuclear vacuolation (NV); The blue arrows indicate the irregular shaped nucleus (ISN). The scale bar represents 20 µm. The enlarged graph is
displayed at a triple scale. Data are shown as mean ± SE (n = 3). Statistical results are derived from F-test and independent sample t-tests. "* " indicates a significant
difference compared to group C (p < 0.05).

in all MPs groups (p < 0.05) (Fig. 4a, b), but concentration-dependent
increases were not recorded; while, sod3 expression dropped signifi­
cantly (p < 0.05) (Fig. 4c). After PS-MPs for 14 days, Cat liver expression
significantly increased, with the greatest change in the MP100 group
(p < 0.05) (Fig. 4d). Gpx expression decreased slightly in the MP10
group (p > 0.05) but significantly increased in MP100 groups (p < 0.05)
(Fig. 4e). In conclusion, the expression of oxidant and antioxidant genes
in MP100 and MP1000 showed consistent activation or inhibition.
3.4. ERS and apoptosis occurred
ERS-related gene (Grp78, Eif-2α, Xbp-1 and Chop) and apoptosis-
related regulator gene (Bax and Bcl-2) are shown in Fig. 5. Grp78
expression showed a significant concentration-dependent increase
(p < 0.05) (Fig. 5a). Eif-2α liver expression was significantly increased
in MP10 and MP100 groups (p < 0.05), while expression showed a
slight increase in the MP1000 group (p > 0.05) (Fig. 5b). We observed
no differences in Xbp-1 expression between control and MP10 groups
(p > 0.05); however, in MP100 and MP100 groups, Xbp-1 expression
was significantly increased (p < 0.05) (Fig. 5c). Chop expression did not
change significantly in MP10 and MP100 groups (p > 0.05), but
increased significantly in the MP1000 group (p < 0.05) (Fig. 5e). In
MP100 and MP1000 groups, Bax expression was significantly increased,
while Bcl-2 expression was significantly decreased (p < 0.05) (Fig. 5f).
The expressions of ERS-related factors were measured by Western
blot analysis (Fig. 6). After 14 days of exposure to microplastics, the
expression of XBP-1 was significantly increased in the MP100 and
MP1000 groups (p < 0.05). The expression of CHOP was significantly
upregulated in all experimental groups (p < 0.05). The expression of
GRP78 showed a slight up-regulation, and there was no statistical dif­
ference (p > 0.05).

4
F.C. Yao et al. Ecotoxicology and Environmental Safety 249 (2023) 114440

Fig. 2. Photo of TUNEL Cell apoptosis detection. Note: Representative pictures acquired by electron microscopy. (a) Control group; (b) MP10 group; (c) MP100
group; (d) MP1000 group. Normal nucleus is blue. Apoptotic cells exhibit distinct brown nuclei; (e) The numbers of apoptosis in unit field of view. The enlarged
graph is displayed at a triple scale. Data are shown as mean ± SE (n = 3). Statistical results are derived from F-test and independent sample t-tests. "* " indicates a
significant difference compared to group C (p < 0.05). The blue arrows indicate apoptosis.

3.5. Enhanced anaerobic respiration and weakened fat metabolism during
MPs stress
As shown (Fig. S2 a, b), anaerobic respiration indicators included LD
and LDH. LD levels increased significantly in MP100 and MP1000
groups (p < 0.05) (Fig.S2 a). MPs significantly elevated LDH activity but
was not concentration-dependent (p < 0.05) (Fig. S2 b). Also, ATP liver
levels were significantly reduced in a concentration-dependent manner
(Fig. S2 c). As shown (Fig. S2 e, f), anaerobic respiration indicators
included NEFA and TG. NEFA levels were significantly increased in MPs
groups (p < 0.05) (Fig. S2 d). TG liver levels were significantly increased
in MP100 and MP1000 groups (p < 0.05) (Fig. S2 e). In conclusion,
medium and high concentrations of PS-MPs significantly enhanced LD,
and LDH significantly weakened NEFA, TG and ATP.
3.6. Innate immunity is activated under high PS-MPs concentrations
As shown in Fig. S3, innate immunity including ACP, AKP and LZM
has activated under PS-MPs stress. After 14 days of exposure, ACP liver
activity was elevated in MP10 and MP100 groups (p < 0.05) (Fig.S3 a),
and did not significantly change in the MP1000 group (p > 0.05). AKP
activity was increased in MP100 and MP1000 groups (p < 0.05) and
slightly increased in MP10 groups (Fig. S3 b). LZM levels were signifi­
cantly different in the MP1000 group (p < 0.05) (Fig. S3 c) and did not
change significantly in the MP10 and MP100 groups (p > 0.05).
3.7. Expression of genes involved in immune recognition is suppressed
Changes in immune-related genes are shown in Fig. S4. Tlt7
expression levels were significantly reduced in all MPs groups (p < 0.05)
(Fig. S4 a). Tlr8 showed activation in the MP1000 group (p > 0.05);
however, the expression level of Tlr8 gene was significantly suppressed

5
F.C. Yao et al. Ecotoxicology and Environmental Safety 249 (2023) 114440

Fig. 3. Changes in indicators related to oxida­

tion and antioxidants in the liver. Note: The
Oxidation and Anti-oxidant indicators included
malondialdehyde (MDA) levels (a), total su­
peroxide dismutase (T-SOD) levels (b), gluta­
thione peroxidase (GSH-PX) levels (c), catalase
(CAT) levels (d) in liver. The columns with
larger grayscales indicate higher concentrations
of microplastics. Data are shown as mean ± SE
(n = 3). Statistical results are derived from F-
test and independent sample t-tests. "*" in­
dicates a significant difference compared to
group C (p < 0.05).

Fig. 4. The expression of oxidative and anti-oxidative genes. Note: The oxidative and anti-oxidative genes included superoxide dismutase family genes (a: Sod1, b:
Sod2 and c: Sod3), catalase (d: Cat) and glutathione peroxidase (e: Gpx). Data are shown as mean ± SE (n = 3). Statistical results are derived from F-test and in­
dependent sample t-tests. "* " indicates a significant difference compared to group C (p < 0.05).

in the MP10 group(p < 0.05); the expression level was slightly increased
in the MP100 group (p > 0.05) (Fig. S4 b). Tlt9 was suppressed under
MPs stress, was significantly suppressed in the MP10 group (p < 0.05)
and slightly suppressed in the MP100 and MP1000 group (p > 0.05)
(Fig. S4 c). IL-1βnot change significantly in all MPs groups (p > 0.05).
4. Discussion
MPs are extensively found in marine ecosystems, with limited reports
on toxicological mechanisms. Oxidative stress, apoptosis, immune re­
sponses, metabolic disturbances, tissue damage and growth restriction
are common responses to microplastic stress, and were observed in our
research. These common responses, under MPs stress, usually involve
chain reactions. Endoplasmic reticulum stress is reported in this study.
We suspect that ER stress may be a key point in the mechanism of
microplastic toxicity. Here, we sought to explain the toxicological
mechanisms underpinning MPs exposure (Fig. 7). In addition, there are
few reports on the adverse effects of MP in the natural environment. It is

6
F.C. Yao et al. Ecotoxicology and Environmental Safety 249 (2023) 114440

Fig. 5. The expression of apoptosis-related genes. Note: The apoptosis-related genes included Glucose regulated protein 78(Grp78) gene (a), Eukaryotic translation
initiation factor 2 A (Eif-2α) gene(b), X-box binding protein 1(xbp-1) gene (c). C/EBP homologous protein (Chop) gene (d), Bcl-2-associated X protein (Bax) gene (e)
and B-cell lymphoma 2 (Bcl-2) gene (f). Data are shown as mean ± SE (n = 3). Statistical results are derived from F-test and independent sample t-tests. "* " indicates
a significant difference compared to group C (p < 0.05).

Fig. 6. The effect of microplastics on the

expression of proteins related to endoplasmic
reticulum stress. Note: (a) Expression levels of
XBP-1, CHOP and GRP78, β-actin as internal
reference. (b) Relative densitometric analysis of
XBP-1 protein expression. (c) Relative densito­
metric analysis of CHOP protein expression. (d)
Relative densitometric analysis of GRP78 pro­
tein expression. Data are shown as mean ± SE
(n = 3). Statistical results are derived from F-
test and independent sample t-tests. "*" in­
dicates a significant difference compared to
group C (p < 0.05).

well known that endoplasmic reticulum stress and apoptosis are pro­
tective mechanisms of the body. This may be related to biological self-
regulation to adapt to MPs stress.
4.1. Oxidative stress occurs under MPs stress
It was repeatedly shown that MPs caused oxidative stress (Capó
et al., 2021). To explore toxicity mechanisms during MPs stress, oxida­
tive stress indicators and related gene expression were examined. MDA
is one of the final products of polyunsaturated fatty acid peroxidation

7
F.C. Yao et al.
Fig. 7. Toxicity and mechanisms of microplastics in this study. Note: The substances inside the circles were measured in this study: Solid arrows represent facili­
tation. dashed arrows represent potential facilitation; Black circles indicate up-regulation; Grey circles indicate down-regulation.
and is commonly used to assess oxidative stress status (Thirupathi et al.,
2021). In our study, MDA levels were significantly up-regulated and
indicated hepatic oxidative stress induction after 14 days of MPs expo­
sure in all MPs groups. Many enzymes and low-molecular-weight com­
pounds help maintain low ROS levels to eliminate the dangers of ROS
overproduction (Pisoschi et al., 2021). The SOD family, as the first de­
fense line in the ROS scavenging system, convert superoxide anions to
hydrogen peroxide in the cytosol (SOD1), mitochondria (SOD2), and
extracellular matrix (SOD3) (Kim et al., 2022). In the second defense
line, hydrogen peroxide is converted to water and oxygen via redox
reactions catalyzed by CAT and GSH. In our study, after 14 days expo­
sure to 100 μg/L and 1000 μg/L PS-MPs, T-SOD and GSH-PX activities
were significantly increased, indicating defense mechanisms were acti­
vated to reduce ROS-induced damage. Increased Sod1, Sod2, Cat, and
Gpx expression levels also supported this. The results are in agreement
with Oxidative stress of other species as zebrafish (Umamaheswari et al.,
2021a) and medaka (Feng et al., 2021). However, in the 10 μg/L MPs
group, SOD and GSH-PX activity were similar to the control group,
therefore low MPs concentrations may not activate the antioxidant
system. After Nile Tilapia exposed to MPs for 15 days, the activity of
total antioxidant capacity decreased (Hamed et al., 2020). In this study,
oxidative stress occurred, but the antioxidant capacity of Golden Pom­
pano did not appear to be compromised. The response to MPs may be
related to individual organisms and the concentration of microplastics.
4.2. Oxidative stress under PS-MPs stress activates endoplasmic reticulum
stress and apoptosis
Oxidative stress alters protein structures (Abramov et al., 2020), with
the ER folding and processing immature proteins. Glucose regulated
protein 78 (GRP78), which is an important UPR modulating component,
is released from UPR sensors under ER stress to maintain properly folded
proteins (Alshammari et al., 2021). UPR stimulates apoptosis when
8
Ecotoxicology and Environmental Safety 249 (2023) 114440
faced with unresolved unfolded protein (UR) stress. UPR sensor acti­
vation activates downstream XBP-1 to participate in RNA degradation,
and it also activates downstream Eif-2α and CHOP to reduce protein
translation (Iurlaro and Muñoz-Pinedo, 2016). ERS is a complex regu­
latory process, and upregulated XBP-1 also induces CHOP expression
(Alshammari et al., 2021). CHOP is also called growth arrest and DNA
damage-inducible gene 153,with important roles in ERS-induced cell
death (Zhou et al., 2019). CHOP up-regulates pro-apoptotic BAX gene
and down-regulates anti-apoptotic BCL2 gene, changing BAX/BCL2 ra­
tios and causing apoptosis (Iurlaro and Muñoz-Pinedo, 2016). In our
study, after 14 days exposure to 1000 μg/L MPs, Grp78, Eif-2α, Xbp-1,
Chop, Bax, and Gpx expression levels were up-regulated, while Bcl-2
expression was down-regulated. The results of TUNEL assay show that
apoptosis occurred in the liver upon medium and high MPs concentra­
tion exposure. Under high MPs concentrations, oxidative stress appeared
to activate GRP78 and the UPR sensor. XBP-1 and Eif-2α mediated CHOP
was activated. Finally, CHOP up-regulated BAX and down-regulated
BCL leading to apoptosis. In addition, western blot results showed that
the increase of XBP-1 and CHOP protein content also confirmed the
occurrence of ER stress. In the study of microplastic toxicity, several
apoptosis-inducing pathways including PTEN/PI3K/AKT/mTOR (Lu
et al., 2022), p53 (Qiang and Cheng, 2021), Wnt/β-catenin (An et al.,
2021) and NLRP3/Caspase-1 (Hou et al., 2021) signaling pathway were
reported. In this study, the ER stress pathway was confirmed in fish. it is
reported that the anti-oxidant system imbalance regulates the caspase
family to induce apoptosis under MPs stress in birds (Lu et al., 2022).
The oxidative stress induced by MPs is the main cause of inflammation,
cell death and disorder of metabolome in targeted tissues (Shengchen
et al., 2021). Our results support this view and are discussed further.
Both ER stress and apoptosis are protective mechanisms. In our study,
the stress period was only 14 days. Longer-term microplastic stress needs
to be implemented to explore the adaptation mechanism of organisms to
microplastics.
F.C. Yao et al.
4.3. Immune disorders occur under microplastic stress
LZM is crucial for peptidoglycan destruction in pathogens, and used
as an immune index to evaluate immune levels in aquatic organisms
(Ahmadifar et al., 2021). After immune system activation, LZM is
released which promotes AKP and ACP release from Lysosomes, thereby
reducing MPs-induced inflammatory reactions (Zhao et al., 2022). LZM
levels increased slightly in the MP100 group (p > 0.05), but were
significantly increased in the MP1000 group (p < 0.05). In our study,
ACP activity in MPs group was higher than in the control group, while
AKP activity showed concentration-dependent increases.
Concentration-dependent increases in LZM and AKP indices suggested
that innate immunity activation was related to MPs concentrations. The
same immune mechanism was reported in tilapia during PS-MPs stress
(Ahmadifar et al., 2021). Lysosomes also undertake important functions
in cellular degradation (Li et al., 2019). Notably, in the high concen­
tration group, severe apoptosis occurred in hepatocytes, we believe that
the enhancement of innate immunity represented by LZM, ACP, AKP is
related to autophagy. Additionally, the Il-1β gene and the toll-like re­
ceptors Tlr7, Tlr8, and Tlr9 were not activated significantly. TLR7, TLR8,
and TLR9 activate the immune system by recognizing pathogen associ­
ated nucleic acids (K. L. Wang et al., 2021a,2021b). We hypothesize that
MPs inhibit recognition functions in the immune system. Further
research is needed in this area.
4.4. MPs-induced cytotoxicity alters liver cell structure and function
In terms of MPs-mediated cell phenotypes, we identified severe liver
damage concentration-dependently. Cytoplasmic vacuolation and
irregular shaped cells were observed and did not alter normal tissue
functions, while nuclear vacuolation did. This observation implies that
high PS-MPs (5 µm size) concentrations may have affected liver function
in the golden pompano. Vacuolation were also detected in Zebrafish
hepatocytes after 5 µm MPs treatments (Lu et al., 2016). In addition,
organelle vacuolation was reportedly tightly linked to apoptosis which is
a programmed cell death mechanism in response to environmental
stimuli and protects different cell populations (Shubin et al., 2016).
The liver is the central tissue for fatty acid metabolism. When liver
cells undergo apoptosis and structural changes, normal liver metabolic
functions may be affected (Feng et al., 2022). In our study, we observed
increased lipid TG and NEFA metabolites in MPs groups, thus MPs
promoted their accumulation in the liver. LDH is enzymatically impor­
tant for anaerobic energy processes and indicates tissue stress, energy
status, and metabolism (Limonta et al., 2021). LDH was activated in all
MPs groups, and suggested that high MPs concentrations induced cell
anaerobic energy production pathways. This was also confirmed by
increased LD levels. ATP levels exhibited a concentration-dependent
reduction under MPs pressure. It is known that anaerobic respiration
produced less ATP. Zebrafish studies also confirmed our results showing
that PS-MPs exposure altered energy and lipid metabolism (Lu et al.,
2016). In addition, negative weight gain in this study may be related to
liver dysfunction and energy allocation. This requires further research.
5. Conclusions
Marine cages aquaculture is the primary golden pompano production
method but faces serious MPs pollution issues. We used the fish as a
model organism to study stress resistance, and identified hepatocyte
damage, oxidative stress, ERS, altered immune responses, metabolic
disorders, and apoptosis under high MPs concentrations. MPs-mediated
toxicity exhibited a certain degree of concentration dependence. ER
stress in fish maybe a new finding for MPs. we speculate that ER stress
may play an important role in the mechanism of microplastic toxicity.
Notably, endoplasmic reticulum stress and apoptosis are a protective
mechanism of the body. In the context of global microplastic pollution,
more attention needs to be focused on ER stress.
9
Ecotoxicology and Environmental Safety 249 (2023) 114440
Funding
This work was supported by the Hainan Province Natural Science
Foundation of China (322QN236), the National Natural Science Foun­
dation of China (No. 32160862), and the project of Hainan Yazhou Bay
Seed Laboratory (B21HJ0105).
Ethics statement
All experiments were performed according to the Guidelines for the
Care and Use of Laboratory Animals in China. All experimental pro­
cedures and sample collection were approved by the Institutional Ani­
mal Care and Use Committee (IACUC) of the College of Ocean of Hainan
University, Hainan, China, under permit No. HNUAUCC-2022–00045.
CRediT authorship contribution statement
Jun Long Sun, Zhi Zhou and Jian Luo complete the experimental
design. Fu Cheng Yao, Yue Gu and Peng Fei Wang completed the rearing
and stressing experiment. Fu Cheng Yao completed the indicators test,
drawing and writing. Yue Gu and Tian Jiang assist with data analysis
and writing, Jun Long Sun and Fei Biao Song proofread the article.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
The authors were grateful to all the laboratory members for contin­
uous technical advice and helpful discussion. Sincere thanks to Chun Xiu
Jin, Da Zheng, De Cai Zheng, Jie Huang, Kai Xi Zhang, Li Ping Shi, Min
Yang, Shu Kui Sun, Tian Jiang, Tian Xiu Li, Xian Zhang, Xin Fan and Yu
Zhang for helping with sample collection.
Consent for publication
Not applicable.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2022.114440.
References
Abramov, A.Y., Potapova, E.V., Dremin, V.V., Dunaev, A.V., 2020. Interaction of
oxidative stress and misfolded proteins in the mechanism of neurodegeneration. Life
10, 101.
Ahmadifar, E., Kalhor, N., Dawood, M.A.O., Ahmadifar, M., Moghadam, M.S.,
Abarghouei, S., Hedayati, A., 2021. Effects of polystyrene microparticles on
inflammation, antioxidant enzyme activities, and related gene expression in Nile
tilapia (Oreochromis niloticus). Environ. Sci. Pollut. Res. 28, 14909–14916.
Alshammari, G.M., Al-Qahtani, W.H., AlFaris, N.A., Albekairi, N.A., Alqahtani, S., Eid, R.,
Yagoub, A.E.A., Al-Harbi, L.N., Yahya, M.A., 2021. Quercetin alleviates cadmium
chloride-induced renal damage in rats by suppressing endoplasmic reticulum stress
through SIRT1-dependent deacetylation of Xbp-1s and eIF2α. Biomed.
Pharmacother. 141, 111862 https://doi.org/10.1016/j.biopha.2021.111862.
An, R., Wang, X., Yang, L., Zhang, J., Wang, N., Xu, F., Hou, Y., Zhang, H., Zhang, L.,
2021. Polystyrene microplastics cause granulosa cells apoptosis and fibrosis in ovary
through oxidative stress in rats. Toxicology 449, 152665. https://doi.org/10.1016/j.
tox.2020.152665.
Capó, X., Company, J.J., Alomar, C., Compa, M., Sureda, A., Grau, A., Hansjosten, B.,
Lopez-Vazquez, J., Quintana, J.B., Rodil, R., 2021. Long-term exposure to virgin and
F.C. Yao et al.
seawater exposed microplastic enriched-diet causes liver oxidative stress and
inflammation in gilthead seabream Sparus aurata, Linnaeus 1758. Sci. Total Environ.
767, 144976.
Chen, X., Cubillos-Ruiz, J.R., 2021. Endoplasmic reticulum stress signals in the tumour
and its microenvironment. Nat. Rev. Cancer 21, 71–88.
Courtene-Jones, W., Martínez Rodríguez, A., Handy, R.D., 2022. From microbes to
ecosystems: a review of the ecological effects of biodegradable plastics. Emerg. Top.
Life Sci.
Feng, S., Zeng, Y., Cai, Z., Wu, J., Chan, L.L., Zhu, J., Zhou, J., 2021. Polystyrene
microplastics alter the intestinal microbiota function and the hepatic metabolism
status in marine medaka (Oryzias melastigma). Sci. Total Environ. 759, 143558.
Feng, Z., Zhong, Y., He, G., Sun, H., Chen, Y., Zhou, W., Lin, S., 2022. Yeast culture
improved the growth performance, liver function, intestinal barrier and microbiota
of juvenile largemouth bass (Micropterus salmoides) fed high-starch diet. Fish.
Shellfish Immunol. 120, 706–715.
Hale, R.C., Seeley, M.E., La Guardia, M.J., Mai, L., Zeng, E.Y., 2020. A Global Perspective
on Microplastics. J. Geophys. Res.: Oceans 125, 1–40. https://doi.org/10.1029/
2018JC014719.
Hamed, M., Soliman, H.A.M., Osman, A.G.M., Sayed, A.E.-D.H., 2020. Antioxidants and
molecular damage in Nile Tilapia (Oreochromis niloticus) after exposure to
microplastics. Environ. Sci. Pollut. Res. 27, 14581–14588.
Hou, J., Lei, Z., Cui, L., Hou, Y., Yang, L., An, R., Wang, Q., Li, S., Zhang, H., Zhang, L.,
2021. Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian
granulosa cells via NLRP3/Caspase-1 signaling pathway in rats. Ecotoxicol. Environ.
Saf. 212, 112012 https://doi.org/10.1016/j.ecoenv.2021.112012.
Iurlaro, R., Muñoz-Pinedo, C., 2016. Cell death induced by endoplasmic reticulum stress.
FEBS J. 283, 2640–2652.
Kim, J.H., Jeong, H.D., Song, M.J., Lee, D.H., Chung, J.H., Lee, S.-T., 2022. SOD3
Suppresses the Expression of MMP-1 and Increases the Integrity of Extracellular
Matrix in Fibroblasts. Antioxidants. https://doi.org/10.3390/antiox11050928.
Kim, J.-H., Yu, Y.-B., Choi, J.-H., 2021. Toxic effects on bioaccumulation, hematological
parameters, oxidative stress, immune responses and neurotoxicity in fish exposed to
microplastics: A review. J. Hazard. Mater. 413, 125423 https://doi.org/10.1016/j.
jhazmat.2021.125423.
Laskar, N., Kumar, U., 2019. Plastics and microplastics: A threat to environment.
Environ. Technol. Innov. 14, 100352 https://doi.org/10.1016/j.eti.2019.100352.
Lemmer, I.L., Willemsen, N., Hilal, N., Bartelt, A., 2021. A guide to understanding
endoplasmic reticulum stress in metabolic disorders. Mol. Metab. 47, 101169
https://doi.org/10.1016/j.molmet.2021.101169.
Li, S.-S., Zhang, M., Wang, J.-H., Yang, F., Kang, B., Xu, J.-J., Chen, H.-Y., 2019.
Monitoring the changes of pH in lysosomes during autophagy and apoptosis by
plasmon enhanced Raman imaging. Anal. Chem. 91, 8398–8405.
Li, Z., Zhu, S., Liu, Q., Wei, J., Jin, Y., Wang, X., Zhang, L., 2020. Polystyrene
microplastics cause cardiac fibrosis by activating Wnt/β-catenin signaling pathway
and promoting cardiomyocyte apoptosis in rats. Environ. Pollut. 265, 115025
https://doi.org/10.1016/j.envpol.2020.115025.
Limonta, G., Mancia, A., Abelli, L., Fossi, M.C., Caliani, I., Panti, C., 2021. Effects of
microplastics on head kidney gene expression and enzymatic biomarkers in adult
zebrafish. Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol. 245, 109037 https://
doi.org/10.1016/j.cbpc.2021.109037.
Liu, X.-J., Wang, Y.-Q., Shang, S.-Q., Xu, S., Guo, M., 2022. TMT induces apoptosis and
necroptosis in mouse kidneys through oxidative stress-induced activation of the
NLRP3 inflammasome. Ecotoxicol. Environ. Saf. 230, 113167 https://doi.org/
10.1016/j.ecoenv.2022.113167.
Lu, H., Yin, K., Su, H., Wang, D., Zhang, Y., Hou, L., Li, J. bo, Wang, Y., Xing, M., 2022,
Polystyrene microplastics induce autophagy and apoptosis in birds lungs via PTEN/
PI3K/AKT/mTOR. Environmental Toxicology.
Lu, Y., Zhang, Y., Deng, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H., 2016. Uptake
and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic
effects in liver. Environ. Sci. Technol. 50, 4054–4060.
Ma, Y., Liu, J., Liu, H., Han, X., Sun, L., Xu, H., 2022. Podocyte protection by Angptl3
knockout via inhibiting ROS/GRP78 pathway in LPS-induced acute kidney injury.
Int. Immunopharmacol. 105, 108549 https://doi.org/10.1016/j.
intimp.2022.108549.
Pisoschi, A.M., Pop, A., Iordache, F., Stanca, L., Predoi, G., Serban, A.I., 2021. a. Eur. J.
Med. Chem. 209, 112891 https://doi.org/10.1016/j.ejmech.2020.112891.
10
Ecotoxicology and Environmental Safety 249 (2023) 114440
PlasticsEurope, E., 2019, Plastics—The Facts 2019. An Analysis of European Plastics
Production, Demand and Waste Data. PlasticEurope https://www. plasticseurope.
org/en/resources/publications/1804-plastics-facts-2019.
Qiang, L., Cheng, J., 2021. Exposure to polystyrene microplastics impairs gonads of
zebrafish (Danio rerio). Chemosphere 263, 128161. https://doi.org/10.1016/j.
chemosphere.2020.128161.
Qiao, R., Sheng, C., Lu, Y., Zhang, Y., Ren, H., Lemos, B., 2019. Microplastics induce
intestinal inflammation, oxidative stress, and disorders of metabolome and
microbiome in zebrafish. Sci. Total Environ. 662, 246–253. https://doi.org/
10.1016/j.scitotenv.2019.01.245.
Rangasamy, B., Malafaia, G., Maheswaran, R., 2022. Evaluation of antioxidant response
and Na+-K+-ATPase activity in zebrafish exposed to polyethylene microplastics:
Shedding light on a physiological adaptation. J. Hazard. Mater. 426, 127789 https://
doi.org/10.1016/j.jhazmat.2021.127789.
Rashid, F., Dzakah, E.E., Wang, H., Tang, S., 2021. The ORF8 protein of SARS-CoV-2
induced endoplasmic reticulum stress and mediated immune evasion by
antagonizing production of interferon beta. Virus Res. 296, 198350.
Shengchen, W., Jing, L., Yujie, Y., Yue, W., Shiwen, X., 2021. Polystyrene microplastics-
induced ROS overproduction disrupts the skeletal muscle regeneration by converting
myoblasts into adipocytes. J. Hazard. Mater. 417, 125962.
Shubin, A.V., Demidyuk, I.V., Komissarov, A.A., Rafieva, L.M., Kostrov, S.V., 2016.
Cytoplasmic vacuolization in cell death and survival. Oncotarget 7, 55863.
Su, L., Sharp, S.M., Pettigrove, V.J., Craig, N.J., Nan, B., Du, F., Shi, H., 2020.
Superimposed microplastic pollution in a coastal metropolis. Water Res. 168,
115140 https://doi.org/10.1016/j.watres.2019.115140.
Sumithra, T.G., Sharma, K.S.R., Gangadharan, S., Suresh, G., Prasad, V., Amala, P.V.,
Sayooj, P., Gop, A.P., Anil, M.K., Patil, P.K., 2022. Dysbiosis and Restoration
Dynamics of the Gut Microbiome Following Therapeutic Exposure to Florfenicol in
Snubnose Pompano (Trachinotus blochii) to Aid in Sustainable Aquaculture
Production Strategies. Front. Microbiol. 1384.
Tang, J., Wu, Z., Wan, L., Cai, W., Chen, S., Wang, X., Luo, J., Zhou, Z., Zhao, J., Lin, S.,
2021. Differential enrichment and physiological impacts of ingested microplastics in
scleractinian corals in situ. J. Hazard. Mater. 404, 124205 https://doi.org/10.1016/
j.jhazmat.2020.124205.
Thirupathi, A., Wang, M., Lin, J.K., Fekete, G., István, B., Baker, J.S., Gu, Y., 2021. Effect
of different exercise modalities on oxidative stress: a systematic review. BioMed. Res.
Int. 2021.
Umamaheswari, S., Priyadarshinee, S., Bhattacharjee, M., Kadirvelu, K., Ramesh, M.,
2021a. Exposure to polystyrene microplastics induced gene modulated biological
responses in zebrafish (Danio rerio). Chemosphere 281, 128592.
Umamaheswari, S., Priyadarshinee, S., Kadirvelu, K., Ramesh, M., 2021b. Polystyrene
microplastics induce apoptosis via ROS-mediated p53 signaling pathway in
zebrafish. Chem. -Biol. Interact. 345, 109550 https://doi.org/10.1016/j.
cbi.2021.109550.
Wang, C., Zhao, J., Xing, B., 2021a. Environmental source, fate, and toxicity of
microplastics. J. Hazard. Mater. 407, 124357 https://doi.org/10.1016/j.
jhazmat.2020.124357.
Wang, K.L., Chen, S.N., Huo, H.J., Nie, P., 2021b. Identification and expression analysis
of sixteen Toll-like receptor genes, TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S,
TLR7− 9, TLR13a− c, TLR14, TLR21− 23 in mandarin fish Siniperca chuatsi. Dev.
Comp. Immunol. 121, 104100 https://doi.org/10.1016/j.dci.2021.104100.
Wang, Y., Zhou, X., Li, D., Ye, J., 2022. Role of the mTOR-autophagy-ER stress pathway
in high fructose-induced metabolic-associated fatty liver disease. Acta Pharmacol.
Sin. 43, 10–14.
Yu, Q., Hu, X., Yang, B., Zhang, G., Wang, J., Ling, W., 2020. Distribution, abundance
and risks of microplastics in the environment. Chemosphere 249, 126059. https://
doi.org/10.1016/j.chemosphere.2020.126059.
Zhao, Z., Wang, H., Zhang, D., Guan, Y., Siddiqui, S.A., Feng-Shan, X., Cong, B., 2022.
Oral vaccination with recombinant Lactobacillus casei expressing Aeromonas
hydrophila Aha1 against A. hydrophila infections in common carps. Virulence 13,
794–807.
Zhou, W., Fang, H., Wu, Q., Wang, X., Liu, R., Li, F., Xiao, J., Yuan, L., Zhou, Z., Ma, J.,
2019. Ilamycin E, a natural product of marine actinomycete, inhibits triple-negative
breast cancer partially through ER stress-CHOP-Bcl-2. Int. J. Biol. Sci. 15, 1723.