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RESEARCH ARTICLE
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Exposure to Polystyrene Nanoplastics Led to Learning and
Memory Deficits in Zebrafish by Inducing Oxidative
Damage and Aggravating Brain Aging
Weishang Zhou, Difei Tong, Dandan Tian, Yingying Yu, Lin Huang, Weixia Zhang,
Yihan Yu, Lingzheng Lu, Xunyi Zhang, Wangqi Pan, Jiawei Shen, Wei Shi,
and Guangxu Liu*
Nanoplastics (NPs) may pass through the blood–brain barrier, giving rise to
serious concerns about their potential toxicity to the brain. In this study, the
effects of NPs exposure on learning and memory, the primary cognitive
functions of the brain, are assessed in zebrafish with classic T-maze
exploration tasks. Additionally, to reveal potential affecting mechanisms, the
impacts of NPs exposure on brain aging, oxidative damage, energy provision,
and the cell cycle are evaluated. The results demonstrate that NP-exposed
zebrafish takes significantly longer for their first entry and spends markedly
less time in the reward zone in the T-maze task, indicating the occurrence of
learning and memory deficits. Moreover, higher levels of aging markers
(𝜷-galactosidase and lipofuscin) are detected in the brains of NP-exposed fish.
Along with the accumulation of reactive free radicals, NP-exposed zebrafish
suffer significant levels of brain oxidative damage. Furthermore, lower levels
of Adenosine triphosphate (ATP) and cyclin-dependent kinase 2 and higher
levels of p53 are observed in the brains of NP-exposed zebrafish, suggesting
that NPs exposure also results in a shortage of energy supply and an
arrestment of the cell cycle. These findings suggest that NPs exposure may
pose a severe threat to brain health, which deserves closer attention.
1. Introduction
Learning and memory are fundamental functions of the brain,[1]
and are important for animals to adapt to living environments,
from locating appropriate food sources and mates to avoiding
predators and parasites.[2] In humans, learning and memory
deficits are currently an enormous social and economic burden,
which could not only lead to decreased labor ability but also
pose a serious threat to individual health and even survival.[3]
W. Zhou, D. Tong, D. Tian, Y. Yu, L. Huang, W. Zhang, Y. Yu, L. Lu,
X. Zhang, W. Pan, J. Shen, W. Shi, G. Liu
College of Animal Sciences
Zhejiang University
Hangzhou 310058, P. R. China
E-mail: guangxu_liu@zju.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.202301799
DOI: 10.1002/adhm.202301799
Although maintaining robust learning and
memory is critical,[4] accumulating evi-
dence indicates that these abilities could
be undermined by various environmen-
tal contaminants, such as air pollution,[5]
pesticides,[6] and heavy metals.[7] With sig-
nificant toxic impacts on a series of physio-
logical processes increasingly documented,
a group of emerging pollutants, such as
micro- and/or nanoplastics,[8] have been
identified in recent years. For instance, it
has been demonstrated that exposure to
polystyrene nanoplastics (PS-NP) could in-
terrupt physiological processes such as glu-
cose metabolism, cortisol homeostasis, and
embryonic development.[9] However, to the
best of our knowledge, the toxic effects
these emerging pollutants may have on
brain functions, such as learning and mem-
ory, remain largely unknown to date.
Due to the massive production (annual
global production is ≈350–400 million tons)
and usage of plastic products (predicted
to exceed 1 billion tons by 2050), huge
amounts of plastic waste have been dumped
into the environment, creating a pervasive global environmental
threat: plastic pollution.[10] Whether originally produced in tiny
pieces or derived from the fragmentation of larger pieces, a sub-
stantial proportion of plastics in the environment have a diam-
eter smaller than 100 nm and are collectively called nanoplas-
tics (NPs).[8] In addition to being prevalent in various natural
environments,[11] plastic particles have also been detected in
foodstuffs such as milk, fruit, and eggs as well as table salt and
bottled water.[12] More recently, plastic particles have even been
found in humans in samples from the blood,[13] placenta,[14] and
lung.[15] Compared to plastic particles at larger sizes, it has been
suggested that NPs may be able to penetrate the blood–brain bar-
rier (BBB) due to their extremely small sizes, giving rise to a se-
rious concern about their toxic impacts on the brain.[16]
Normal cognitive functions such as learning and memory de-
pend on the intactness of the brain and the abundant supply
of energy; however, all these functions could be severely threat-
ened by the ineffective removal of free radicals in the brain.[17]
On the one hand, excessive free radicals such as hydrogen
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peroxide (H2O2) and superoxide anion (O2−) can attack biomacro-
molecules, causing DNA damage, protein carbonylation, and
lipid peroxidation.[18] These changes might aggravate brain ag-
ing and thus make the brain less efficient in its cognitive
functions.[19] For instance, oxidative stress-induced DNA dam-
age could activate p53 and thus arrest the cell cycle by modu-
lating cyclin-dependent kinase 2 (CDK2), which may boost the
proportion of senescent cells in the brain.[20] Hence, brain aging
represents a pivotal mechanism through which oxidative stress
exerts its influence on brain function. On the other hand, the
brain requires a huge amount of energy to function normally;
for example, with a mass of ≈2% of the body weight, the human
brain consumes ≈25% and 20% of the total consumption of glu-
cose and oxygen, respectively.[21] However, oxidative damage to
metabolic enzymes as well as to the mitochondria, the power-
house of eukaryotic cells, could put a severe constraint on energy
production, which may disturb brain function as well.[22]
Recently, a growing number of in vitro and in vivo studies
carried out in vertebrate model species such as zebrafish and
mice indicated that exposure to plastic particles may induce
oxidative stress in the brain.[16,23 ] Considering the established
link between oxidative stress and brain aging, which is associated
with cognitive impairments in vertebrates,[24] it is theoretically
plausible that exposure to nanoplastics (NPs) could disrupt
cognitive functions, specifically learning and memory processes,
through imposition of oxidative stress and exacerbation of brain
aging. However, to the best of our knowledge, this inference
has never been verified with empirical data. Due to ease of
manipulation and high homology to humans, zebrafish (Danio
rerio) are widely used as a model fish species in toxicological,
pharmacological, and pathological studies.[25] Notably, zebrafish
share similar pathways of oxidative stress-induced brain aging
with other vertebrates, including humans, and exhibit compa-
rable aging phenotypes (i.e., accumulation of 𝛽-galactosidase
(𝛽-gal) and lipofuscin (lipo) in the brain, as well as decline in
cognition).[19a,26 ] Moreover, maze-exploration tasks have been
well developed in zebrafish to assess cognition, particularly
learning and memory.[27] For example, the T-maze exploration
tasks are well-developed behavioral paradigms to evaluate learn-
ing and memory capacities in zebrafish, in which the latency to
enter and the cumulative time spent in the reward zone can serve
as effective indicators for learning and memory, respectively.[28]
All these factors make zebrafish an ideal animal to explore the
impacts of environmental stressors on cognition as well as brain
aging.[25]
In the present study, to better understand the health risks of
plastic pollution, the impacts of environmentally realistic levels
of NPs on learning and memory were evaluated by classic T-maze
exploration tasks in zebrafish. Moreover, the status of brain aging
was assessed by quantifying the two brain aging-specific mark-
ers, 𝛽-gal and lipo. To further reveal potential mechanisms un-
derlying the hampered learning and memory as well as the ag-
gravated brain aging observed, oxidative stress (levels of reactive
oxygen species (ROS), H2O2,andO
2−along with the total antioxi-
dant capacity) and degrees of oxidative damage (levels of lipid per-
oxidation, DNA damage, and protein carbonylation) in the brain
were also analyzed. Furthermore, energy provision (in vivo con-
tent of ATP) as well as the status of the cell cycle (expression of
p53 and CDK2), the other two possible causes for brain dysfunc-
tion that might be induced by oxidative damage, were also deter-
mined.
2. Results and Discussion
2.1. Physicochemical Characterization of Polystyrene
Nanoplastics
According to scanning electron microscopical (SEM, ZEISS
Gemini300, Germany) analysis, the polystyrene nanoplastics
(PS-NPs) under investigation were almost perfectly sphere-
shaped at a primary diameter of 50.00 ±3.00 nm (Figure 1A, and
the data of size, shape, density, and dynamic swelling rate were
provided in Table S1 (Supporting Information). Using a nanopar-
ticle size and zeta potentiometer (Zetasizer Nano, Malvern Pan-
alytical, UK), the zeta potential of the PS-NPs in water was de-
termined to be −27.51 ±1.86 mV (Figure 1B). Furthermore, the
chemical composition of the PS-NPs was verified with Fourier-
transform infrared spectroscopy (NICOLET iS50FT-IR, Thermo
Scientific, USA) at the wavenumber range of 4000–400 cm−1
(Figure 1C). As a consequence of methylene’s (CH2) symmetri-
cal bending vibration (𝛿s), symmetric stretching vibration (𝜎s),
and antisymmetric stretching vibration (𝜎as), peak values were
observed at 1452.39, 2848.86, and 2922.16 cm−1, respectively. In
addition, a peak at 3024.28 cm−1caused by the unsaturated hydro-
carbon group stretching vibration (𝜎) of the benzene ring (═CH)
and peaks at 1492.90 and 1600.91 cm−1produced by the bend-
ing vibration (𝛿) of the benzene ring skeleton (C═C) were also
identified. Furthermore, peaks at 700.16 and 756.09 cm−1indi-
cating the unsaturated hydrocarbon groups ( ═C─H) during out-
of-plane bending vibration (𝛿) on the benzene ring were detected
as well.
2.2. Impacts of PS-NPs Exposure on Learning and Memory in
Zebrafish
On the 5th day of testing (after 4 days of reward learning), the re-
constructed swimming tracks demonstrated that zebrafish from
the control group manifested an evident preference for the re-
ward zone (end of the right short arm), however, this prefer-
ence was found to be eliminated to a certain degree for those
treated with NPs (Figure 2A). According to the statistics, com-
pared to the control zebrafish, the NP-exposed zebrafish (both
male and female) exhibited significantly longer latency times in
the T-maze exploration tasks after 4 days of training with food
rewards (Figure 2B,C). In addition, the latency time was shown
to decrease with increasing training time for both males and fe-
males (Figure 2B,C, p<0.05).
Similarly, the heatmaps of zebrafish on the 5th day of testing
illustrated that fish individuals from the control group stayed in
the reward zone for a longer time in the T-maze exploration tasks
compared to those exposed to NPs (Figure 3A). Based on the sta-
tistical analysis, after 4 days of training with food rewards, ze-
brafish from the control group had significantly longer cumula-
tive time in the reward zone than those from the NP-exposure
groups (Figure 3B,C). In addition, the cumulative time in the re-
ward zone was found to increase with the training time after 4
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Figure 1. Physicochemical characterizations of PS-NPs. The representative image of PS-NPs obtained by scanning electron microscopy A). The scale
bar =1.00 μm for 10 cells (100 nm per cell). The zeta potentials of PS-NPs in distilled water (n=3) determined by the zeta potentiometer B). Result of
the Fourier-transform infrared spectroscopy (FTIR) analysis of the PS-NPs at the wavenumber range of 4000–400 cm−1C).
days of reward learning for both males and females (Figure 3B,C,
p<0.05).
In this study, although the latency decreased and the cumu-
lative time increased with training time for zebrafish from both
the control and NP-exposure groups, after 4 days of reward learn-
ing, the NP-exposed zebrafish took significantly longer to reach
the food pellets (Figure 2) and spent markedly less time in the
reward zone (Figure 3) than the control zebrafish. This delay in
latency and reduction in cumulative time indicated that both the
learning and memory capacity of the fish were significantly ham-
pered by NP exposure.
2.3. Effects of PS-NPs Exposure on Brain Aging in Zebrafish
Along with the learning and memory deficits observed, it was
also shown that NP-exposed zebrafish had significantly higher
levels of 𝛽-gal and lipo in their brains than the control zebrafish
(Figure 4,p<0.05). Compared to that of the corresponding con-
trol, significantly higher levels of the brain aging marker 𝛽-gal
were observed in the brains of male and female zebrafish from
the NP-exposure groups (Figure 4A–C, p<0.05). Similarly, af-
ter 28 days of exposure to the PS-NPs tested, zebrafish had sig-
nificantly more lipo in their brains, which were ≈1.25 and 1.22
times the corresponding control for males and females, respec-
tively (Figure 4D,E, p<0.05).
𝛽-gal is a hydrolase inside lysosomes, the amount of which in-
creases with the senescence of the cell and thus is widely used
as an indicator of aging.[29] Being famous as the "aging pig-
ment," lipo is a complex product of the oxidation of proteins and
lipids and is regarded as another reliable biomarker for brain
aging in vertebrates, including zebrafish.[30] Thus, higher levels
of these two aging markers detected in the brain indicated that
NP-exposed fish might suffer significant degrees of brain aging,
which offers a potential explanation for the learning and memory
deficits observed.
2.4. Impacts of PS-NPs Exposure on the Level of Oxidative Stress
in the Brains of Zebrafish
ROS-specific dihydroethidium (DHE) staining (Figure 5A)
demonstrated that zebrafish exposed to NPs for 28 days had
significantly higher ROS-specific fluorescence intensity in their
brains, which was ≈2.40 and 4.23 times that of the corresponding
control for males (Figure 5B, p<0.01) and females (Figure 5C, p
<0.05), respectively. After 28 days of NP exposure, significantly
lower levels of total antioxidant capacity (T-AOC) were detected
in the fish brains, which were ≈89.52% and 87.67% of that of the
corresponding control for male and female individuals, respec-
tively (Figure 5D,E, p<0.05). In addition, compared to those
of the corresponding control, zebrafish treated with NPs had
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Figure 2. Representative reconstructed swimming tracks A) on the 5th
day of testing (the day after the 4 day reward learning) and the quantified
latency values for male B) (n=15) and female C) (n=15) zebrafish after
28 days of exposure to control and PS-NPs. Latency data (means ±SDs)
in (B,C) with different superscripts above were significantly different (*
and ** indicate p<0.05 and p<0.01 compared to the control; # and ##
indicate p<0.05 and p<0.01 compared to Day 1). RZ: reward zone.
markedly higher levels of H2O2(Figure 5F,G, p<0.05) and O2−
(Figure 5H,I, p<0.05) in their brains.
These data suggested that NP-induced brain aging may be due
to the induction of oxidative stress and subsequent damage to the
brain. On the one hand, compared to that of the control, signif-
icantly higher levels of free radicals (ROS, H2O2,andO
2−)were
detected in the brains of NP-exposed fish (Figure 5), indicating
an evident induction of oxidative stress by the NPs tested. In ad-
dition, since excessive free radicals should be removed by the an-
tioxidant system under normal conditions, the accumulation of
free radicals in the brains of NP-exposed fish may arise from the
constrained antioxidant capacity (lower T-AOC, Figure 5D,E) ob-
served.
2.5. Effects of PS-NPs Exposure on the Level of Brain Oxidative
Damage
Both the immunofluorescent staining (Figure 6A) and subse-
quently quantified numbers of 𝛾-H2AX foci per cell (Figure 6B,C,
p<0.01) showed that NP-exposed fish had markedly higher lev-
els of 𝛾-H2AX in their brains, which were ≈2.33 and 1.67 times
the corresponding control for males and females, respectively.
Similarly, compared to the corresponding control, significantly
higher 8-hydroxydeoxyguanosine (8-OHdG) contents were de-
tected in the brains of zebrafish from the NP-exposure groups
(Figure 6D,E, p<0.05). Moreover, the brain malondialdehyde
(MDA) contents were ≈21% and 93% higher than those of the
corresponding controls for NP-exposed male (Figure 6F, p<0.05)
and female (Figure 6G, p<0.01) zebrafish, respectively. In addi-
tion, compared to the control, zebrafish treated with NPs had sig-
nificantly higher levels of protein carbonyl in their brain, which
were ≈1.98 and 1.10 times the control for males (Figure 6H, p<
0.01) and females (Figure 6I, p<0.05), respectively.
The 8-OHdG is a product produced by the attack of the 8th
carbon atom of the guanine base in DNA by active oxygen
radicals.[31] 𝛾-H2AX is produced by the phosphorylation of his-
tone H2A variant H2AX upon oxidative stress-induced double-
strand breaks of DNA.[32] Similarly, MDA and protein carbonyl
are oxidation products of lipids and proteins, respectively.[33]
Thus, the boost of these markers (Figure 6) indicated that ex-
posure to NPs resulted in marked oxidative damage to the fish
brain, a well-known potential cause for brain aging.[22,24b,34 ]
2.6. Impacts of PS-NPs Exposure on Energy Provision and the
Cell Cycle of the Brain
After 28 days of PS-NPs exposure, significantly less Adenosine
triphosphate (ATP) was detected in the fish brains, which were
≈65.83% and 50.34% of the ATP amounts in the correspond-
ing control for male and female individuals, respectively (Figure
7A,B, p<0.05). Compared to the corresponding control group,
zebrafish treated with NPs had markedly higher contents of p53
protein (≈1.21 and 1.23 times the amount in the male and fe-
male controls, respectively, Figure 7C,D, p<0.05) but lower lev-
els of CDK2 (≈93.65% and 94.03% of the amount in the male
and female controls, respectively, Figure 7E,F, p<0.05) in their
brains. These data indicated that the brains of NP-exposed fish
may suffer energy deficits and cell cycle arrestment. Because suf-
ficient energy supply and normal brain cell populations are cru-
cial for the cognitive function of the brain,[19b,35 ] these alterations
could be another cause for the NP-induced learning and mem-
ory deficits observed. According to previous studies,[22,36 ] the dis-
ruption of energy supply and the cell cycle may result from the
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Figure 3. Representative heatmaps A) of zebrafish in the T-maze on the 5th day of testing (the day after the 4-day reward learning) and the cumulative
time in the reward zone for male B) (n=15) and female C) (n=15) zebrafish after 28 days of exposure to control and PS-NPs. Cumulative time data
(means ±SDs) in (B,C) with different superscripts above were significantly different (* and ** indicate p<0.05 and p<0.01 compared to the control;
# and ## indicate p<0.05 and p<0.01 compared to Day 1). RZ: reward zone.
induction of oxidative stress as well. For instance, oxidative dam-
age to key cellular organelles (i.e., mitochondria) and enzymes
may impair mitochondrial function and lead to insufficient en-
ergy supply.[35,37] However, this inference awaits further verifica-
tion with direct evidence.
Finally, although some behavioral and physiological dif-
ferences have been documented between male and female
zebrafish,[38] no such difference was observed in the response of
learning and memory to NPs in this study. This result is in accor-
dance with those reported for the response of zebrafish to other
exogenous stimuli,[39] indicating that the learning and memory
function of the brain might be independent of sex.
Compared to those reported for terrestrial species (i.e., nema-
tode and mice) [40] our study represents the first empirical evi-
dence to establish a causal relationship between PS-NPs expo-
sure and learning and memory impairment in aquatic species.
Furthermore, the finding that brain aging, oxidative damage, in-
sufficient energy supply, and cell cycle arrestment might be the
causes for the learning and memory impairment detected should
contribute to a better understanding of corresponding mecha-
nism of action. Although microplastics are extremely diverse (i.e.,
in terms of polymer composition and particle size) and their con-
centrations differ dramatically among different regions in natural
environments, only PS-NPs at a regular size (50 nm) and a sin-
gle exposure dose (1 mg L−1) was investigated in this study due
to constrains of labor and finance. In future research, the corre-
sponding effects of different concentrations, sizes, and types of
microplastics can be given priority consideration to further en-
rich our understanding of this topic.
3. Conclusion
With the increasingly aging population (people older than 80 are
predicted to surpass 400 million in 2050), learning and memory
deficits, a representative cognitive impairment of many neurode-
generative diseases accompanied by aging, are currently a ma-
jor challenge to human health worldwide.[41] Our results demon-
strated that NP exposure may lead to learning and memory
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Figure 4. Images of 𝛽-galactosidase (𝛽-gal) staining A), quantified 𝛽-gal
stained area to that of the whole tissue observed B,C) (n=3), and lipo-
fuscin (lipo) contents D,E) (n=6) in the brain of zebrafish after 28-day
exposure to control and PS-NPs, respectively. Magnification at 400×and
scale bar =50 μm for (A). Representative 𝛽-gal signals are highlighted by
white arrows in (A). Data (means ±SDs) with different superscripts above
were significantly different at p<0.05 in (B–E).
deficits in zebrafish by inducing oxidative stress, constraining en-
ergy supply, arresting the cell cycle, and aggravating brain aging.
Considering the ubiquitous characteristics of NPs (the overall in-
take of plastic particles by human adult is predicted to be 2.93 ×
1010 particles per year)[42] and the high homologies of zebrafish
to other vertebrates, including humans, the potential risk of NPs
deserves more attention.
4. Experimental Section
Experimental Animals and Materials:According to previous
surveys,[43] PS-NPs were adopted as representative NPs in this study
due to their ubiquitous presence in the environment. Commercial PS-
NPs were obtained from the Regal Nanoplastic Engineering Research
Institute (Jiangsu, China). Before the experiments, the shape, size,
zeta potential, and chemical composition were verified with scanning
electron microscopy (SEM, ZEISS Gemini300), nanoparticle size and
zeta potentiometer (Zetasizer Nano, Malvern Panalytical, UK), and
Fourier-transform infrared spectroscopy (FTIR, NICOLET iS50FT-IR,
Thermo Scientific, USA), respectively.
Adult zebrafish (wild type, TU strain, 4 months old and 3.5 ±0.4 cm
in body length) were purchased from FishBio Co.; Ltd. (Shanghai, China)
and acclimated in dechlorinated tap water (aeration for 72 h before use)
for 2 weeks before the experiments. During the acclimation, the tempera-
ture and pH of the water were maintained at 28.2 ±0.2 and 7.2 ±0.3 °C,
respectively. Commercial food pellets (FishBio Co.; Ltd.; Shanghai, China)
were provided at a rate of ≈5% body weight and the water was renewed 1
h after feeding every day. In addition, a light cycle of 14 h light/10 h dark
was adopted for the acclimation. All experiments were approved by the
Animal Care Committee of Zhejiang University, and all methods were per-
formed following the Guidelines for the Care and Use of Animals for Re-
search and Teaching at Zhejiang University (ETHICS CODE Permit NO.
ZJU20220031). After corresponding exposure, the zebrafish were anes-
thetized in 0.02% Tricaine (E10521, Sigma-Aldrich) and sacrificed in ice
water before obtaining the tissue specimen.[44]
Exposure Experiments:In total, four experimental groups were set
up in triplicate, namely, a male control group, a male PS-NPs exposure
group, a female control group, and a female PS-NPs exposure group.
In brief, after the acclimation, 840 zebrafish individuals (420 males and
females, respectively) were randomly assigned to corresponding control
and NP-exposure groups (4 experimental groups ×3 replicates/group
×70 zebrafish/replicate). According to previous surveys, 1.0 mg L−1
was adopted as the exposure concentration for NPs in this study, which
is equivalent to those reported in the Miri River and the Amsterdam
canals.[45] In this study, 800 μL of the commercial PS-NPs solution
(250 mg 10 mL−1, without inclusion of any additives) was added into 20
L of dechlorinated tap water to attain the desired exposure concentration
(1.0 mg L−1). The exposure was conducted in glass tanks filled with 20
L dechlorinated tap water for 4 weeks, during which the same conditions
(i.e., temperature, pH, and light cycle) described for acclimation were
adopted.
Evaluation of Learning and Memory Capability of Zebrafish by T-Maze Ex-
ploration Tasks:To evaluate the learning and memory capability of the
zebrafish, classic T-maze exploration tasks were performed according to
previously reported methods.[46] A glass T-maze with a long arm and two
short arms (L: W: H =40 cm: 10 cm: 10 cm and 20 cm: 10 cm: 10 cm for
the long and short arms, respectively) was custom made. A start zone and
arewardzone(L:W:H=10 cm: 10 cm: 10 cm for both) were defined at
the far ends of the long arm and the right short arm, respectively (T-maze
schematic representation is provided in Figure S1, Supporting Informa-
tion). After the corresponding exposure treatment, 30 individual fish were
randomly collected from each experimental group and used for the T-maze
exploration tasks. According to the testing paradigm, a 4-day habituation
was conducted first by introducing fish into the T-maze (filled with water
at 28 °C to a height of 6 cm) for 10 min every day to minimize potential
novelty stress. During this process, the number of fish habituated once
gradually decreased every day (15, 10, 5, and 1 individual for the 4 consec-
utive days, respectively). Fifteen zebrafish from each experimental group
that exhibited normal swim behavior after habituation were then individu-
ally subjected to reward learning. Specifically, after depositing food pellets
(≈20.0 mg, FishBio Co.; Ltd.) in the reward zone, one fish was introduced
into the start zone of the T-maze and allowed to explore for 5 min, dur-
ing which the swimming behavior of the fish was recorded by an overhead
digital camera (1920 ×1080 pixels @30 fps, C930e, Logitech, Lausanne,
Switzerland). Reward learning was conducted with each zebrafish once a
day for 4 consecutive days. The day after the last day of reward learning
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Figure 5. Images of ROS-specific fluorescent staining A), quantified ROS fluorescent intensities B,C) (n=3), the total antioxidant capacity (T-AOC) D,E)
(n=6), and in vivo contents of H2O2F,G) (n=6) and O2−H,I) (n=6) in the brain of zebrafish after 28 days of exposure to control and PS-NPs. Brain
cells and ROS were stained blue (DAPI) and red (DHE) in (A), respectively (magnification at 100×and scale bar =100 μm). Data (means ±SDs) with
different superscripts above in (B–I) were significantly different between groups at *p <0.05 and **p <0.01.
(the 5th day of testing), each fish was tested one more time as described
above except without adding the food pellets in the reward zone.
Following the methods reported,[47] video data collected were trans-
formed into grayscale image stacks and then analyzed using ImageJ soft-
ware. In brief, by adjusting the threshold, the position of the zebrafish in
the T-maze was identified in the grayscale images of the video. The time
it took for the first entry of the fish into the reward zone after release
(latency) and the total duration (cumulative time) that fish spent in the
reward zone were then determined using the grayscale image stacks ob-
tained. To visualize the results, after defining the region of interest (ROI),
threshold (Thd), and frame parameters (number and rate), the fish posi-
tion was identified in each frame of the video and then used to construct
the swimming track and heatmap in MATLAB (the program code written
for the analysis is provided in Text (S1) of the Supporting Information).
Assessment of Brain Aging Status:The accumulation of NPs in the brain
of zebrafish was verified with confocal microscopy (detailed methods (Text
S2, Supporting Information) and results (Figure S2, Supporting Informa-
tion) are provided in the Supporting Information ). Then, the status of
brain aging of the fish was assessed by quantifying the two brain aging-
specific markers, 𝛽-gal and lipo, in the brain using corresponding com-
mercial 𝛽-gal staining kit (C0602, Beyotime, China) and lipo ELISA kit
(JL51902, Jonln Biotechnology, China), respectively.
To estimate 𝛽-gal in the brain, after the corresponding exposure treat-
ment, three fish were randomly selected from each experimental group (n
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Figure 6. Images of 𝛾-H2AX immunofluorescence staining A), quantified numbers of 𝛾-H2AX foci per cell B,C), (n=3), and in vivo contents of 8-OHdG
D,E) (n=6), malondialdehyde (MDA, F,G) (n=6), and protein carbonyl H,I) (n=6) in the brains of zebrafish after 28 days of exposure to control and
PS-NPs. Brain cells and 𝛾-H2AX were stained blue (DAPI) and green (𝛾-H2AX), respectively, as shown in (A) (magnification at 200×and scale bar =
100 μm). Data (means ±SDs) with different superscripts above in (B–I) were significantly different at *p<0.05 and ** p <0.01.
=3) and fixed in 4% paraformaldehyde (PFA, 4 °C for 12 h). Brains were
subsequently dissected from the fish, washed three times with phosphate-
buffered saline (PBS, P1010, Solarbio, China). The telencephalon of the
brain was cryosectioned longitudinally to a thickness of 20 μmwithacryo-
stat microtome (CM 1950, Leica, Germany), and then stained with 𝛽-gal-
specific staining (C0602, Beyotime, China) following the manufacturer’s
instructions. Images of the stained sample were subsequently captured
using an Olympus BX53 microscope fitted with a Qimaging MicroPub-
lisher 5.0 RTV camera. To quantify the level of 𝛽-gal in the brain, the ratio
of the 𝛽-gal stained area to that of the whole tissue observed was deter-
mined using ImageJ software following the methods of Zhao et al.[48]
After corresponding exposure, 18 zebrafish were randomly col-
lected from each experimental group, and the brains dissected
from three individuals out of the same replicate were pooled as
onesample(n=6) for lipo quantification. In brief, brain sam-
ples were homogenized with precooled PBS (4 °C) and then cen-
trifuged at 5000 g for 10 min. The supernatant obtained was
incubated with horseradish peroxidase (HRP) labeled antibodies at
37 °C for an h, followed by another 15 min incubation with the chro-
mogenic reagent of the kit. The absorption value of the sample was then
determined at 450 nm with a microplate reader (Multiskan GO, Thermo
Scientific, USA). After quantifying the protein content using the Bradford
method (P0006, Beyotime, China), the lipo content in the brain was
calculated with the absorption value obtained and standardized to the
protein content of the sample.
Estimation of Oxidative Stress in the Brain:To evaluate the level of ox-
idative stress in the fish brain, total antioxidant capacity (T-AOC) as well as
levels of ROS, H2O2,andO
2−were determined with the 2,2′-azino-bis (3
ethylbenzthiazoline)−6-sulfonic acid (ABTS) T-AOC kit (S0121, Beyotime,
China), the ROS-specific fluorescent dye dihydroethidium (DHE, D7008,
Sigma, USA), the H2O2content kit (BC3595, Solarbio, China), and the O2−
content kit (BC1295, Solarbio, China), respectively.
After corresponding exposure, brains dissected from 18 fish (three from
the same replicate were pooled as one sample, n=6) for each experimen-
tal group were homogenized with precooled PBS and centrifuged (12 000 g
for T-AOC and 8000 g for H2O2and O2−, respectively; 4 °C for 10 min).
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Figure 7. The in vivo contents of ATP A,B) (n=6), p53 protein C,D) (n=6), and cyclin-dependent kinase 2 (CDK2, n=6) E,F) in the brains of zebrafish
after 28 days of exposure to control and PS-NPs. Data (means ±SDs) with different superscripts above were significantly different at *p<0.05 and **
p<0.01.
For T-AOC estimation, the supernatant obtained was incubated with the
working solution (containing ABTS) of the kit at room temperature for
6 min. The absorption value of the sample at 414 nm was then recorded
with a microplate reader (Multiskan GO, Thermo Scientific, USA). To eval-
uate H2O2, the supernatant was incubated with titanium sulfate for 10 min
followed by centrifugation at 4000 g for 10 min. After redissolving the pre-
cipitate obtained with the lysate of the kit, the absorption value of the sam-
ple was determined at 414 nm with a microplate reader. For O2−assess-
ment, the supernatant was incubated with hydroxylamine hydrochloride
at 37 °C for 20 min followed by another 20 min incubation at 37 °Cwith
sulfanilamide and naphthaleneethylenediamine hydrochloride. After cen-
trifugation at 8000 g for 5 min, the absorption value of the supernatant at
530 nm was determined with a microplate reader. The level of T-AOC and
contents of H2O2and O2−in the sample were then calculated with the
absorption values obtained following the manufacturer’s instructions and
standardized by the protein content of the sample.[49]
To estimate ROS, brains collected individually from three fish (n=3)
for each experimental group were fixed with 4% PFA, washed with PBS,
and dehydrated at 4 °C with sucrose (15% and 30% sequentially). After
being embedded in optimal cutting temperature compound (OCT, 4583,
SAKURA), the telencephalon of the brain was cryosectioned longitudinally
to 16 μm thickness, stained with ROS-specific fluorescent dye (DHE) at
37 °C in the dark for 30 min, and subsequently stained with DAPI (D9542,
Sigma, USA) at room temperature in the dark for 10 min. Sample images
(magnification at 100×) were then captured with an Eclipse E100 fluores-
cence microscope (Nikon, Japan) at excitation and emission wavelengths
of 535 and 610 nm, respectively. The ROS-specific fluorescence intensity of
each sample was determined using ImageJ software following the meth-
ods of Qiang and Cheng.[50]
Quantification of Oxidative Damage Levels in the Brain:To assess the
levels of oxidative damage to the brain, two representative DNA oxidative
damage markers, 8-OHdG and 𝛾-H2AX, along with MDA (terminal prod-
uct of lipid peroxidation) and protein carbonyl content (specific marker
for protein oxidative damage), were evaluated with corresponding com-
mercial kits.
Brains dissected from 18 fish for each experimental group (three were
pooled as one sample, n=6) were homogenized and centrifuged at 8000 g
and 4 °C for 10 min, and the supernatants collected were then used to de-
termine the 8-OHdG and MDA levels. For 8-OHdG estimation, according
to the instructions of the commercial 8-OHdG ELISA kit (JL22584, Jonln
Biotechnology, China), after incubation with the HRP-labeled antibody and
the chromogenic reagent of the kit, the absorption value of the sample at
450 nm was measured with a microplate reader (Multiskan GO, Thermo
Scientific, USA). Similarly, following the protocols provided by the MDA
content kit (BC0025, Solarbio, China), after incubation with thiobarbituric
acid (TBA) at 100 °C for 1 h and subsequent centrifugation at 10 000 g for
10 min, the absorption values of the supernatant at 450, 532, and 600 nm
were recorded with a microplate reader.
Brain samples harvested from 18 individuals for each experimental
group (three were pooled as one sample, n=6) were homogenized with
the lysis buffer of the protein carbonyl assay kit (BC1275, Solarbio, China).
Supernatant obtained by centrifugation (8000 g and 4 °C for 10 min) was
then incubated with 2,4-dinitrophenylhydrazine (DNPH) solution for 1 h.
After adding trichloroacetic acid and centrifugation (10 000 g and 4 °Cfor
15 min), the precipitate collected was washed three times with acetone
and dissolved in 200 μL double distilled water (ddH2O). The absorption
value of the sample at 375 nm was then determined with a microplate
reader. After quantifying the protein content in the sample as described
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above, the contents of 8-OHdG, MDA, and protein carbonyl in each sam-
ple were subsequently calculated with the absorbance values obtained and
normalized to the protein content of the sample.
The immunostaining of 𝛾-H2AX, the key protein participating in DNA
repair after damage, was conducted following the methods of Zhao
et al.[48] In brief, after cryosectioning the telencephalon (n=3) longitudi-
nally to a thickness of 16 μm as described above, the sample was washed
with PBS to remove OCT, permeabilized with 0.2% Triton for 15 min, rinsed
with 0.5% bovine serum albumin (BSA) for 5 min, and then blocked with
20% goat serum. The sample was subsequently incubated with the pri-
mary antibody (anti-H2A. XS139ph (GTX127340, Genetex, China) diluted
with 0.5% BSA at a ratio of 1:200) at 4 °C overnight, followed by a 1 h
incubation with the secondary antibody (anti-rabbit IgG H&L Alexa Fluor
647 (ab150143, Abcam, China) diluted at 1:500) at room temperature in
the dark. After three rounds of BSA wash (5 min each) and DAPI (D9542,
Sigma, USA) staining at room temperature in the dark for 10 min, images
of the sample (magnification at 200×) were then captured with an Eclipse
E100 fluorescence microscope (Nikon, Japan) at excitation and emission
wavelengths of 652 and 668 nm, respectively. According to the methods of
Cai et al.,[51] the number of 𝛾-H2AX foci per cell was subsequently counted
with ImageJ to quantify the level of 𝛾-H2AX
Determination of In Vivo ATP Content in the Brain:According to the
manufacturer’s instructions, the energy provision in the brain was esti-
mated by quantifying the in vivo content of ATP using a commercial ATP
content kit (BC0305, Solarbio, China). In brief, after corresponding expo-
sure, brains were dissected from 18 fish for each experimental group (three
were pooled as one sample, n=6), homogenized with the extraction so-
lution of the kit, and then centrifuged at 8000 g and 4 °C for 10 min. The
supernatant obtained was mixed with chloroform and then centrifuged at
10 000 g and 4 °C for 3 min. After incubating the supernatant obtained
with the ATP assay buffer at 25 °C for 10 min, the absorbance value of
the sample at 340 nm was determined with a microplate reader (Thermo
Multiskan Go, USA). The ATP content in the sample was then calculated
with the absorption value obtained and standardized to the sample protein
content.
Assessment of the Cell Cycle Status of the Brain:To assess the cell cy-
cle status of the brain, two cell cycle-specific markers, p53 and CDK2,
were quantified with corresponding commercial ELISA kits (JL39151 and
JL44898, Jonln Biotechnology, China, respectively). Briefly, collected brains
(18 fish per experimental group, brains from 3 individuals were pooled as
one sample, n=6) were homogenized with PBS and then centrifuged at
3000 g and 4 °C for 10 min. The supernatant obtained was subsequently
incubated with the corresponding enzyme-labeled antibodies at 37 °Cfor
1 h followed by another 15 min incubation (37 °C) with the corresponding
chromogenic reagent in the dark. After determining the absorption value
of the sample at 450 nm with a microplate reader, the contents of p53 and
CDK2 were calculated with the absorption values obtained and normalized
to the protein content of the sample.
Statistical Analysis:After verifying the prerequisite of the analysis with
the Shapiro–Wilk test, all parameters were then compared using Student’s
t-test. Values are presented as the mean ±standard deviation (SD) in the
present study. The sample size (n) and testing level (pvalue) were noted
in corresponding legends of the figures. All analyses were conducted us-
ing OriginPro 8.0 and GraphPad Prism 9.0 software, and statistical signif-
icance was set at p<0.05.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
This work was funded by the National Key R&D Program of China
(No. 2018YFD0900603), the Key R&D Program of Zhejiang Province
(No. 2021C02048), the Science and Technology Ombudsman Project of
Huzhou, Zhejiang Province (No. 2021KT46), the Natural Science Foun-
dation of Zhejiang Province (No. LQ21C190003), and the Open Fund of
Zhejiang Key Laboratory of Exploration and Preservation of Costal Bio-
resources (No. J2021001).
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
W.S.Z., W.S., and G.X.L. contributed to experimental design, statistical
analysis, and manuscript preparation. W.S.Z., D.F.T., D.D.T., and Y.Y.Y. car-
ried out the experiments. L.H., W.X.Z., Y.H.Y., W.Q.P., J.W.S., L.Z.L., and
X.Y.Z. contributed to experiment preparation and data analysis. G.X.L.
contributed to substantive discussion of the results and revision of the
manuscript.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
brain aging, learning and memory deficits, nanoplastics, oxidative stress,
zebrafish
Received: June 8, 2023
Revised: August 16, 2023
Published online:
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