Ecotoxicology and Environmental Safety 218 (2021) 112296

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Ecotoxicology and Environmental Safety

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

Preliminary study on impacts of polystyrene microplastics on the

hematological system and gene expression in bone marrow cells of mice
Rongli Sun a, *, Kai Xu a, Linling Yu a, Yunqiu Pu a, Fei Xiong a, Yuhong He b, Qingchen Huang a,
Mingjie Tang b, Minjian Chen c, d, Lihong Yin a, Juan Zhang a, Yuepu Pu a, *
a
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, Jiangsu, China
b
School of Medicine, Southeast University, Nanjing 210009, Jiangsu, China
c
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, China
d
Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China

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

Edited by Dr Yong Liang Microplastics (MPs) are currently a global environmental pollutants and health hazards that caused by MPs
cannot be ignored. However, studies on MP toxicity in mammals are scare. Here, we investigated the effects of
Keywords: two doses (0.1 mg and 0.5 mg) of 5 µm polystyrene microplastic (PS-MP) particles on the hematological system
Microplastics of mice through traditional toxicology experiments and assessed the related potential biological mechanisms
Toxicity
using transcriptome sequencing analysis. The toxicological examinations showed that the 0.5 mg dose signifi­
Hematological system
cantly decreased white blood cell count, increased Pit count, and inhibited the growth of colony-forming unit
Transcriptomics analysis
Mice CFU-G, CFU-M and CFU-GM. Compared with the control group, there were 41 differentially expressed genes
(DEGs) in the 0.1 mg-treated group and 32 significantly changed genes in 0.5 mg-treated group. Of note, eight
genes were found to be significantly altered in both the PS-MP-treated groups. Gene ontology analysis showed
that DEGs were mainly involved in T cell homeostasis, response to osmotic stress, extracellular matrix and
structure organization, and metabolic process of NADP and nucleotides. In addition, pathway analysis revealed
that the Jak/Stat pathway, pentose and glucuronate interconversions, nicotinate and nicotinamide metabolism,
biosynthesis of unsaturated fatty acids, and the pentose phosphate pathway were involved in PS-MP-induced
toxicity in mice. These results indicated that PS-MP exposure can cause hematotoxicity to some extent, impact
gene expression, and disturb related molecular and biological pathways in mouse bone marrow cells. Our study
provides fundamental data on the hematotoxicity of PS-MPs in terrestrial mammals that will help to further
assess the corresponding health risks in these mammals.

1. Introduction
Microplastics (MPs) are generally considered to be plastic particles
or fragments with a particle size of less than 5 mm (Wright and Kelly,
2017), and are divided into two categories: primary MPs and secondary
MPs (Zhao et al., 2018). Owing to their low production cost, good
ductility, and durability, plastics are widely used worldwide. Between
1950 and 2015, approximately 6300 MT of plastic waste was generated,
of which around 4900 MT was discarded in landfills or the natural
environment (Geyer et al., 2017). Microplastic pollution exists widely,
not only in the ocean, estuaries, rivers, lakes, and atmosphere, but also
in the soil and indoor air (Rillig and Lehmann, 2020; Sekudewicz et al.,
2021; Zhang et al., 2020). Excessive MPs that enter the ocean and soil
environments through multiple channels can affect the animals, plants,
and microorganisms who live in these systems, and can even endanger
human health through the delivery effect of the food chain/net (Jiang
et al., 2020). Current research shows that MP inhalation (Enyoh et al.,
2019), and digesting MP-polluted water and food (marine fish, shellfish,
and vegetables) are the main pathways through which MPs enter the
human body (Huerta Lwanga et al., 2017; Santana et al., 2017).
Microplastic pollution is listed as an important scientific issue in the
fields of environment and ecology (Horton et al., 2017), and the health
risks caused by it have attracted increasing attention (Prata et al., 2020).
At present, most studies on the biological toxicity of MPs have mainly
focused on marine and aquatic animals, such as various fish species and
invertebrates (Akdogan and Guven, 2019). However, there are a few

* Corresponding authors.
E-mail addresses: 101012172@seu.edu.cn (R. Sun), yppu@seu.edu.cn (Y. Pu).

https://doi.org/10.1016/j.ecoenv.2021.112296
Received 3 January 2021; Received in revised form 31 March 2021; Accepted 24 April 2021
Available online 4 May 2021
0147-6513/© 2021 The Author(s). 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/).
R. Sun et al.
studies on the health risks of MPs to organisms at the highest trophic
level, such as marine mammals and terrestrial mammals, and even
humans. Mice are a commonly used mammalian model in medical
research. Deng et al. (2017) conducted a study and found an accumu­
lation of 5 µm and 20 µm fluorescent and pristine polystyrene micro­
plastic (PS-MP) particles in mouse liver, kidney, and gut. In addition,
biochemical and metabolomics analyses indicated that MP exposure
disturbs energy and lipid metabolism and causes oxidative stress and
alterations in neurotoxicity biomarkers. Two other studies (Jin et al.,
2019; Lu et al., 2018) also revealed that micron-level MPs could accu­
mulate in the gut of mice and induced intestinal barrier dysfunction, gut
microbiota dysbiosis, and metabolism disorder in mice. Recent studies
also showed that MPs caused pyroptosis and apoptosis of ovarian
granulosa cells (Hou et al., 2021), and inflammatory effects (Zheng
et al., 2021), and lead to reproductive toxicity or behavioral disorders
(da Costa Araújo and Malafaia, 2021; Jin et al., 2021). As orally
administered MPs can be detected in the intestine, liver, and kidney,
they likely enter the body’s organs through blood circulation; however,
no study has presently reported the effect of MPs on the hematological
system.
Influences of environmental pollutants on the blood system can be
investigated via traditional toxicological assays. Traditional toxicolog­
ical methods for evaluating blood toxicity of exogenous chemical sub­
stances using animal experiments that include blood parameters, bone
marrow examination, coagulation function test, and histological exam­
ination, etc. However, it should be noted that the identification of re­
lations between such toxic effects and active molecular pathways likely
enables elucidation of the mode of action and is therefore essential.
Transcriptomics is the study of gene function and gene structure at the
overall level, and reveals the specific biological processes and molecular
mechanisms that occur during the course of a disease. It has been widely
used in basic research, clinical diagnosis, and drug development. As a
new efficient and fast transcriptome research method, RNA-seq tech­
nology can provide people with more biological transcription informa­
tion more quickly and accurately (Shao et al., 2014; Stark and Grzelak,
2019). Bone marrow (BM) is the main hematopoietic organ that pro­
duces red blood cells, granulocytes, monocytes, lymphocytes, and
platelets, and includes cells at different developmental stages. Poly­
styrene microplastic (PS-MP) is one of the main sources of marine drifts
and microplastics. Given the lack of information concerning the effects
and mechanisms of PS-MPs on the hematological system, RNA-seq
analysis was used to investigate the impact of PS-MPs on the gene
expression and biological pathways of mouse BM cells.
In the present study, traditional toxicology detection was combined
with transcriptome sequencing to explore the effect of 5 µm PS-MPs on
the hematological system of mice and the gene expression in their BM
cells. Our efforts will be helpful to comprehensively understand the toxic
effects and mechanisms induced by PS-MPs at the transcriptome level in
the mouse hematological system.
2. Materials and methods
2.1. Materials and reagents
Pristine and fluorescent (Ex 488 nm/Em 518 nm) 5 µm PS-MP par­
ticles were purchased from the BaseLine ChromTech Research Centre
(Tianjin, China). The former was used for the animal toxicologic tests
and the latter for examining the distribution of MPs in mice. The PS-MPs
were dissolved in ultrapure water and prepared by ultrasonic dispersion
(50 Hz, 5 min) to the required concentration. The characterization of the
5 µm polystyrene microplastic (PS-MP) particles by optical microscopy,
transmission electron microscopy (TEM), and coulter particle size
analyzer was shown in Fig. S1.
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Ecotoxicology and Environmental Safety 218 (2021) 112296
2.2. Experimental animals and treatment
Five-week-old male C57BL/6 mice were obtained from Beijing Vital
River Laboratory Animal Technology Co., Ltd. (Beijing, China). They
were housed in the SPF animal house of Southeast University, China and
were allowed to adapt to their new surroundings for a week before the
experimentation. Animals were housed in independent cages with ad
libitum access to food and water at 25 ± 2 ◦ C, 50 ± 5% relative hu­
midity, and a 12/12 h light/dark cycle. For toxicological experiments,
18 mice were randomly assigned to three groups: the control group (C,
ultrapure water, n = 6), the 0.1 mg 5 µm PS-MPs particles (1.46 × 106
items) group (MP1, n = 6), and the 0.5 mg 5 µm PS-MPs particles (7.3 ×
106 items) group (MP2, n = 6) group. To study the subacute toxicity of
PS-MPs, mice were provided with water or PS-MPs-water solution once
daily (0.2 mL per mouse) by oral gavage for 28 days. For tissue distri­
bution examination, another ten mice were divided into two cages (n =
5 per cage). Mice were treated with ultrapure water or 0.5 mg 5 µm
fluorescent PS-MPs once daily for 28 days by oral gavage at a volume of
0.2 mL per mouse. All animal experiments conducted in this study were
approved by the Welfare and Ethical Treatment of Animals Committee
of Southeast University, China (Approval number: 20181224007). The
general condition of mice was observed every day and the body weight
of mice was measured and recorded once a week.
2.3. Distribution of MPs in mice
To examine the existence of PS-MPs in mice organs, in vivo fluo­
rescence imaging was conducted using the IVIS® Lumina LT Series III
Quantitative Fluorescence and Bioluminescence Imaging system (Per­
kinElmer, Waltham, MA, USA) (Excitation wavelength: 488 nm; Emis­
sion wavelength: 518 nm) on mice after they were exposed to 0.5 mg 5
µm fluorescent PS-MPs for 28 days.
2.4. Blood routine and bone marrow cell count
After PS-MPs exposure, the mice were anesthetized via carbon di­
oxide inhalation and blood was acquired from the orbital sinuses.
Twenty microliters of ethylene diamine tetra acetic acid was added to
180 μL of whole blood, gently mixed, and allowed to stand at room
temperature before routine blood testing. A Sysmex XE-2100 fully
automatic hematology analyzer (Sysmex, Kobe, Japan) was used to
examine major blood indicators, including white blood cell (WBC)
counts, red blood cell (RBC) counts, hemoglobin (Hgb), and platelets
(Pit). The mouse femur and tibia were aseptically peeled off, and the BM
cells were flushed out with a syringe containing 1 mL of pre-cooled
phosphate buffered saline (2% fetal bovine serum). The BM cells were
suspended and centrifuged for 5 min (1000 rpm, 4 ◦ C). Finally, cells
were counted and prepared for the follow-up experiments.
2.5. Colony-forming assay
The MethoCult™ GF M3434 (STEMCELL Technologies) medium was
used to perform the colony-forming assay. The product has been
formulated to support optimal growth of granulocyte-macrophage pro­
genitor cells (CFU-GM, CFU-G and CFU-M), and multi-potential gran­
ulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-
GEMM). Briefly, 0.1 mL of diluted mouse BM cells (4 × 104) were mixed
with 1 mL of MethoCult™ culture medium and vortexed vigorously.
Then, the tube was allowed to stand for 5 min to exclude bubbles. A
sterile disposable 3 mL syringe fitted with a new 16 gauge blunt-end
needle was used to dispense the mixture onto 35 mm culture dishes.
These dishes were then incubated at 37 ◦ C in 5% CO2 with ≥ 95% hu­
midity. Different colonies were counted under a microscope on day 12 of
culture according to their morphology.
R. Sun et al.
2.6. RNA extraction
BM samples from three mice in each group were randomly taken for
transcriptome sequencing. Total RNA from mouse BM cells (1 × 107)
was extracted using the Trizol reagent (Invitrogen, CA, USA) following
the manufacturer’s protocol. The RNA Nano 6000 Assay Kit of the
Agilent 2100 Bioanalyzer system (Agilent Technologies, CA, USA) was
used to assess RNA integrity. The RNA purity was checked with a
NanoPhotometer spectrophotometer (Implen, Inc., CA, USA) and RNA
concentration was measured using a Qubit RNA Assay Kit in Qubit 2.0
Fluorometer (Life Technologies, CA, USA).
2.7. cDNA library construction and sequencing
First, ribosomal RNA was removed from total RNA, which was then
degraded into short 250–300 bp fragments. Fragmented RNA was used
as a template to synthesize cDNA. After end repair, A-tailing addition,
and ligation of sequencing adapters, 200 bp cDNA was screened using
AMPure XP beads. Then, the USER enzyme was used to degrade the
second strand of cDNA containing U, and finally PCR amplification was
carried out to obtain the library. Once the library was constructed, Qubit
2.0 was used for preliminary quantification and the library was diluted
to 1.5 ng/μL. Agilent 2100 was used to detect the insert size of the li­
brary. The real-time quantitative reverse transcription polymerase chain
reaction (qRT-PCR) method was performed to determine the effective
concentration of the library (higher than 3 nM) for accurate quantifi­
cation to ensure the quality of the library. After the library was qualified,
Illumina PE150 sequencing was conducted after pooling according to
the effective concentration of the library and data output requirements.
2.8. Alternative splicing(AS) analysis
Alternative splicing refers to the process of generating different
mRNA splice isomers by selecting different splice site combinations from
an mRNA precursor through different splicing methods. The rMATS
software was used to compare the results of alternative splicing (AS)
classification and to obtain differentially expressed AS analysis. Alter­
native splicing events can be classified into the following five categories:
skipped exon (SE), mutually exclusive exon (MXE), alternative 5′ splice
site (A5SS), alternative 3′ splice site (A3SS), and retained intron (RI).
Each variable splicing event corresponds to two isoforms, namely exon
inclusion isoform and exon skipping isoform. The expression levels of
the two isoforms were divided by their effective length to obtain the
corrected expression level. Then, the ratio of the exon inclusion isoform
in two isoforms was calculated, and, finally, a significant difference
analysis was conducted.
2.9. Bioinformatics analysis
After the original data were obtained by sequencing, sequencing data
quality assessment and information mining and analysis were per­
formed. The gene expression value was expressed using fragments per
kilobase of transcript sequence per millions base pairs sequenced
(FPKM). It is the number of fragments obtained per kilobase length from
a certain gene/transcript per million fragments. It also considers the
impact of sequencing depth and gene length on the fragment count.
After the quantitative analysis, the Cuffdiff software was used to analyze
the significance of the expression difference. An adjusted p-value of less
than 0.05 was used as the difference significance standard to screen the
differentially expressed genes (DEGs).
In organisms, different genes coordinate with each other to perform
their biological functions. Through pathway significant enrichment, the
main biochemical metabolic pathways and signal transduction path­
ways involved in DEGs can be explored. The widely used annotation
gene database, Gene Ontology (GO), Kyoto Encyclopedia of Genes and
Genomes (KEGG) and clusterProfiler (http://www.bioconductor.org/
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Ecotoxicology and Environmental Safety 218 (2021) 112296
packages/release/bioc/html/clusterProfiler.html).
Software tools were used for pathway enrichment analysis. GO (gene
ontology) is a database established by the Gene Onotology Consortium,
which is used to describe and classify gene and protein functions based
on three aspects: molecular function (MF), biological process (BP), and
cellular component (CC). Studying the distribution of gene sets in GO
may clarify the biological functions of differential gene enrichment.
Adjusted p-value of less than 0.05 was considered as a threshold to
identify significant GO and KEGG enrichment.
2.10. Quantitative reverse transcriptase polymerase chain reaction (RT-
qPCR)
Quantitative reverse transcriptase polymerase chain reaction (RT-
qPCR) was performed to quantify and validate the transcriptome
sequencing data. For each sample, 500 ng of total RNA was added to a
10 μL RT reaction system using PrimeScriptTM RT Master Mix
(TAKARA, Dalian, China) to synthesize cDNA. The primers were
designed and synthesized by Generay Biotech Co., Ltd. (Shanghai,
China) (Table S1). The RT-qPCR analysis was performed on a StepO­
nePlus™ Real-Time PCR System (Version 2.2.2, Applied Biosystems,
Carlsbad, CA, USA) using the One-Step TB Green PrimeScript RT-PCR
Kit (TAKARA, Beijing, China) according to the following thermal
cycling conditions: 95 ◦ C for 30 s, 40 cycles of: 95 ◦ C for 5 s and 60 ◦ C for
30 s, followed by a default stage of melting curve analysis. The relative
expression of target genes was normalized against the mRNA of refer­
ence gene β-actin using the 2-ΔΔCT method. Three replicates were set for
each experimental group.
2.11. Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Sta­
tistical significance was determined using Student’s t-test or one-way
analysis of variance (ANOVA) followed by Student-Newman-Keuls
post-hoc test using the SPSS 19.0 software (SPSS, Chicago, Illinois,
USA). The level of significance was set at P < 0.05.
3. Results
3.1. Distribution of fluorescent PS-MPs in mice
Fluorescent molecular imaging has many advantages such as con­
venience, simplicity, high sensitivity, and absence of substrates. This
technology can be used to track and observe the distribution and
metabolism of fluorescently labeled polypeptides, antibodies, and small-
molecule drugs in the body. We used in vivo fluorescence imaging to
investigate the distribution of 5 µm fluorescent PS-MPs in mice after
entered the stomach through oral administration. The results revealed
that stronger signals in the abdomen and the limb bones of mice after
treatment with fluorescent PS-MPs compared to those in controls
(Fig. S2A- S2B).
3.2. Effect of PS-MP particles on body weight, blood parameters, and BM
cell count
No significant differences were observed in the mice body weights of
mice between the PS-MP-treated groups and the control group (Fig. 1A).
Routine blood test (Fig. 1B–1E) showed that no obvious differences
between the MP1 group and the control group. However, significantly
decreased WBC count (p < 0.05) and increased Pit count (p < 0.001)
were observed in the MP2 group. As peripheral blood cells are derived
from the differentiation of BM cells, we isolated BM cells from the mouse
femur and tibia and counted the number. No significant difference was
found in the BM cell count of the three groups (Fig. 1F).
R. Sun et al.
3.3. Effect of 5 µm PS-MP particles on the colony-forming ability of
mouse HSCs and HPCs
We performed the colony-forming assay to examine whether treat­
ment with 0.1 mg and 0.5 mg 5 µm PS-MP particles would impact the
clonogenicity of mouse HSCs and HPCs. On the 12th day of culture, the
number of CFUs was counted and characterized according to the
morphological features of different colonies under a microscope
(Fig. 2B). As shown in Fig. 2A, 0.5 mg 5 µm PS-MPs particles signifi­
cantly inhibited colony growth in CFU-GM, CFU-G, and CFU-M. How­
ever, the CFU-GEMM counts did not change significantly in this MP2
with control. **: p < 0.01 compared with control. ***: p < 0.001 compared with control.
4
Ecotoxicology and Environmental Safety 218 (2021) 112296
Fig. 1. The effect of PS-MPs particles on
body weight, blood routine and bone
marrow cell count. Mice were given ultrapure
water, 0.1 mg or 0.5 mg 5 µm PS-MPs once
daily by oral gavage for 28 days. Mice were
weighed and recorded every week (A). At the
end of the studies, the peripheral blood WBC
count (B), RBC count (C), Hgb content (D) and
Pit count (E) were examined. Mouse femurs and
tibias were separated and the number of bone
marrow cells of each mouse was counted (F).
**: p < 0.01 compared with control. #:
p < 0.05 compared with 0.1 mg 5 µm PS-MPs
group. ###: p < 0.001 compared with 0.1 mg
5 µm PS-MPs group.
group compared with those in the control group. No significant effect on
CFU counts was observed in the MP1 group as compared with that in the
control.
3.4. Alternative splicing analysis
The analysis results of differentially expressed alternative splicing
(AS) caused by PS-MPs are summarized in Fig. S3. The results showed
that the highest proportion of happened AS type was retained intron (RI)
(25.80% in the MP1 group and 23.21% in the MP2 group).
Fig. 2. The effect of PS-MPs particles on
colony forming ability of mouse HSCs and
HPCs. (A) Male C57BL /6 mice were treated
with 0.1 mg (MP1) and 0.5 mg (MP1) 5 µm PS-
MPs particles for 28 days. The number of CFUs
were counted after 12 days of culture under
microscope. Data are presented as the
mean ± SD (n = 6). (B) Images for various
types of colony-forming units (CFUs) for mouse
HSCs and HPCs. CFU-GM (colony-forming unit-
granulocyte and macrophage), CFU-G (colony-
forming unit-granulocyte), CFU-M (colony-
forming unit-macrophage), and CFU-GEMM
(colony-forming unit-granulocyte, erythroid,
macrophage and megakaryocyte). (Magnifica­
tion: 200 ×). MP1 represents 0.1 mg/day 5 µm
PS-MPs group and MP2 and represents 0.5 mg/
day 5 µm PS-MPs group. *: p < 0.05 compared
R. Sun et al.
3.5. Changes in gene expression levels after 5 µm PS-MPs exposure
The analysis results of differential gene expression showed that,
compared with those in the control group, a total of 41 genes were
significantly altered in the MP1 group, of which 25 genes were signifi­
cantly upregulated and 16 genes were downregulated (Fig. 3A and C). In
the MP2 group, 32 genes changed significantly (17 upregulated and 15
downregulated) (Fig. 3B and D). After comparing the results from MP1
group with those from the control group, the top 10 significantly
upregulated and down-regulated genes were collated and are listed in
Table S2. Table S3 summarizes the top 10 upregulated and down­
regulated genes in the MP2 group.
Subsequently, we compared the differential genes of the two PS-MP
particles groups. We found that compared with those in the control
group, the expression change trends of eight genes were consistent in
both the 5 µm PS-MP particles treatment groups, of which seven were
upregulated and one was downregulated at the same time (Fig. 3E). The
heatmap of clustering molecules based on these eight genes showed that
the expression levels of these genes in the treatment group were similar,
but there were significant changes compared with gene expression levels
in the control group (Fig. 3F).
3.6. Gene Ontology enrichment analysis
The GO annotation analysis was performed on the BM cells of mice
exposed to 5 µm PS-MP. At the same time, the enrichment analysis of the
gene set was also recognized. In general, GO items with small p-values
are likely to be related to the toxicity of 5 µm PS-MPs. On comparing the
MP1 group to the control, we found that DEGs were widely distributed
in 512 BP items, 77 CC items, and 145 MF items. Among these, there
were 47 BP items, 77 CC items, and 145 MF items with p-values less than
0.05, and the top 15 significant GO items are shown in Fig. 4A (when the
significant items were less than 15, all are shown). On comparison be­
tween the MP2 group and the control group (Fig. 4B), GO analysis
revealed that the DEGs were distributed in 448 BP items, 40 CC items,
and 80 MF items. According to the p-value, 63 BP items, 5 CC items, and
10 MF items were considered to be significantly altered GO items
induced by 0.5 mg 5 µm PS-MP exposure.
3.7. Kyoto encyclopedia of genes and genomes enrichment analysis
In the present study, the changed biological pathways caused by
5 µm PS-MPs treatment were identified using the KEGG database by
annotating all DEGs. Fig. 5A summarizes the pathways enriched by
DEGs between the MP1 group and the control group, whereas Fig. 5B
displays the pathways enriched by DEGs between the MP2 group and the
control group. When these identified pathways were classified, multiple
processes were found to be disturbed by 5 µm PS-MPs, including envi­
ronmental information processing, cellular processes, organismal sys­
tems, and metabolism.
3.8. Validation of gene expression by RT-qPCR
Finally, we randomly selected upregulated and downregulated DEGs
with different fold changes from two comparison groups and verified by
qRT-PCR. Consequently, the relative expression of upregulated DEGs
(Spag17, Kcnt2, Kcns3, Rfx8, Pifo, Spon1, Cpz, and GM4952) and
downregulated DEGs (Nnt, Efch2, Sema3e, Leng1, Marchf8, and Tex11)
was consistent with the trend of transcriptome analysis results
(Fig. 6A–D). Therefore, qRT-PCR results verified the reliability of the
high-throughput transcriptome results.
4. Discussion
As a new type of environmental pollutant, ecological and health risks
caused by MPs have attracted the attention of researchers (Akdogan and
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Ecotoxicology and Environmental Safety 218 (2021) 112296
Guven, 2019; Geyer et al., 2017; Wright and Kelly, 2017). MPs are
widely distributed in the ocean, freshwater systems, and soil. They can
be detected in aquatic and terrestrial animals and plants, and even in
human biological samples (Jiang et al., 2020). Therefore, it is critical to
study the toxicity of MPs in mammals. In this study, traditional toxico­
logical examinations and transcriptome sequencing analysis were car­
ried out to comprehensively explore the effects of PS-MPs on the
hematological system of mice and on related genes and pathways. For
mouse toxicology experiments, dosage is the first issue to consider. We
reviewed the literature on the richness of MPs in various environments
and the existing studies on MPs in mice (Deng et al., 2017;
Eerkes-Medrano et al., 2015; Jin et al., 2019), and selected the current
experimental doses (0.1 mg/day and 0.5 mg/day of 5 µm PS-MPs par­
ticles). The reason for choosing a low dose is that it is equivalent to the
environmental concentration of MPs in rivers (about 106 items m− 3)
(Eerkes-Medrano et al., 2015). The high dose in this study was five times
higher than the low dose. Thus, the dose in our experiment was carefully
designed and rational.
There have been some studies on the accumulation of MPs in mouse
tissues. Deng et al. (2017) treated mice with 5 µm and 20 µm fluorescent
PS-MPs particles by oral gavage for 28 days and found that PS-MPs
accumulated in mouse liver, kidney and gut. Jin et al. (2019) exposed
mice to fluorescent PS-MPs for six weeks and observed a clear fluores­
cent signal in gut sections of mice using a high-resolution confocal mi­
croscope. In our study, we used a fluorescence imaging system to
observe the distribution of PS-MPs in mice after 28 days of intragastric
administration of fluorescently labeled PS-MPs. Compared with those in
the control mice, enhanced fluorescence signals were observed in the
abdomen and lower limb bones of the mouse in the supine position. In
the prone position, stronger fluorescence signals were observed in the
bones of the mouse limbs. Our results investigated the accumulation of
PS-MPs in mice, suggesting that orally ingested PS-MPs may be
distributed to the stomach, intestines, liver, and other digestive organs
and bones of mice through the blood circulation. Blood is the medium
through which exogenous substances enter the body for distribution.
To explore the impact of MPs on the blood system of mice, we first
performed traditional toxicology tests, such as blood routine, BM cell
count, and colony-formation experiments. Our results showed that the
0.5 mg dose of the 5 µm PS-MPs particles caused a significantly
decreased WBC count and elevated Pit count. In addition to playing a
role in material transportation, body fluid regulation, and maintenance
of homeostasis, the hematological system also has a defense function. Its
defense function is mainly reflected in immunity and hemostasis. In the
hematological system, WBCs form one of the defensive components of
the body against harmful factors in the internal and external environ­
ment. Thus, a lower level of WBC count may be related to immune re­
sponses caused by PS-MPs particles. Platelets are small pieces of
biologically active cytoplasm formed from the mature megakaryocyte
cytoplasm of mammalian BM. They play an important role in hemo­
stasis, wound healing, inflammation and other physiological and path­
ological processes (Guo and Rondina, 2019; Stegner et al., 2019).
Thrombocytosis is divided into two types: primary and secondary. Pri­
mary thrombocytosis is common in chronic myelogenous leukemia,
polycythemia vera and other myeloproliferative diseases. Secondary
thrombocytosis is common in acute and chronic inflammation, iron
deficiency anemia and tumors. Therefore, elevated Pit counts suggest
that PS-MPs damaged the blood system.
BM hematopoietic stem cells refer to immature cells that can pro­
liferate and differentiate into various types of blood cells, precursor cells
and mature blood cells, and are released into the peripheral blood to
perform their respective tasks, such as red blood cells, white blood cells
and platelets. Given the results of the peripheral blood routine, a colony-
forming assay was performed to detect the potential of multi-lineage
differentiation of HSCs. As displayed in Fig. 3, the colony number of
CFU-GM, CFU-G, and CFU-M was lower in the MP2 group than that in
the control. These results indicated that PS-MPs exposure could affect
R. Sun et al. Ecotoxicology and Environmental Safety 218 (2021) 112296

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R. Sun et al. Ecotoxicology and Environmental Safety 218 (2021) 112296

Fig. 3. DEGs between the control and 0.1 mg 5 µm PS-MPs treatment group (A, C) and control and 0.5 mg 5 µm PS-MPs treatment group (B, D). Volcano
map for DEGs between the control and 0.1 mg 5 µm PS-MPs treatment group (A), and between the control and 0.5 mg 5 µm PS-MPs treatment group (B). Red
represents upregulated gene, green represents downregulated gene and blue color represents non- DEGs. (C) A heatmap of DEGs after hierarchical cluster analysis
(control group VS 0.1 mg 5 µm PS-MPs group) (n = 3). (D) A heatmap of DEGs after hierarchical cluster analysis (n = 3) (control group VS 0.5 mg 5 µm PS-MPs
group). Red represents upregulated gene and blue represents downregulated gene. (E) Venn diagram showed that there were 8 common DEGs between when the
0.1 mg 5 µm PS-MPs treatment or 0.5 mg 5 µm PS-MPs treatment was compared with the control, respectively. The left circle represents 41 DEGs in 0.1 mg 5 µm PS-
MPs treatment group. The right circle represents 32 DEGs in 0.5 mg 5 µm PS-MPs treatment group. The intersection of two circles displays 8 common DEGs. (F) A
heatmap of 8 common DEGs in two PS-MPs treated groups after hierarchical cluster (Control, 0.1 mg 5 µm PS-MPs group and 0.5 mg 5 µm PS-MPs group) (n = 3).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the peripheral blood cell count by inhibiting the differentiation of BM
HSCs into granulocytes and megakaryocytes.
To further investigate the genes and related biological processes
altered in the hematological system after PS-MP treatment, we per­
formed transcriptome analysis of mice BM cells using RNA-seq tech­
nology. In this study, 41 and 32 DEGs were found in the 0.1 mg and
0.5 mg 5 µm PS-MP-treated groups, respectively. Surprisingly, there was
no obvious dose-effect relationship between the two doses (0.1 mg and
0.5 mg 5 µm PS-MPs). We speculate that the differential changes in the
gene expression profile of bone marrow cells caused by PS-MPs occur
earlier than the changes in colony forming ability and blood routine. Of
note, eight genes were found to be critical because of significant changes
in their expression in two PS-MP-treated groups, such as Spag17, Nnt,
Marchf8, and Zfp329.
AS is an important mechanism for regulating gene expression and
generating proteome diversity and complexity. By comparing the results
of DEGs and differentially expressed AS, we found that the gene
expression levels of Osgin1 and Ccnd3 may be regulated by RI, A3SS and
A5SS after PS-MPs exposure, respectively, which leads to a disturbance
in the corresponding biological processes.
Bioinformatics analysis was then performed to reveal biological
processes or pathways associated with toxicity of PS-MPs in mouse BM
cells using GO and KEGG databases. GO analysis showed that exposure
to a 0.1 mg dose of 5 µm PS-MPs significantly affected biological pro­
cesses including T cell homeostasis, response to osmotic stress, extra­
cellular matrix and structure organization, and negative regulation of
angiogenesis. The T cells are derived from the lymphoid stem cells in the
BM. After differentiation and maturation in the thymus, T cells are
distributed to the immune organs and tissues of the body through the
lymphatic and blood circulation to exert immune functions. T cell ho­
meostasis is crucial for the adaptive immune system as it regulates the
size of the lymphocytes (Wong et al., 2020). Infection or other external
stimuli can initiate expansion or contraction of a specific population of T
cells during an immune response (Jameson, 2002). Analysis of MF
showed that identified genes were mainly related to the activity of gated
channels, potassium channels, and bioactive lipid receptors, whereas the
cellular components revealed that the affected genes were mostly
distributed in several channel complexes and transporter complexes. Ion
channels are an important way for living cells to exchange materials
with the surrounding environment to maintain metabolic activities. The
activity of ion channels is of great significance for the realization of
various functions of cells. When genes encoding ion channel subunits are
mutated or expressed abnormally, their functions can be weakened or
enhanced, leading to disorders of the physiological functions. In addi­
tion, certain diseases or drugs can also cause changes in the number,
function, and even structure of one or several ion channels, such as
Alzheimer’s disease (AD) (Villa et al., 2020), and cerebral ischemia
(Woo et al., 2020). In this study, Kcnt2/Kcns3, two channel complex
genes, were significantly upregulated after exposure to a 0.1 mg dose of
5 µm PS-MPs. Thus, the expression of genes related to ion channels and
complexes was significantly affected by 0.1 mg 5 µm PS-MPs treatment,
which in turn causes changes in the structure and function of various ion
channels.
In the MP2 group, DEGs mainly function in the metabolic process of
NADP and nucleotide, mammary gland development, and post-
embryonic development. NADP serves as an electron carrier in a
number of reactions, being alternately oxidized (NADP+) and reduced
(NADPH). It is involved in biosynthetic pathways, energy, and signal
transduction by participating in redox homeostasis and synthesis of
signaling compounds. The expression of Nnt and Rpe, two genes of the
NADP metabolic process, changed significantly after treatment with
0.5 mg of 5 µm PS-MPs, indicating a disturbance in redox homeostasis.
Meanwhile, the identified DEGs were found to be associated with
various subcellular locations, such as lytic vacuole, lysosome, myelin
sheath, axoneme, and vacuole. Lysosomes can not only digest the local
cytoplasm or organelles of the cell itself, but can also decompose sub­
stances that enter the cell from the outside. Because the lysosomal
membrane is sensitive to reactive oxygen and nitrogen oxides, lysosomal
damage occurs under oxidative stress (Olsson et al., 1989). A previous
study showed that the dilatation of the endosomal-lysosomal system was
decreased and the activity of lysosomal sulfatases was reduced after
exposure to 20 nm polystyrene particles for 24 h (Fröhlich et al., 2012).
Other studies also indicated that nano-sized polystyrene particles
induced lysosomal dysfunction. As micron-sized polystyrene particles
can only be observed on the exterior of cells (Sipos and Kim, 2019) it
suggests that 5 µm PS-MPs are likely to cause indirect damage to lyso­
somes through oxidative stress induced by its exposure. In summarize,
consistent with the results that we observed using traditional toxicology
methods, GO analysis suggested that low doses of PS-MPs (0.1 mg)
mainly caused changes in the body’s immune response and adaptation
to exogenous substances, whereas high-dose exposure (0.5 mg) affected
metabolism and developmental processes by causing alterations in
subcellular organelles.
Based on the KEGG database, the Jak/Stat pathway was the most
significantly enriched pathway for DEGs in the MP1 group. The Jak/Stat
pathway, as an evolutionarily conserved pathway of eukaryotes, can
regulate many important biological processes such as cell proliferation,
differentiation, apoptosis, and immune regulation (Müller et al., 2012;
Mughal et al., 2014; Trivedi and Starz-Gaiano, 2018). Dysregulation of
Jak/STAT signaling plays a key role in various types of blood cell dis­
orders and cancers in humans (Mughal et al., 2014). For example, Xiang
et al. (2008) screened the entire coding region of JAK1 and identified
two mutations in the JAK1 gene in acute myeloid leukemia cases,
indicating the involvement of the Jak/STAT pathway in the develop­
ment of this disease. Here，upregulated Epo and downregulated Ccnd3
in the Jak/Stat pathway were observed. We speculated that the increase
in Epo expression induced by 5 µm PS-MPs (0.1 mg) could activate the
JAK1 receptor Cntfr and cause a state change of JAK1. The transcription
factor STAT1 is phosphorylated, so that it enters the nucleus as a dimeric
and regulates the transcription of Ccnd3. Cyclin, encoded by CCND3,
can form a complex with CDK4 or CDK6 and act as its regulatory sub­
unit. Its activity is necessary for the G1/S transition of the cell cycle.
Thus, exposure to 0.1 mg of 5 µm PS-MPs is likely to impact the G1/S
phase of the cell cycle through the Jak/Stat pathway.
In the MP2 group, KEGG analysis revealed that several metabolic
pathways were significantly changed, including pentose and glucuro­
nate interconversions, nicotinate and nicotinamide metabolism,
biosynthesis of unsaturated fatty acids and pentose phosphate pathway.
Studies have shown that metabolic disturbances play an important role
in the occurrence and development of diseases and even various types of
tumors (Faubert and Solmonson, 2020; Li et al., 2020; Rinaldi et al.,
2018). Metabolic reprogramming allows cells to adapt to changes in the

7
R. Sun et al. Ecotoxicology and Environmental Safety 218 (2021) 112296

Fig. 4. GO classification of the differentially expressed genes between the control and 0.1 mg 5 µm PS-MPs treatment (A) and control and 0.5 mg 5 µm PS-
MPs treatment (B). All GO items were sorted by p value from small to large and GO items with p value less than 0.05 were displayed in this figure. Only the first 15
items are listed if the number is greater than 15, and all the items are listed when the number is less than 15.

8
R. Sun et al.
Fig. 5. KEGG pathways of DEGs between the control and 0.1 mg 5 µm PS-MPs treatment (A) and control and 0.5 mg 5 µm PS-MPs treatment (B). Vertical
axis lists the names of the pathways in the KEGG database. Horizontal axis shows the p-value of each pathway.
body’s microenvironment caused by exogenous stimulation or various
pathological processes, such as oxygen partial pressure and nutrients.
The pentose phosphate pathway is a process of glucose turnover. This
pathway can provide reducing equivalents in the form of NADPH and
maintain the reduced state of glutathione (GSH) (Alfarouk et al., 2020).
As ROS detoxification, the metabolites (NADPH and GSH) in the pentose
phosphate pathway can alleviate oxidative stress to a certain extent
(Faubert and Solmonson, 2020). Hence, the changes in the pentose
phosphate pathway may imply that the metabolic reprogramming is
initiated to respond to oxidative stress caused by exposure to 0.5 mg of
5 µm PS-MPs.
Considering the existence of false-positive results in RNA-seq, we
used RT-PCR to verify the results randomly. Eight genes with different
trends in the two PS-MPs exposure groups were respectively selected to
ensure validation. We noted that the trend of RT-PCR results was basi­
cally in line with the results from the sequencing analysis. Of note, the
Nnt expression levels were reduced in both the treated groups. Nico­
tinamide nucleotide transhydrogenase (Nnt) plays a role in mitochon­
drial redox balance by generating high concentrations of NADPH
(Meimaridou et al., 2018). This process is required for antioxidant en­
zymes to detoxify reactive oxygen species, such as the glutathione and
thioredoxin systems. The deletion of Nnt leads to a significant increase
in lipid peroxidation (LPO) in vivo, higher NADP/NADPH ratio in vitro,
9
Ecotoxicology and Environmental Safety 218 (2021) 112296
Fig. 6. RT-qPCR validation for the gene
expression of bone marrow cells in mouse
exposed to 0.1 mg and 0.5 mg 5 µm PS-MPs
(A-D). Eight DEGs with different fold changes
from two comparison groups were randomly
selected and verified by RT-qPCR. Data from
RT-qPCR showed the mean ± SD for six sam­
ples. MP1, 0.1 mg 5 µm PS-MPs group; MP2,
0.5 mg 5 µm PS-MPs group. RT-qPCR, quanti­
tative reverse transcription-polymerase chain
reaction. *: p < 0.05 compared with control. **:
p < 0.01 compared with control. ***: p < 0.001
compared with control.
and reduced levels of mitochondrial antioxidant genes (Prdx3 and
Txnrd2) (Meimaridou et al., 2018). Therefore, decreased Nnt after
PS-MP exposure may be related to impaired antioxidant capacity and
increased susceptibility of the mitochondria to oxidative damage.
5. Conclusion
In this study, we found that PS-MPs caused a significant decrease in
the peripheral blood WBC count and the inhibition of the colony-
forming ability of BM cells in mice (an advanced mammal). At the
same time, the transcriptome analysis results suggested that the genes
interfered by PS-MPs treatment are mainly distributed in immune re­
sponses, oxidative stress, Jak/STAT pathway, pentose phosphate
pathway and other metabolic pathways. In the future, systematic and in-
depth studies should be conducted to investigate the hematotoxicity of
PS-MPs based on the candidate gene targets and pathways provided by
the present study and other –omics research. Collectively, our study not
only provides data about the adverse effects of MPs in mice but also
support further studies that will focus on these candidate biological
molecules.
R. Sun et al.
CRediT authorship contribution statement
Rongli Sun: Conceptualization, experiment execution, Writing -
review & editing, All authors have read and agreed to the published
version of the manuscript. Kai Xu: Experiment execution. Linling Yu:
Experiment execution. Yunqiu Pu: Experiment execution. Fei Xiong:
Experiment execution. Yuhong He: Experiment execution. Qingchen
Huang: Data analysis. Mingjie Tang: Data analysis. Minjian Chen:
Review. Lihong Yin: Supervision. Juan Zhang: Supervision. Yuepu Pu:
Supervision.
Declaration of Competing Interest
The authors declare no potential conflict of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (81703265), the Fundamental Research Funds for the Central
Universities (2242018K40019, 2242018K3DN25), Zhishan Youth
Scholar Program Of SEU (2242019R40050).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2021.112296.
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