An examination of the short- and long-term effects of
microplastics in mammals using a mouse model

By

Laura Janiaski

Thesis submitted in partial fulfilment of the requirements for the degree of
Master of Science
Environmental and Life Sciences
Brandon University

© Laura Janiaski
2021

Brandon University
FACULTY OF SCIENCE
The undersigned certify that they have read, and recommended to the Senate for acceptance, a
MASTER’S THESIS entitled:
An examination of the short- and long-term effects of microplastics in
mammals using a mouse model
Submitted by: Laura Janiaski
In partial fulfillment for the requirements for the degree of

MASTER OF SCIENCE (ENVIRONMENTAL AND LIFE SCIENCES)

ii

Co-authorship
CO-AUTHORSHIP STATEMENT
The Statement concerns the following publication:
Chapter 2: In prep: “Short-term exposure to microbeads and microfibers pose minimal risk of
inflammation and tissue injury in mice”
Author: Laura Janiaski
Contribution: Study design, Data curation, Methodology, Analysis, Writing (including
review/editing)
Co-author: Bernadette Ardelli
Contribution: Study Design, Data curation, Methodology, Analysis, Writing (including
review/editing)

CO-AUTHORSHIP STATEMENT
The Statement concerns the following publication:
Chapter 3: In prep: “Sex-based and time-dependent effects of microplastic exposure in mice”
Author: Laura Janiaski
Contribution: Study design, Data curation, Methodology, Analysis, Writing (including
review/editing)
Co-author: Bernadette Ardelli
Contribution: Study Design, Data curation, Methodology, Analysis, Writing (including
review/editing)

iii

Abstract
The United Nations has recognized plastic pollution as the second most concerning
environmental problem after climate change. Photo-oxidation and other natural processes can
fragment plastics into smaller particles, giving rise to another problem: microplastics. Despite
many studies showing that humans are exposed to microplastics, their toxic effects are
uncertain. The aim of this study was to contribute to the understanding of microplastic health
impacts on mammals, using a mouse model. For the manuscript 1 study, we exposed female
mice to two different shapes of microplastics (microbeads, microfibers) for up to four weeks.
Although microbead and microfiber-treated groups showed a slightly altered food
consumption, body weight was not affected in both groups. Microbeads, but not microfibers,
were able to translocate from gut lumen to the liver, however no histopathological changes
were observed. The expression levels of genes associated with inflammation (Apcs, Crp, Cfb)
and tissue injury repair (Ccn1) did not show a statistically significant difference after
exposure to microbeads and microfibers. For the manuscript 2 study, we exposed female and
male mice to microbeads for up to sixteen weeks, followed by a four-week recovery period.
Although microbeads translocated to deeper organs in both female and male mice, only
females showed significant changes in gene expression. Overall, longer exposure caused
more aggravated effects than short exposure; some of these effects were reversed after the
recovery period, while others showed a delayed manifestation. Biological response to
microplastic exposure has proven complex, depending on time of exposure and showing
remarkable sex differences.

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Acknowledgements
First and foremost, I would like to thank my supervisor, Dr. Bernadette Ardelli, for
welcoming me into her lab. She has inspired me both personally and professionally
throughout these years. I have learned tremendously from her knowledge, experience, and
dedication. I would also like to thank my advisory committee members, Dr. Christophe
LeMoine, Dr. Chenkuan Li and Dr. Peter Pieroni, for their insightful feedback and assistance.
Throughout these years as a BU grad student, I have received a great deal of support and
words of encouragement from staff and faculty members. I would like to express my sincere
thanks to Ms. Heather Teeple, Ms. Linda Krieser, Dr. Neal Melvin, Dr. Bryan Cassone, Dr.
David Greenwood, Dr. Ellen Boudreau, Dr. Mousumi Majumder, and Dr. Steffi LaZerte.
I am also grateful to my MELS colleagues and my lab mates that helped me with animal care,
sample collection, use of equipment, and cheering me up after my experiments have failed.
Thanks Nick Kurchaba, Glenda, Markus Sudermann, Jessica Kalyniuk, Chantel Lim, Olivia
Pieroni and Ezinne Nwadike. Special thanks to Claudia Rodriguez for helping me with the
sample collection for manuscript 1.
Finally, I would like to thank my husband, Diogo Janiaski, for always being kind and
supportive. And my family that even thousands of miles away is my strong rock. My deep
and sincere gratitude for their continuous and unparalleled love, help and support.

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Table of contents
1-

Introduction ...................................................................................................................... 1
1.1

Plastic pollution and microplastics .............................................................................. 1

1.2

Microplastic exposure and potential health risks ........................................................ 3
1.2.1 Exposure routes and translocation………………………………….…………..3
1.2.2 Microplastic toxicity studies…………………………………….………….….4
1.2.3 Effects of plastic additives………………………………………………….….7

1.3

Microplastics and the immune system ........................................................................ 9
1.3.1 Microplastics and inflammation…………….………………………………….9
1.3.2 Acute-phase reactants…………………………………………………………13
1.3.3 The Hippo pathway and tissue injury repair………………………………….14

2(In prep) Short-term exposure to microbeads and microfibers pose minimal risk of
inflammation and tissue injury in mice .................................................................................... 16
2.1

Chapter summary ...................................................................................................... 16

2.2

Introduction ............................................................................................................... 17

2.3

Methods ..................................................................................................................... 21

2.4

Results ....................................................................................................................... 29

2.5

Discussion ................................................................................................................. 37

2.6

Conclusions ............................................................................................................... 42

3-

4-

(In prep) Sex-based and time-dependent effects of microplastic exposure in mice ....... 44
3.1

Chapter summary ...................................................................................................... 44

3.2

Introduction ............................................................................................................... 45

3.3

Methods ..................................................................................................................... 49

3.4

Results ....................................................................................................................... 52

3.5

Discussion ................................................................................................................. 69

3.6

Conclusions ............................................................................................................... 79
Conclusions .................................................................................................................... 80

References ................................................................................................................................ 82
Appendices ............................................................................................................................... 92

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List of figures and tables
Figure 2-1: Microfiber length. ................................................................................................. 22
Figure 2-2: Experimental design. ............................................................................................. 23
Figure 2-3: Food consumption, body weight and microplastic egestion. ................................ 32
Figure 2-4: Microplastics in feces............................................................................................ 32
Figure 2-5: Histological analysis after microplastic exposure. ................................................ 33
Figure 2-6: MB found in mouse tissues. .................................................................................. 34
Figure 2-7: Gene expression in the liver. ................................................................................. 36
Figure 3-1: Experimental design .............................................................................................. 50
Figure 3-2: Female food consumption, body weight and microplastic egestion. .................... 55
Figure 3-3: Male food consumption, body weight and microplastic egestion. ........................ 57
Figure 3-4: Relative organ weight. .......................................................................................... 60
Figure 3-5: Histological analysis after microplastic exposure. ................................................ 62
Figure 3-6: MB found in mouse tissues. .................................................................................. 64
Figure 3-7: Gene expression in the liver. ................................................................................. 66
Figure 3-8: Gene expression in the large intestine................................................................... 67
Figure 3-9: Gene expression in the spleen. .............................................................................. 68

Table 2-1: TaqMan probe details ............................................................................................. 27
Table 3-1: MB accumulation in different tissues. .................................................................... 63

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List of appendices
Appendix 1: Histological preparations .................................................................................... 92
Appendix 2: Total RNA extraction protocol (TRIzol method) ............................................... 95
Appendix 3: Reverse transcription (cDNA synthesis) ............................................................. 97
Appendix 4: TaqMan RT-PCR ................................................................................................ 99

viii

List of acronyms
Acute-phase reactants (APR)
Cellular communication network factor 1 (CCN1)
Complement factor B (CFB)
C-reactive protein (CRP)
Damage-associated molecular patterns (DAMPs)
Dextran sodium sulfate (DSS)
Extracellular matrix (ECM)
Glyceraldehyde-3-phosphate dehydrogenase (GADPH)
Hematoxylin and eosin (H&E)
Immunoglobulin (Ig)
Inflammatory bowel disease (IBD)
Interferon (IFN)
Interleukin (IL)
Lipopolysaccharides (LPS)
Membrane attack complex (MAC)
Microbeads (MB)
Microfibers (MF)
Nasal associated lymphoid tissue (NALT)
Pathogen-associated molecular pattern (PAMPs)
Pattern recognition receptors (PRRs)
Peripheral blood mononuclear cells (PBMCs)
Polyamide (PA)
Polycarbonate (PC)
Polyethylene (PE)
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Polyethylene terephthalate (PET)
Polymethylmethacrylate (PMMC)
Polypropylene (PP)
Polystyrene (PS)
Polyurethane (PU)
Polyvinyl alcohol (PVA)
Polyvinylchloride (PVC)
Reactive oxygen species (ROS)
Serum amyloid P-component (SAP)
Transcriptional co-activator with PDZ-binding motifs (TAZ)
Tumor necrosis factor (TNF)
Yes-associated protein (YAP)

x

1-

Introduction

1.1

Plastic pollution and microplastics

Plastic pollution has become a global environmental issue and a growing concern since the
rise of the plastic industry in the mid-1950s. The long durability of plastics in the
environment and deficient recycling policies contribute to environmental pollution, especially
in aquatic ecosystems. Once in nature, plastics are exposed to a variety of physical and
chemical processes, such as photolysis, thermal-oxidation, and hydrolysis, and can be
fragmented into smaller pieces known as microplastics (Thompson, 2015). Microplastics are
defined as plastic particles smaller than 5mm and are categorized as primary or secondary
according to their source. Primary microplastics are originally manufactured to be that size,
while secondary microplastics result from the breakdown of larger plastic items. Examples of
primary microplastics include pellets/powders and engineered plastic microbeads used in
cosmetic formulations, cleaning products and industrial abrasives (EFSA, 2016; GESAMP,
2016).
In the environment, microplastics differ in shape, size, source and chemical composition. The
most common microplastic composition are polyethylene (PE), polypropylene (PP),
polystyrene (PS), polyvinylchloride (PVC), polyamide (PA), polyethylene terephthalate
(PET), polycarbonate (PC), polyvinyl alcohol (PVA), polyurethane (PU),
polymethylmethacrylate (PMMC) and plastic additives such as stabilizers, flame retardants,
plasticizers, fillers and pigments (Hirt and Body-Malapel, 2020). There is still some debate
over the most appropriate upper and lower size limit, but usually researchers classify
microplastics as particles <5mm and nanoplastics as particles ≤0.1µm. Microplastics can be
1

subcategorized according to their shape and includes beads, nurdles, fibers, foam, fragments,
pellets, films, filaments, cylindrical, disk, flat, ovoid, spheruloid, elongated, rounded and
irregular (Hirt and Body-Malapel, 2020).
Microplastics have been reported on a global scale. Many studies have reported microplastics
in marine environments (Eriksen et al., 2014), freshwater ecosystems (Dris et al., 2015;
Imhof et al., 2013), soil (Rillig, 2012), ground water (Mintenig et al., 2018), sewage and the
atmosphere (Dris et al., 2015) and even in remote areas such as in the Arctic Sea ice (Obbard
et al., 2014), Mount Everest (Napper et al., 2020) and Swiss Alps (Bergmann et al., 2019).
Microplastics have also been found in bottled mineral water (Schymanski et al., 2018), tap
water (Kosuth et al., 2018), beer (Kosuth et al., 2018; Liebezeit and Liebezeit, 2014), tea
(Hernandez et al., 2019), cow milk (Kutralam-Muniasamy et al., 2020), table salt (Yang et
al., 2015; Kosuth et al., 2018), honey (Liebezeit and Liebezeit, 2015, 2013), sugar (Liebezeit
and Liebezeit, 2013), fruits and vegetables (Conti et al., 2020) and seafood (Mathalon and
Hill, 2014; Murray and Cowie, 2011; Smith et al., 2018).
A growing body of evidence suggests that humans are exposed to microplastics. Recently
microplastics were found in eight human stool samples (on average 20 particles/10g of feces,
size range: 50 - 500µm) with PP and PET microparticles being the most abundant (Schwabl
et al., 2019). Similarly, microplastics were found in eleven human colectomy samples (on
average 331 particles/individual or 28.1 particles/g of colon tissue, size range: 0.8 - 1.6mm)
of which 90% were PC, 50% PA and 40% PP (Ibrahim et al., 2021). In addition, pigmented
microplastics were observed in four out of six human placenta samples (12 particles, size
range: 5-10µm) where most of them were PP microplastics (Ragusa et al., 2021). Washes
from hand, face, hair and mouth showed a remarkable inter and intra-individual variability of
microplastic count within a 24-hour and 7-day period. This heterogeneity suggests that
2

human exposure to microplastics depends on the individual habits and environment, such as
household furnishings, clothing, cleaning frequency, use of cosmetics and urbanization
(Abbasi and Turner, 2021).

1.2

Microplastic exposure and potential health risks

1.2.1 Exposure routes and translocation
The routes of human exposure might be oral, inhalation and dermal, although the latter seems
to have little effect on an intact skin and is only relevant for nanoparticles (Revel et al.,
2018). Synthetic textiles, the erosion of rubber tires and city dust are thought to be the most
important source of airborne microplastics (Hirt and Body-Malapel, 2020). Dermal contact
occurs through the use of cosmetics and hygiene products that contain microbeads, such as
facial cleansers, toothpaste, shampoos and scrubs (Hirt and Body-Malapel, 2020). The
estimated annual consumption of microplastics for a North American diet ranges from 39,000
to 52,000 depending on age and sex. Inhalation contributes up to 35,000 - 62,000 additional
particles and these numbers escalate even more if the source of water is exclusively plastic
bottled water (Cox et al., 2019).
Microplastics can cross the intestinal and pulmonary epithelial barrier, causing systemic
exposure. Transcytosis via M-cells in the Peyer’s patch seems to be the main entry pathway
of nano/microplastics across the intestinal wall, followed by endocytosis, paracellular
transport and persorption (Powell et al., 2010; Stock et al., 2019). Approximately 10% of the
PS nanoplastic dose administered to rats was recovered from the gastrointestinal tract, and
60% of the particle uptake occurred through the Peyer’s patch (Hillery et al., 1994). In rats,
3

translocation of PS nanoparticles from intestinal lumen to the lymph occurred within minutes
after exposure (Seifert et al., 1996) and these particles were observed in the stomach,
intestines, kidneys, heart, spleen, lungs, and testes within 6 hours of oral administration
(Walczak et al., 2015). Inhaled microplastics might escape mechanical and mucociliary
clearance in the upper airways and advance to deeper respiratory tract reaching the alveoli
(Dong et al., 2020; Pauly et al., 1998). After intranasal application, PS microplastics were
found in the mouse nasal associated lymphoid tissue (NALT) and bronchopulmonary
deposits were observed within 15 minutes. These particles were found in the spleen and liver
10 days after exposure (Eyles et al., 2001). In rats, PS nanoplastics accumulated in maternal
lungs, heart and spleen and translocated to fetal liver, lungs, heart, kidney and brain 24 hours
after intratracheal instillation (Fournier et al., 2020).

1.2.2 Microplastic toxicity studies
Many studies using in vivo analyses showed that microplastic exposure causes harmful
effects, such as genotoxicity, neurotoxicity, oxidative stress, hepatotoxicity, metabolic
disorder, disturbed immune response, endocrine system dysfunction, microbiome dysbiosis,
intestinal barrier dysfunction, reproductive impairment, reduced feeding activity and growth,
altered behaviour and reduced survival rate. These adverse effects have been shown in algae,
amphipods (Au et al., 2015), Daphnia (Besseling et al., 2014; Ogonowski et al., 2016),
waterfleas (Ziajahromi et al., 2017), copepods (Cole et al., 2015), mussels (Avio et al., 2015;
von Moos et al., 2012), crabs (Watts et al., 2015), lugworms (Browne et al., 2013),
Caenorhabditis elegans (Dong et al., 2018; Lei et al., 2018b, 2018a), fish (Ding et al., 2018;
Jin et al., 2018b; Lu et al., 2016; Mattsson et al., 2017, 2015; Rochman et al., 2014), tadpoles
(Araújo et al., 2020b, 2020a), rats (Amereh et al., 2020; Brown et al., 2001; Donaldson et al.,
4

2000; Fournier et al., 2020; J. Hou et al., 2021; Porter et al., 1999; Xu et al., 2004) and mice
(Araújo and Malafaia, 2021; Deng et al., 2021, 2020, 2018, 2017; B. Hou et al., 2021; Jin et
al., 2021, 2018a; Li et al., 2020; Lu et al., 2018; Luo et al., 2019a, 2019b; Park et al., 2020;
Xie et al., 2020; Zheng et al., 2021). Other studies have found that microplastics might pose a
minimum health risk.
Some results suggest that the exposure to microplastics at environmental concentrations does
not bear a remarkable potential to cause toxic effects (Stock et al., 2019). After a short
depuration period, microplastics were quickly egested by Xenopus tadpole (Hu et al., 2016),
sea bass larvae (Mazurais et al., 2015) and Daphnia (Rosenkranz et al., 2009) and were
almost completely eliminated after 6 days, 2 days and 4 hours, respectively. Zebrafish larvae
exposed to PE fragments showed no histopathological signs, no oxidative stress, and no
significant difference in the expression of genes related to neurotransmission and apoptosis
(Karami et al., 2017). Similarly, PS microplastic exposure did not cause intestinal
dysfunction and immune response in rainbow trout (Ašmonaite et al., 2018). Mice exposed to
PS microparticles three times per week for 28 days showed no histological alterations and
only a few particles were detected in the intestinal wall, while no particles were found in the
liver, spleen, and kidney. In addition, there was no activation of the heme oxygenase 1
promoter, which is associated with oxidative stress and the inflammatory response (Stock et
al., 2019). The variation among studies could be explained by the different microplastic
characteristics (concentration, size, and chemistry), administration method, exposure time,
and the use of different animal models, strains, or different stages of their life cycle.
Exposure to microplastics affects gene expression by downregulating or upregulating a
variety of genes. Whole animal transcriptomics showed that larval zebrafish exposed to PE
microplastics for 48 hours presented a transient and extensive change in up to 1,734 genes,
5

most of them were downregulated and are associated with the central and peripheral nervous
systems (LeMoine et al., 2018). Zebrafish embryo and larvae injected with PS microplastics
showed 26 and 51 differently expressed genes, respectively, mostly related to lipid
metabolism, oxidative stress and complement pathway (Veneman et al., 2017). Hepatic
transcriptome of mice exposed to PS microplastics showed that 200 genes were
downregulated and 218 upregulated, most of them related to locomotor rhythm, insulinactivated receptor activity, negative regulation of insulin secretion, heat generation and
energy metabolism, such as lipid metabolism and gluconeogenesis (Luo et al., 2019a). The
gut transcriptome in mice showed that di- (2-ethylhexyl) phthalate (DEHP)-contaminated PE
microplastics had a stronger effect than exposure to virgin PE microplastics or DEHP alone.
Most of the 703 differentially expressed genes are related to oxidative response, hormone and
lipid metabolism and immune response (Deng et al., 2020).
Microplastics can cause reproductive impairments and the adverse effects might persist
across generations. Blood-testis barrier disruption, abscission, disordered arrangement of
spermatogenic cells, multinucleated gonocytes, reduced testosterone serum levels along with
lower sperm cell count and increase in sperm cell abnormalities were observed in male mice
after PS microplastic exposure (B. Hou et al., 2021; Jin et al., 2021; Xie et al., 2020).
Moreover, the expression of the anti-apoptotic protein Bcl2 was decreased while the proapoptotic protein Bax increased, which explains the increased number of apoptotic cells in
the mouse testes (B. Hou et al., 2021). Exposure to PS microplastics induced oxidative stress
and apoptosis in ovarian granulosa cells, leading to reduced number of growing follicles and
reproductive toxicity in female rats (J. Hou et al., 2021). Mice dams exposed to PS
microparticles during gestation and lactation showed metabolism disorder that persisted for
the F1 and F2 generations (Luo et al., 2019a, 2019b). In addition, exposure to PE
microplastics affected the number of live births, sex ratio and body weight as well as spleen
6

lymphocyte count of the offspring (Park et al., 2020). After intratracheal instillation of PS
nanoparticles in pregnant rats, particles were observed in the fetal lungs, liver, heart, kidney,
and brain 24 hours after the exposure, suggesting translocation across the maternal
pulmonary barrier to placental and fetal tissues (Fournier et al., 2020). Recently a pilot study
reported the presence of microplastics in four human placentas, drawing attention to the risks
microplastics might pose to pregnancy and fetal development (Ragusa et al., 2021).
Microplastics represent a new substrate for microbial colonization and transportation.
Genome sequence data showed that the microbial community living on plastics from the
North Atlantic Subtropical Gyre was distinct from the microorganisms in the surrounding
seawater and included opportunistic pathogens (Zettler et al., 2013). Likewise, potentially
pathogenic Vibrio spp. were found on microplastics collected in the North and Baltic Sea
(Kirstein et al., 2016). Interestingly, the biofilms on the microplastic surface are structurally
different compared to those on natural substrates and might favor the propagation of
antibiotic resistant microorganisms. The complex microbial consortia in microplastic biofilms
may promote horizontal gene transfer between phylogenetically distinct microbes more
rapidly than in free-living microorganisms (Wu et al., 2019). Therefore, microplastics may
function as a vector for dispersion of invasive species and pathogenic organisms.

1.2.3 Effects of plastic additives
The adverse effects on organisms exposed to microplastics are categorized by physical or
chemical effects (Campanale et al., 2020). Physical damage is associated with microplastic
shape, size, and concentration while chemical damage is caused by leaching of environmental
pollutants adsorbed on their surfaces and the release of plastic additives into the tissues

7

(Bouwmeester et al., 2015; Browne et al., 2013; Campanale et al., 2020). As microplastics
have a high surface area to volume ratio, the concentration of these adsorbed chemicals can
be orders of magnitude greater than those in the surrounding environment. These pollutants
include heavy metals and persistent organic pollutants, such as polychlorinated biphenyls,
organo-halogenated pesticides, nonylphenol, polycyclic aromatic hydrocarbons and dioxins
(Endo et al., 2005; Heskett et al., 2012; Hirai et al., 2011; Ogata et al., 2009). More than a
pathway to transfer pollutants into tissues, microplastics might augment their toxic effects,
synergizing with the adverse effects of the toxics that they contain or have adsorbed (Deng et
al., 2021, 2020, 2018; Luís et al., 2015). A possible explanation is that microplastics augment
the exposure and accumulation of these chemicals and slowly release them into the tissues,
increasing their bioavailability (Deng et al., 2018).
Plastic additives account on average for 4% of the plastic composition (EFSA, 2016) and
usually are not chemically bound to the plastic polymer, being easily released into the tissues
(Campanale et al., 2020). Plasticizers, such as phthalates and bisphenol A, are used to confer
flexibility to plastic products and are well known endocrine disrupting chemicals. These
chemicals interfere with receptor binding and hormone signaling, disrupting endocrine
functions and leading to reproductive abnormalities (Rattan et al., 2017). Phthalates can
affect ovarian follicles at different stages of folliculogenesis, either activating apoptosis
(atresia), inducing follicle arrest or accelerating their development (Hannon and Flaws,
2015). Each of these outcomes can affect the production of ovarian hormones, leading to
reproductive and non-reproductive detrimental effects (Hannon and Flaws, 2015). A
significant reduction of ovarian weight was observed in mice after bisphenol A treatment,
likely due to the inhibition of follicular growth during puberty (Nah et al., 2011).

8

Microplastics were able to adsorb, transport and accumulate phthalate esters into mouse
tissues, indicating that microplastics are a route of exposure to plasticizers (Deng et al., 2021,
2020). Moreover, the accumulation of phthalates in the gut and in the liver was higher when
transported by PE microplastics compared with phthalates alone (Deng et al., 2021, 2020).
Microplastics enhanced the reproductive toxicity caused by phthalates in male mice, as
demonstrated by spermatogenesis disorder, testicular oxidative stress, reduced sperm cell
quality and altered testicular transcriptome (Deng et al., 2021). Microplastic and phthalate coexposure also caused aggravated adverse effects on mouse gut, leading to microbiota
dysbiosis, intestinal inflammation and altered transcriptome (Deng et al., 2020). Similarly,
organophosphorus flame retardants in the presence of PE and PS microplastics led to worse
harmful effects in mice, such as oxidative stress, neurotoxicity and metabolism disorder
(Deng et al., 2018). Due to their toxic effects, some additives have been replaced by
alternatives. However, as they have similar structure, the alternatives are thought to cause
equivalent toxicological effects (Rattan et al., 2017).

1.3

Microplastics and the immune system

1.3.1 Microplastics and inflammation
It is reasonable to expect the immune system to be affected by microplastic exposure, as its
main function is to provide protection against foreign particles. Inflammation is an immune
response against damaged cells, irritants and pathogens that is highly coordinated by genes
expressed in immune cells, particularly neutrophils. Inflammation can be acute (short-term)
followed by healing or chronic (long-term) and characterized by persistent and increased
9

expression of inflammatory cytokines (e.g. interleukin [IL]-1, IL-6, and tumor necrosis factor
[TNF]-α). Physical damage to tissue induces the secretion of inflammatory cytokines and
other mediators typical of the innate immune response, leading to chronic inflammation if the
damage is not resolved (Owen et al., 2018). Therefore, the microplastic potential to trigger an
acute or chronic inflammation will depend upon the cell damage caused by microplastics, if
they are eliminated/neutralized by the organism or if they persist in damaging cells.
Many in vitro studies have shown the nano/microplastic potential to induce an inflammatory
response. Exposure to PS and PC nanoparticles induced phagocytosis of particle aggregates
and individual particles by neutrophils. In addition, these particles induced degranulation of
primary granules of neutrophils, respiratory burst and extracellular trap release (Greven et al.,
2016). Exposure to PP microparticles caused hemolysis of red blood cells (RBCs) and
increased reactive oxygen species (ROS), IL-6, TNF-α and histamine levels in dermal
fibroblasts, peripheral blood mononuclear cells (PBMCs), macrophages and mast cells, while
no effect was observed in IL-2, IL-10 and IL-1β secretion levels (Hwang et al., 2019).
Human lung epithelial cells exposed to PS microparticles showed oxidative stress, loss of
epithelial barrier integrity, and increased secretion of proinflammatory cytokines IL-6 and IL8 (Dong et al., 2020). Mouse testicular cells (germ cell-1, Leydig and Sertoli cells) showed
increased levels of TNF-α, IL-6, monocyte chemoattractant protein-1 (MCP-1) and
chemokine (C-X-C motif) ligand 10 (CXCL10) after PS exposure, indicating a strong
testicular inflammation (Jin et al., 2021).
Microplastics have been reported to cause an inflammatory response in both invertebrate and
vertebrate species. Mussels showed a sharp increase in hyalinocytes (precursor of
granulocytes), decreased lysosomal membrane stability and granulocytoma formation after
exposure to microplastics (Avio et al., 2015; von Moos et al., 2012). Gut inflammatory cell
10

infiltration was observed in mice after oral exposure to PE microplastics (Deng et al., 2020)
as well as in adult zebrafish exposed to PS microparticles (Qiao et al., 2019b). After an
intraperitoneal injection of PS microplastics, mice showed a strong inflammatory response,
characterized by the influx of monocytes, macrophages and neutrophils into the particleinfiltrated area. Macrophages rapidly engulfed small and medium sized particles (1.2 6.2µm), whereas larger particles (12µm) were surrounded by macrophages that apparently
fused to each other to create giant cells (Tomazic-Jezic et al., 2001). After oral PE exposure,
mice showed a decreased number of leukocytes in the blood with an elevated proportion of
neutrophils, increased helper T cell to cytotoxic T cell ratio and decreased proportion of
mature dendritic cells in the spleen; some of these effects were also observed in the offspring
(Park et al., 2020). Inhaled microplastics that have escaped clearance mechanisms in the
upper airways interact with lung cells and lung fluid triggering an inflammatory response,
usually mediated by macrophages (Dong et al., 2020; Pauly et al., 1998).
Adult zebrafish exposed to PS microplastics presented increased mRNA levels of the proinflammatory cytokines IL-1α, IL-1β, IL-8 and interferon (IFN) in the gut (Jin et al., 2018b).
Mice exposed to PE microplastics showed altered serum concentration of the cytokines IL1α, granulocyte colony-stimulating factor (G-CSF), IL-2, IL-5, IL-6, IL-9, interferon gammainduced protein 10 (IP-10), and RANTES (regulated on activation, normal T cell expressed
and secreted, also known as CCL5) (Li et al., 2020) and higher levels of immunoglobulin (Ig)
A, while no change was observed for IgM, IgE and IgG (Park et al., 2020). Caspase-3, NFκB, TNF-α, IL-1β and IL-6 levels were increased while the anti-inflammatory molecules
Nrf2 and HO-1 were decreased in the testicular tissue of mice exposed to PS microplastics
(B. Hou et al., 2021; Xie et al., 2020). These effects were reversed by the use of an
antioxidant agent and a p38 MAPK inhibitor, suggesting that PS microplastic toxicity
involves oxidative stress and activation of the MAPK signaling pathway (Xie et al., 2020).
11

Moreover, microplastics can exacerbate inflammation in a pre-existing inflammatory
condition, as demonstrated in dextran sodium sulfate (DSS)-induced colitis mice exposed to
PS microplastics (Zheng et al., 2021). This study showed that microplastics can have a
stronger adverse effect on people with chronic inflammation, such as inflammatory bowel
disease (IBD) patients (Zheng et al., 2021).
Microplastic exposure seems to activate the complement system. The complement system
includes 50-plus serum proteins that cooperate with both innate and adaptive immune
systems to eliminate pathogens, dying cells and immune complexes (Owen et al., 2018).
Once in biological fluids, nanoparticles can bind proteins and form protein coronas within 30
seconds of exposure; most of these corona proteins are complement components (Tenzer et
al., 2013). The complement system can be initiated through three different ways: classical,
lectin and alternative pathways. The classical and lectin pathways rely on the binding of
pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) or
damage-associated molecular patterns (DAMPs) while the alternative pathway is initiated by
the spontaneous hydrolysis of the complement protein C3 (Thorgersen et al., 2019; Owen et
al., 2018). Larval zebrafish injected with PS microparticles showed altered transcriptome
profile, including the upregulation of genes of the complement pathway (cfb, cfh, c3a.2,
c3a.3, c3a.6 and c9), indicating that microplastics can activate the complement system,
mainly through the alternative pathway (Veneman et al., 2017). All three initiation pathways
converge in the generation of a C5 convertase, which cleaves C5 molecule into C5a and C5b
and ultimately leads to the formation of the membrane attack complex (MAC) and cell death
(Owen et al., 2018). It is important to notice that although the MAC formation is the final
product of the complement cascade, the molecules that are generated in the process also play
a major role on the immune response, such as opsonins (C3b, C4b) and anaphylatoxins (C3a,
C5a) that mediate inflammation.
12

1.3.2 Acute-phase reactants
Acute-phase reactants (APR) are serum proteins that rapidly increase or decrease in response
to infection, tissue damage or inflammation. These proteins are commonly recognized
inflammation markers and are extensively used in diagnostics and prognostics in a variety of
clinical conditions. C-reactive protein (CRP) is a major APR in humans. During an
inflammatory response, blood levels of CRP can rapidly increase up to 1,000-fold and
decrease just as rapidly when the stimuli end. Studies have shown that CRP opsonize
pathogens and apoptotic cells and activate the classical complement pathway (Sproston and
Ashworth, 2018). The name CRP arose because it was first identified as a substance that
reacted with the C-polysaccharide of pneumococcal cell wall in patients with acute
pneumonia. Similarly, CRP binds to exposed or denatured chromatin and
phosphatidylcholine present on damaged cell membranes, promoting clearance by
macrophages and neutrophils (Du Clos, 2000). By contrast, CRP in mice is less inducible by
inflammatory stimuli and is deemed a minor APR. Serum amyloid P-component (SAP),
however, is the major inflammation marker in mice (Pepys et al., 1979).
Similar to CRP, SAP is a pattern recognition molecule secreted by the liver and modulates
the innate immune response (Cox et al., 2014). Its concentration in the serum increased up to
50-fold in mice within 24-48 hours of injection of lipopolysaccharides (LPS) and returned to
the basal level after 32 days (Pepys et al., 1979). The main roles of SAP are the regulation of
neutrophil migration and spreading, inhibition of monocyte differentiation into fibrocyte,
opsonization of cell debris, regulation of macrophage polarization and activation of the
classical complement cascade (Cox et al., 2014). Studies have shown that SAP binds to a
variety of microbial polysaccharides and toxins as well as chromatin and DNA in circulation,
enhancing the clearance of autoantigens to prevent autoimmunity (Mold et al., 2001).
13

1.3.3 The Hippo Pathway and tissue injury repair
As studies have reported that microplastics can potentially cause tissue damage, it is expected
that injury repair mechanisms to be engaged following microplastic exposure. The Hippo
Signaling Pathway is a protein kinase cascade that modulates cell proliferation,
differentiation, growth and death (Yu and Guan, 2013). Cell contact, mechanical stress,
cytoskeletal tension, cell polarity, and growth factor receptor signaling are known upstream
regulators of this pathway. Yes-associated protein (YAP) and transcriptional co-activator
with PDZ-binding motifs (TAZ) are the major downstream effectors of the Hippo pathway in
mammals. When the Hippo pathway is activated, YAP/TAZ is phosphorylated and unable to
enter the nucleus, resulting in inhibition of target gene transcription. On the other hand,
inactive upstream kinases result in nonphosphorylated YAP/TAZ that translocate into the
nucleus and interact with DNA-binding transcription factors, promoting target gene
transcription (Yu and Guan, 2013). Abnormal YAP/TAZ overexpression leads to
uncontrolled cell growth and cancer development (Wang et al., 2013). This pathway regulates
the transcription of a diverse range of growth-promoting and apoptosis-inhibiting genes,
including cellular communication network factor 1 (CCN1), previously named cysteine-rich
61 (Cyr61) (Lee et al., 2019).
Cellular communication network proteins are conserved matricellular proteins that regulate
cell adhesion, migration, differentiation, proliferation, and connective tissue remodeling (Jun
and Lau, 2013; Lee et al., 2019). These proteins modulate extracellular signal molecules,
such as growth factors, chemokines, cytokines and enzymes, or directly bind to integrin
receptors to activate pathways in a cell-type dependent manner (Jun and Lau, 2013). While in
adults CCN1 expression has been associated with inflammation and tissue injury repair,
during embryogenesis it is essential for cardiovascular and skeletal development (Jun and
14

Lau, 2013; Kim et al., 2018). Wound healing and injury repair consist of three temporarily
overlapping but biologically distinct phases - inflammation, proliferation and maturation –
and CCN1 has been shown to play an important role in each of these phases (Kim et al.,
2018). Immune cells, particularly neutrophils, infiltrate the damaged area during
inflammation. However, neutrophils are short-lived and rapidly undergo apoptosis. The
clearance of apoptotic neutrophils is facilitated by CCN1, that functions as a bridging
molecule between the phosphatidylserine molecules of apoptotic cells and integrins in
macrophages (Jun et al., 2015). During the proliferation phase, myofibroblasts are activated
to produce extracellular matrix (ECM) components to promote tissue integrity. Treatment
with CCN1 induced cell proliferation and differentiation after bile duct injury (Kim et al.,
2015) and DSS-induced colitis (Choi et al., 2015) leading to biliary repair and intestinal
mucosal healing, respectively. During the maturation phase, CCN1 triggers myofibroblast
senescence, ceasing ECM production in order to avoid fibrosis while upregulating ECM
degradation enzymes to promote matrix remodeling (Kim et al., 2018).
This research tested the ability of microplastics to affect the health of mammals using a
mouse model. We assessed the microplastic effects on food consumption, body and organ
weights, the effects on the expression levels of genes related to inflammation (complement
factor B - Cfb, C-reactive protein - Crp, serum amyloid P-component - Apcs) and tissue
injury repair (cellular communication network factor 1 - Ccn1) and microplastic tissue
distribution, accumulation and associated histopathology. Manuscript 1 summarizes the pilot
project results, where two different shapes of microplastics were used (microbeads and
microfibers). Manuscript 2 discusses the follow-up project, where the exposure time was
increased and sex-based differences were observed.

15

2-

(In prep) Short-term exposure to microbeads and microfibers pose

minimal risk of inflammation and tissue injury in mice

2.1

Chapter summary

Microplastics are ubiquitous pollutants, traveling in the atmosphere and depositing all over
the world, whether at sea or land. Recent studies have found microplastics in human stools,
colectomy samples and placenta. Despite considerable evidence suggesting that microplastics
cause negative outcomes in model organisms and wild species, the risks to human health are
unknown. Moreover, most researches use manufactured single-shaped microplastics while in
reality humans are exposed to a variety of shapes and sizes of microplastics. Here we
investigated the biological effects of two different shapes and sizes of microplastics
(microbeads and microfibers) using a mouse model. Female mice were fed microplasticcontaining food for two and four weeks, followed by a four-week recovery period.
Microplastic exposure did not affect body weight and only minor changes were observed in
food consumption for both microbead and microfiber-treated groups. Microfibers were not
able to translocate from the gut lumen into tissues, while microbeads were observed in the
liver and small intestinal wall. Although microbeads remained embedded in the liver after the
recovery period, no histopathological signs were observed. Similarly, microplastic exposure
did not affect the expression levels of genes related to inflammation (Apcs, Crp, Cfb) and
tissue repair (Ccn1). Although we did not find any significant change for the biomarkers used
in the present study, other researches using different endpoints have reported a broad range of
adverse effects. Further research is needed to investigate the fate and effects of different types
of microplastics and the impact of long-term exposure on biological systems.

16

2.2

Introduction

The distribution and abundance of plastics into the world are so extensive that they have
earned a historical epoch: The Plasticene (Campanale et al., 2020). Microplastics are plastic
particles smaller than 5mm that are purposefully manufactured in this size (primary
microplastics) or derived from the fragmentation of larger pieces of plastic (secondary
microplastics) due to natural processes, such as photo-oxidation, hydrolysis, abrasion, and
biodegradation. Microplastics are ubiquitous, have been reported in aquatic systems (Dris et
al., 2015; Eriksen et al., 2014; Imhof et al., 2013), soil (Rillig, 2012), ground water (Mintenig
et al., 2018), sewage and the atmosphere (Dris et al., 2015). In addition, studies have found
microplastics in drinking water (Kosuth et al., 2018) and food destined for human
consumption (Conti et al., 2020; Kosuth et al., 2018; Smith et al., 2018; Yang et al., 2015;
Liebezeit and Liebezeit, 2015; 2013; Mathalon and Hill, 2014; Murray and Cowie, 2011).
Emerging evidence suggests that humans are exposed to microplastics mainly through diet
and inhalation. Recently, microplastics were found in human stools (Schwabl et al., 2019),
colectomy samples (Ibrahim et al., 2021) and placenta (Ragusa et al., 2021). However,
microplastic exposure risk to humans is unresolved. A remarkable inter and intra-individual
variability of microplastic abundance in washes from hand, face, hair and mouth suggests that
human exposure to microplastics depends on the individual habits and environment (Abbasi
and Turner, 2021). A recent study found that the dietary exposure to microplastics depends
on sex and age, with an annual consumption ranging from 39,000 to 52,000 particles (Cox et
al., 2019).
Mouse models have been used to elucidate the microplastic potential risk to mammalian
health. Studies have shown that microplastics can translocate across the gut barrier into
different organs, being found in the mouse gut, liver and kidney (Araújo and Malafaia, 2021;
17

Deng et al., 2018, 2017; Jin et al., 2018a). Some studies have shown that microplastics can
lead to intestinal homeostasis disturbance, as revealed by gut inflammation, oxidative stress,
reduced mucus secretion, increased intestinal permeability and microbiota dysbiosis (Jin et
al., 2018a; Li et al., 2020; Lu et al., 2018; Luo et al., 2019a). Neurotoxic effects have also
been observed in mice exposed to microplastics, as indicated by inhibition of
acetylcholinesterase activity (Deng et al., 2017) and behavioural disorders (Araújo and
Malafaia, 2021). Microplastic exposure caused changes in serum and hepatic metabolite
levels, suggesting lipid, amino acid and energy metabolism disorders (Deng et al., 2017; Jin
et al., 2018a; Lu et al., 2018); some of these adverse effects persisted across generations (Luo
et al., 2019b, 2019a). Additionally, microplastics caused reproductive impairment in male
mice, as shown by testis inflammation, reduced serum testosterone and decrease in sperm cell
quality (B. Hou et al., 2021; Jin et al., 2021; Xie et al., 2020).
In the environment, microplastics differ in shape, size and composition; this is not always
reflected in laboratory studies. Microplastics can be subcategorized according to their shape
and includes beads, nurdles, fibers, foam, fragments, pellets, films, filaments, cylindrical,
disk, flat, ovoid, spheruloid, elongated, rounded, and irregular. (Hirt and Body-Malapel,
2020). While spherical particles (microbeads) are most commonly used in laboratory studies,
fibers and fragments are the most common types detected in field samples (de Sá et al.,
2018). Filaments or fibers accounted for 96.1% of microplastics found in human colon
samples (Ibrahim et al., 2021) while fragments and films were the most abundant in stool
samples (Schwabl et al., 2019). Spherical and irregular shapes were predominant in human
placenta samples (Ragusa et al., 2021).
Although in the natural environment organisms are exposed to different types of
microplastics, their toxicity is more dependent on their size and shape than their chemical
18

composition (Lei et al., 2018b), with fibers showing greater adverse effects in comparison to
beads. Waterflea (Ceriodaphnia dubia) exposed to PET microfibers showed a more
pronounced reduction in growth and brood size along with body deformations compared with
PE microbead exposure (Ziajahromi et al., 2017). Similarly, PP microfiber exposure caused
higher mortality, slower egestion time and reduced growth compared with PE microbead
exposure in amphipod (Hyalella azteca) (Au et al., 2015). Secondary irregular shaped
microplastics caused elevated mortality, decreased reproduction and longer egestion time
compared with microbeads in Daphnia magna (Ogonowski et al., 2016). It should be noted
that the microfiber adverse impacts reported in small zooplankton might be related to the
physical stress promoted by entanglement rather than ingestion (Ziajahromi et al., 2017). In
zebrafish, however, PP microfibers had higher tissue accumulation and caused more
intestinal damage than PS microbeads and PS fragments. Additionally, microfibers caused
body weight loss and gut dysbiosis, along with stronger oxidative stress and higher IL-1α
level compared with microbeads and fragments (Qiao et al., 2019a). Rounded shapes might
be transported through the gut more easily while non-spherical particles have a longer gut
passage and may be easily embedded in the tissues (Au et al., 2015; Qiao et al., 2019a).
Considering the knowledge gap concerning the impact of different shapes of microplastics on
vertebrate species, particularly mammals, the aim of the present study was to evaluate the
health risks posed by microbeads (MB) and microfibers (MF) using a mouse model. BALB/c
female mice were exposed to MB or MF via diet and sacrificed at three different time points:
after two weeks of exposure, after four weeks of exposure and after four weeks of exposure
followed by a four-week recovery period. We monitored food consumption, body weight,
gene expression and tissue distribution, accumulation, and associated histopathology. Our
hypothesis is that microplastics will cross the intestinal barrier and reach other organs
triggering an immune response. The expression levels of genes related to inflammation (Apcs,
19

Cfb, Crp) and tissue repair (Ccn1) are expected to be high after microplastic exposure and
return to normal levels after the recovery period. If there is a difference between MB and MF,
we hypothesize that MF will cause more aggravated outcomes.

20

2.3

Methods

Care of animals
Five to six-week old female BALB/c mice (n=27) were ordered from Charles River
Laboratories (Pointe-Claire, Quebec, Canada). The mice weighed approximately 20 grams.
The animals were housed in the Animal Care Facility, Brandon University, Brandon, Canada,
on a 12-hour light/dark period with food and water provided ad libitum. Each cage housed
three mice. Temperature and relative humidity were maintained between 20°C - 25°C and
50% - 70%, respectively. The cages used were clear polycarbonate with stainless steel lids.
The composition of the bedding used for nesting was corn cob and natural paper. Paper rolls
were used for environmental enrichment. Upon arrival, the mice were acclimated for two
weeks before beginning the experiment to ensure there were no signs of stress or illness. The
animals were randomly divided into three groups: nine were assigned to the control group,
nine were assigned to the microbead (MB)-treated group, and nine were assigned to the
microfiber (MF)-treated group. The cages were cleaned once a week, when the feces were
collected and the mice were weighed. Mice were anesthetized with 5% isoflurane until
unconscious and then immediately euthanized with CO2 at a flow rate of 20% chamber air
displacement per minute. The animal care protocol was approved by the Brandon University
Animal Care Committee (BUACC) in accordance with guidelines of the Canadian Council on
Animal Care (BUACC Certificate #2019R04-1).

Microplastic exposure and sampling
Fluorescent red polyethylene microbeads (PE MB) were purchased from Cospheric
(UVPMS-BR-0.995, size range: 10 - 45 µm, mean diameter: 36.4µm, density: 0.995g/cc,
21

excitation: 300-550 nm, peak emission of 607 nm when excited at 575nm). The source of
microfibers (MF) was a red felt fabric composed by 15% acrylic and 85% of a polyester fiber
made from recycled plastic bottles (Ecofi®, size range: 36.20 - 2953.33µm, density:
0.213g/cc). The fabric was cut into 2 x 2 cm pieces and rinsed with deionized (DI) water and
96% ethanol to remove possible surface contamination. The material was dried and cut into
smaller pieces and then placed in a Petri dish with DI water to allow the fibers to spread. The
coarse parts were removed from the dish and the remaining floating MF were filtered, dried
and stored until use. MF were counted and measured using Nikon Eclipse Ti inverted
microscope with a Nikon DS-Ri1 camera and NIS-Elements Advanced Research Software
(version 4.13.00). The MF mean length was 609.06± 413.98 µm ranging from 36.20µm to
2953.33µm (Figure 2-1).

Figure 2-1: Microfiber length.
Felt fabric was manually cut, washed and dried. The MF mean length was 609.06 ±
413.98µm and size range 36.20 - 2953.33µm. a) MF length histogram. b) MF
photomicrograph, scale bar = 250µm.

The mice were exposed to microplastics via food intake. Commercial mouse feed (LabDiet
Rodent 5001) was ground and mixed with microplastics at a concentration of 100µg/Kg,
except for the control food that was microplastic-free. It is worth mentioning that we chose to
22

use the same mass/mass concentration for both microplastic exposures (100µg/Kg), however
the number of microplastic particles differed between MB and MF as they have different
sizes and densities (approximately 3,960 MB particles/Kg and 150 MF particles/Kg). After
mixing the microplastics with the ground feed, distilled water was added until the dough
became smooth and uniform (approximately 1L of water to 1Kg of food). The dough was cut
into a square shape and dried for 48 hours at 45°C.
The experimental procedure was conducted as outlined in the Figure 2-2. The animals were
randomly divided into three groups: nine were assigned to the control group, nine were
assigned to the MB-treated group and nine were assigned to the MF-treated group. Upon
arrival, the mice were fed microplastic-free food and the experiment began after two weeks
of acclimation. After two weeks of exposure, three mice from each group were sacrificed and
samples collected. To sacrifice the animals, they were first sedated and anaesthetized with
isoflurane gas, followed by carbon dioxide euthanasia. Kidney, liver, spleen, small and large
intestines were harvested. Another three mice from each group were sacrificed after four
weeks of exposure. At this point, all the remaining mice were fed microplastic-free food and
were sacrificed after four weeks (recovery period).
Exposure start

Exposure end

Acclimation
2 weeks

2 weeks

4 weeks

2 weeks
Sacrifices:
3 Control mice
3 MB-treated mice
3 MF-treated mice

Sacrifices:
3 Control mice
3 MB-treated mice
3 MF-treated mice

Sacrifices:
3 Control mice
3 MB-treated mice
3 MF-treated mice

Figure 2-2: Experimental design.
Microplastic exposure began after two weeks of acclimation. The sacrifices occurred after
two and four weeks of exposure. The exposure was terminated and all remaining mice were
fed microplastic-free food and sacrificed after the four-week recovery period.

23

Microplastic intake and egestion
The microplastic intake and egestion were estimated by the amount of ingested food and the
number of microplastics present in the feces, respectively. Feces were collected from each
cage once a week and separated from the bedding using test sieves. Feces were weighed and
reconstituted in DI water (0.3g of feces in 150mL of DI water). The homogenate was filtered
through a Whatman® qualitative filter paper (grade 595, pore size: 4 -7µm) using vacuum
filtration (Millipore® vacuum pump). The filter papers were placed in a Petri dish and all MB
or MF particles were counted with the use of an Olympus MVX10 stereomicroscope with XCite® 120 fluorescence mercury lamp and U-MRFPHQ/XL mirror unit (excitation: 545 +/10nm, emission: 597.5 +/- 27.5nm, dichroic mirror: 565nm). All samples were analyzed in 3
replicates.

Food consumption and body weight
Food and water were provided at libitum and the amount of food in the cages was measured
daily. When the amount of food in each cage was low (around 30 grams), more food was
added to completely fill the feed hopper (approximately 75 grams). Food was added to all
cages at the same time to prevent differences in food consumption that could be caused by the
better palatability of fresh food. Daily food consumption was calculated by the difference in
the amount of food left in the cage from the previous day, e.g. food consumed on Day 1 =
amount of food left in the cage on Day 1 – amount of food left in the cage on Day 2 (all
measurements were taken around 8:00am). The individual mice were weighed weekly.

24

Histological analysis and tissue accumulation
Samples from liver, kidney, spleen, small and large intestines were collected from control and
experimental groups, fixed in 10% formalin, embedded in paraffin wax, sectioned at 5µm
thickness, and stained with hematoxylin and eosin (H&E) for microscopic observation
(Appendix 1). The “Swiss roll” technique as described by Moolenbeek and Ruitenberg
(1981) was performed for the small and large intestines.
As a section is a two-dimensional image taken from a three-dimensional object (Altunkaynak
et al., 2012), the sections were chosen in a way that different depths of the tissues were
represented. The analyzed sections were cut 10-60µm apart (depending on the thickness of
the whole tissue) until the whole embedded organ had been sampled. At least 50 sections
were analyzed per tissue of each mouse.
The histological sections were analyzed using a Nikon Eclipse Ti inverted microscope and
the photomicrographs were captured using a Nikon DS-Ri1 camera and NIS-Elements
Advanced Research Software. In order to detect the MB fluorescence, the sections from MBtreated group were analyzed using an Olympus BX51 microscope with X-Cite® 120
fluorescence mercury lamp and TRITC filter cube (excitation: 543 +/- 11nm, emission: 591.5
+/- 21.5nm, dichroic mirror: 562nm). The MB photomicrographs were captured using an
Olympus SC100 camera and cellSens Imaging software.

25

Gene expression analysis
•

RNA extraction

Liver samples were harvested and immediately placed in 2mL cryotubes containing
RNAlaterTM RNA Stabilization Solution (Invitrogen, Thermo Fisher Scientific, Lithuania).
Samples were incubated overnight at 2-8°C and stored at -80°C until RNA extraction.
Approximately 100mg of tissue was used for total RNA extraction with TRIzol® Reagent
(Ambion, Life Technologies, USA) according to the manufacturer’s protocol (Appendix 2).
The extracted total RNA was eluted with nuclease-free water and the concentration and
quality were assessed with a NP80 NanoPhotometer (Implen, Munich, Germany) using
nuclease-free water as a blank. Samples with optical density ratios of 260/280nm close to 2.0
and concentrations above 500ng/μl were considered acceptable for downstream application
(Matlock, 2015). The extracted total RNA samples were aliquoted and stored at -80°C until
reverse transcription (cDNA synthesis).

•

Reverse Transcription

Following nuclease digest treatment with DNase I, total RNA (2,000 ng) was reverse
transcribed using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor
(Thermo Fisher Scientific, Vilnius, Lithuania) (Appendix 3). Quantity and quality of
transcribed cDNA samples were measured using a NP80 NanoPhotometer (Implen, Munich,
Germany) using a solution of RT buffer and nuclease-free water as a blank. Samples with
260/280 ratio between 1.6 - 1.8 and concentration above 500ng/μl were considered
acceptable for downstream application. Transcribed cDNA was aliquoted and stored at -20°C
until qPCR.
26

•

Quantitative Real-Time PCR (qPCR)

Gene expression analysis was performed using commercially available gene-specific TaqMan
probes (Applied Biosystems, Pleasanton, CA, USA).
Table 2-1: TaqMan probe details
Gene name

Complement Factor B (Cfb)

Cellular Communication
Network Factor 1 (Ccn1)
C-Reactive Protein (Crp)
Serum Amyloid P-Component
(Apcs)
18S ribosomal RNA (18s)
Glyceraldehyde-3-phosphate
dehydrogenase (Gapdh)

Gene assay

Mm00433909_m1

Amplicon
length

PCR setting

Expression sites
Mainly in the liver, followed by
placenta, kidney, large intestine,
mammary glands, small intestine,
bladder, spleen, lung, ovary, etc.

85

Hold:
95°C 10min

Broad expression including lung,
limb, bladder, placenta, ovary,
heart, spleen, stomach, intestines,
CNS, liver, thymus, etc.

Mm00487498_m1

112

Mm00432689_g1

134

Mm00488099_g1

114

Mm04277571_s1

115

Ubiquitous

Mm99999915_g1

109

Ubiquitous

Cycle:
(x50)
95°C 15sec
58-60°C 60sec

Liver
Liver

For qPCR, cDNA samples were diluted in nuclease-free water (1:80). The qPCR was
performed according to the manufacturer’s instructions with a reaction final volume of 20μl
(forward and reverse primer concentration: 900nM, probe concentration: 250nM, and 4µL of
cDNA) (Appendix 4). The amplification efficiency was determined by constructing standard
curves for each TaqMan assay using a 5-point 2-fold dilution series of pooled cDNA
templates. Efficiencies between 90-105% and R2 above 0.985 were considered acceptable.
All samples and non-template control (NTC) were run in duplicate and 3 independent runs
were performed for each assay. Replicates from the same run were less than 0.5 cycle apart
and replicates from different runs were less than 1.0 cycle apart.
The cycle threshold (Ct) values were calculated using the Corbett Rotor-Gene 6000 (Qiagen)
software using the corresponding standard curve following manufacturer’s instructions.
27

Relative gene expression ratio was calculated according to the ∆∆Ct method (Livak and
Schmittgen, 2001) using the geometric mean of two housekeeping genes (18s and Gapdh) as
normalizers.

Statistical analysis
Food consumption and body weight data were tested using two-way repeated measures
ANOVA (one factor repetition) and multiple comparisons versus control group (Holm-Sidak
method) was used to isolate the group that differed from the others. Gene expression data
were tested using one-way ANOVA. All tests were performed using SigmaPlot 11.0 at a
significance level of p ≤ 0.05 after passing normality and equal variance tests.

28

2.4

Results

Food consumption and body weight
Food consumption was measured by cage daily - each cage housed three mice. The results of
the two-way repeated measures ANOVA revealed that there was no significant main effect of
microplastic exposure on food consumption (df=2, p=0.089). However, there was a
significant interaction between treatment (control, MB, MF) and time (days 1 to 56) (df=110,
p<0.001), i.e. the effect of MB or MF microplastic exposure on food consumption depends
on the day. Multiple comparisons versus control group (Holm-Sidak method) showed
significant difference on days 13, 20, 45 and 51 for MB-treated mice, on day 7 for MFtreated mice and on days 4, 5, 8, 31, and 40 for both MB and MF-treated mice compared to
the control group (marked with * in the graph – see Figure 2-3a). There was no consistency if
the consumption was increased or decreased compared with the control group.
Individual mice were weighed weekly. Two-way repeated measures ANOVA analysis
revealed that there was a statistically significant effect of time on body weight (df=7,
p<0.001), however it was due to the mouse expected growth during puberty phase. There was
no statistically significant effect of microplastic exposure on body weight (df=2, p=0.143)
and there was no statistically significant interaction between treatment (control, MB, MF) and
time (weeks 1 to 8) (df=14, p=0.062) (Figure 2-3b).

Microplastic intake and egestion
Food consumption was measured by cage daily. On average, MB and MF-treated cages
consumed 10.25g/day and 9.46g/day, respectively. As each cage housed three mice, each
MB-treated mouse consumed 3.42g/day and each MF-treated mouse consumed 3.15g/day,
29

assuming equal food consumption. As the microplastic concentration in the mouse food was
3,960MB/Kg or 150MF/Kg, each mouse was exposed to approximately 13.53 MB/day or
0.47 MF/day.
Microplastic egestion was estimated counting the number of MB or MF present in the feces.
Both MB and MF egested in the feces did not show any sign of deterioration (Figure 2-4).
Both MB and MF-treated mice were able to egest microplastics within the first week of
exposure. There was a significant decrease in the amount of microplastics in feces after the
fourth week, when the exposure was terminated for both MB and MF-treated cages (df=7,
p=0.003). MB-treated mice continued to eliminate MB until the eighth week, while no MF
was visualized in MF-treated mouse feces in week 3 and in the recovery period (Figure 2-3c).

30

a

b

c

31

Figure 2-3: Food consumption, body weight and microplastic egestion.
a) The presented values are the average food consumption per cage. Statistically significant
difference was observed on days 13, 20, 45 and 51 for MB-treated group, on day 7 for MFtreated group and on days 4, 5, 8, 31 and 40 for both MB and MF-treated groups compared to
the control group (marked with *). Linear regression for control group: y = -0.0041x + 9.281
R² = 0.0034, MB group: y = -0.0355x + 10.933 R² = 0.0565, and MF group: y = -0.0107x +
9.8547 R² = 0.0064.
b) Individual mouse weight. The presented values are the mean±SEM. No statistically
significant difference was observed between control and microplastic-treated groups. Linear
regression for control group: y = 0.5181x + 18.891 R² = 0.816, MB group: y = 0.4148x +
20.414 R² = 0.8869, and MF group: y = 0.4579x + 19.43 R² = 0.9224. “Pre” refers to the
body weight before exposure.
c) Microplastic egestion. MB and MF in feces were counted using a stereo microscope. The
presented values are the mean±SEM amount of microplastics found in 1g of feces (each
sample was run in triplicate). There was a sharp drop after the fourth week, when the
exposure was terminated.

Figure 2-4: Microplastics in feces.
Feces were reconstituted in DI water and filtered through a filter paper with pore size of 47µm (Whatman®). The filters were analyzed using a stereo microscope. a) Fluorescent red
PE MB and b) red polyester MF found in the feces. Scale bar = 100µm.

Histological analysis and tissue accumulation
The kidney, liver, spleen and small and large intestine were harvested for histological
analysis. The H&E staining showed normal cellular morphology with no signs of
inflammation in all tissues analyzed after two and four weeks of exposure and recovery
period. There was no evidence of necrosis or immune cell infiltration in control mice as well
as microplastic-treated mice (Figure 2-5).
32

Figure 2-5: Histological analysis after microplastic exposure.
Representative photomicrographs of H&E stained kidney, liver, spleen, small and large
intestines of control, MB and MF-treated mice. There are no histological alterations. Scale
bar = 100µm.
33

No MF was found in MF-treated mouse tissues, while two MB particles were found in the
small intestine after four weeks of exposure and one MB particle was found in the liver after
the four-week recovery period (Figure 2-6).

Figure 2-6: MB found in mouse tissues.
Fluorescent red PE MB in the liver (recovery period) and small intestine (4-week exposure)
of MB-treated mice. Left column: 20x objective (scale bar = 100µm), middle and right
columns: 40x objective (scale bar = 20µm).

34

Gene expression analysis
The expression level of genes related to inflammation (Apcs, Cfb, Crp) and tissue injury
repair (Ccn1) was measured in the liver. Although the expression pattern of microplastictreated groups differed from the control group, the difference was not statistically
significant (p>0.05) (Figure 2-7).

Relative gene expression levels

a

2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl
MB
MF

Apcs

Relative gene expression levels

b

Cfb

Crp

Ccn1

2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl
MB
MF

Apcs

Cfb

Crp

35

Ccn1

Relative gene expression levels

c
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl
MB
MF

Apcs

Cfb

Crp

Ccn1

Figure 2-7: Gene expression in the liver.
Apcs, Cfb, Crp and Ccn1 gene expression profiles of control (Ctrl), MB and MF-treated
groups after a) two weeks of exposure, b) four weeks of exposure, and c) recovery period.
18s and Gapdh were used as normalizer genes. Mean ± SEM (n=3). No statistically
significant differences were observed.

36

2.5

Discussion

Microplastics are a pollutant of emerging concern, being reported in the atmosphere, drinking
water and food destined for human consumption (Wright and Kelly, 2017). Although recent
evidence suggests that humans are exposed to microplastics, their fate and effects on the
human body are still controversial and uncertain (Campanale et al., 2020). Studies have
shown that particles smaller than 150µm might translocate from the gut into the lymph and
circulatory system, causing systemic exposure (Barboza et al., 2018). The adverse effects on
organisms exposed to microplastics might be caused by the physical or chemical microplastic
properties. Physical damage is associated with microplastic shape, size, and concentration
while chemical damage is caused by microplastic monomers and additives as well as the
environmental chemicals they adsorb on their surface (Campanale et al., 2020).
The microplastic effects on feeding activity and growth depend on the biology, physiology
and behaviour of organisms. Species with lower energy demand might be less vulnerable to
low-calorie diet and species able to modulate food intake can avoid plastic-contaminated food
(Watts et al., 2015). Microplastics have been thought to cause false satiation, damage and
blockage of the intestinal tract that could lead to reduced feeding activity and growth. On the
other hand, organisms might show a compensatory response to non-nutritious plastic content
in the food and temporarily increase feeding activity (Watts et al., 2015). Crabs fed PP
microfibers showed reduced food consumption and a significant reduction in energy available
for growth (Watts et al., 2015). Similarly, lugworms showed reduced feeding activity and
depleted energy reserves after exposure to PVC microparticles (Wright et al., 2013).
In the present study, change in food consumption was observed on days 13, 20, 45 and 51 for
MB-treated mice, on day 7 for MF-treated mice and on days 4, 5, 8, 31, and 40 for both MB
and MF-treated mice compared with the control group. However, microplastic exposure did
37

not affect body weight. In contrast, a significant loss of body weight was observed in male
mice after PS (Jin et al., 2021; Lu et al., 2018; Xie et al., 2020) and PE microplastic exposure
(Park et al., 2020) with or without changes in food/water consumption. Other researchers
have reported no effect on body weight and food consumption in male rats (Rafiee et al.,
2018) and male mice (Deng et al., 2017; Stock et al., 2019) after PS exposure. These
contradictory results could be explained by differences in experimental design, such as
microplastic characteristics and route of administration. Most of the studies that reported a
significant reduction in body weight used PS microplastics and an extremely high dose
administered by oral gavage.
Both MB and MF-treated mice were able to egest microplastics in feces within the first week
of exposure. MF were found in MF-treated mouse feces during the exposure phase, except for
week 3, and no MF were found during the recovery period. On the other hand, MB were
found in MB-treated mouse feces throughout the experiment although in a lower
concentration during recovery period. This finding suggests that MB might be retained in the
gastrointestinal tract for a longer time than MF, which could be explained by the higher MB
concentration exposure and/or by the MB properties, such as smaller size and spherical
shape. In contrast, microplastic beads were egested more rapidly than microfibers and
irregular shaped microplastics in amphipods and Daphnia, respectively (Au et al., 2015;
Ogonowski et al., 2016). Plastic microspheres were quickly egested by Xenopus tadpoles (Hu
et al., 2016), sea bass larvae (Mazurais et al., 2015) and Daphnia (Rosenkranz et al., 2009)
and were almost completely eliminated after 6 days, 2 days and 4 hours of depuration,
respectively.
Under visual inspection, the egested microplastics showed no signs of deterioration or
fragmentation, which confirms microplastic bioresistance, i.e. refractory to digestion or
38

dissolution. PP microfibers were significantly altered by the passage through crab foregut,
being excreted with a smaller size and length (Watts et al., 2015). The different result may be
due to the crab foregut complex gastric mill used for grinding carapace shells and animal and
plant tissues (Watts et al., 2015). In humans, digestive enzymes and bile salts are thought to
affect the color of microplastics, as the majority of the microfilaments found in human
colectomy samples was transparent (Ibrahim et al., 2021) whereas all microplastics found in
human placenta were pigmented (Ragusa et al., 2021).
Histological analysis revealed normal tissue morphology without noticeable histopathological
signs and inflammatory response in the liver, kidney, spleen, and intestines in control, MB
and MF-treated groups throughout the experiment (2-week and 4-week exposure and 4-week
recovery period). Similarly mice exposed to PS microplastics had no histopathological
changes in the above mentioned tissues (Stock et al., 2019) and rats exposed to PS
nanoparticles showed no histological alterations in the liver, kidney, spleen, lung, brain,
testes, stomach, small and large intestines (Walczak et al., 2015). Zebrafish showed no
histological changes in the gills, liver, brain, kidney and intestines after exposure to PE micro
fragments (Karami et al., 2017) and PS microbeads (Lei et al., 2018b). Conversely, zebrafish
showed intestinal injury, such as epithelial damage, thinning of the gut wall and congestive
inflammation (Qiao et al., 2019b) as well as cracking of villi and splitting of enterocytes (Lei
et al., 2018b) after exposure to different types of microplastics. In mice, histological
alterations have been reported in the gut, liver and testes. PE microplastic exposure caused
intestinal damage and inflammation, as indicated by edema, loose glands, and infiltration of
lymphocytes and plasma cells into the lamina propria (Li et al., 2020). Accumulation of lipid
droplets and ballooning degeneration of hepatocytes were observed in mouse liver after PS
microplastic exposure (Deng et al., 2017; Luo et al., 2019a). Additionally, PS microplastic
exposure caused histopathological changes in mouse testicular tissue, as shown by disordered
39

arrangement of spermatid cells, pyknosis, nuclear rupture, cell detachment, and reduced
spermatogenic cell number (B. Hou et al., 2021; Xie et al., 2020).
Researches have shown that microplastics can cross the intestinal barrier and accumulate in
different organs (Araújo and Malafaia, 2021; Deng et al., 2018, 2017; Jin et al., 2021). Recent
studies have found that different sizes of microplastics (<40µm) can accumulate in mouse gut
wall, liver, kidney and testes (Araújo and Malafaia, 2021; Deng et al., 2017; Jin et al., 2021,
2018a; Stock et al., 2019). In the present study, histological sections were analyzed to assess
microplastic tissue distribution and accumulation. Only two MB particles were found in the
small intestine after four weeks of exposure and one MB particle was observed in the liver
after the recovery period. No MF was observed in the MF-treated mouse tissues. Studies have
confirmed that only particles smaller than 150µm can translocate across the intestinal
epithelial barrier and only a small fraction is expected to penetrate deeply into organs
(Barboza et al., 2018). This could explain the absence of MF and the small number of MB
found in the tissues, as the MF average length used in this study exceeds the size limit. The
estimate bioavailability of PS nanoplastics (50nm) in rats ranged from 0.2 – 1.7% of the
administered dose, being found mostly in the stomach and intestines, followed by kidney,
spleen, testes, heart and lungs (Walczak et al., 2015).
It is noteworthy that in the present study MB persisted in the liver after four weeks of
recovery, confirming the plastic biopersistence, i.e. ability to escape clearance mechanisms
and reside in the tissues indefinitely. It is also important to note that although MF were not
able to cross the gut barrier, they might have local effects on the gut lumen. MF could be
retained in the mucous layer lining the gastrointestinal tract and interfere with the gut
homeostasis, leading to microbiota dysbiosis, epithelial disruption, oxidative stress and local
inflammatory response (Hirt and Body-Malapel, 2020). In adult zebrafish, microplastic fibers
40

caused more severe intestinal toxicity than microplastic beads and fragments, as
demonstrated by increased histological damage, stronger oxidative stress and more
pronounced decline in mucus secretion in the gut (Qiao et al., 2019a). In addition, cellulosic
and plastic fibers greater than 250µm have been found in human lung specimens and have
been associated with acute and chronic inflammation that can cause a range of respiratory
diseases (Pauly et al., 1998).
In the present study, the expression of genes related to inflammation and tissue injury repair
were measured in the liver. The alternative pathway of complement activation is independent
of antibody-antigen interaction. Complement factor B (CFB) is unique to the alternative
complement pathway and is part of the C3 and C5 convertase complex, which generates
potent opsonins and anaphylatoxins, such as C3b and C5a (Owen et al., 2018). CRP and SAP
are pattern recognition molecules produced by the liver and secreted into the blood to interact
with pathogens and cell debris and promote their clearance by phagocytic cells (Mold et al.,
2001). In addition, SAP can activate the classical complement cascade and interact with
monocytes, neutrophils and macrophages to modulate the immune response and wound
healing (Cox et al., 2014; Pilling and Gomer, 2018). CCN1 is a secreted matricellular protein
that is involved in all tissue repair phases: inflammation, proliferation and maturation. CCN1
binds to specific integrin receptors on distinct cell types to promote clearance of apoptotic
neutrophils by macrophages, induce cell proliferation and differentiation, promote
angiogenesis and matrix remodeling (Kim et al., 2018). In the present study, gene expression
of Cfb, Crp, Apcs, and Ccn1 were measured in the liver. Although the expression pattern in
microplastic-treated groups differed from the control group throughout the experiment, there
was no statistically significant difference.

41

2.6

Conclusions

Microplastics have emerged as one of the most concerning environmental problems in recent
years. A growing body of evidence indicates that humans are exposed mainly through diet
and inhalation. Although a broad range of microplastic types is found in the environment,
most studies have used manufactured single-sized microbeads to assess microplastic health
risks. Moreover, researchers have used an extremely high microplastic concentration in an
attempt to induce more aggravated outcomes. Although several studies have shown the
harmful effects caused by microplastics, research comparing the effects of different shapes of
microplastics is still lacking. In the present study, we used two different shapes of
microplastics (beads and fibers) in a wide range of sizes and a realistic environmental
concentration. We also included a recovery period in our experimental design in order to
evaluate the reversibility of the adverse effects.
Microplastic exposure affected mouse food consumption however did not influence the body
weight. Microbeads were able to cross the gut barrier and only a few particles accumulated in
the liver and small intestine wall. Although microfibers were not able to cross the gut barrier
due to their larger size, it is worth mentioning the potential local effects on the intestinal
lumen. Microfibers could be retained in the mucous layer lining the gastrointestinal tract and
interfere with the gut homeostasis, causing microbiota dysbiosis, oxidative damage and local
inflammatory response. Microbeads remained in the liver even after the recovery period;
however, the biological markers used in this study failed to indicate inflammation or tissue
injury. Other researches using different biomarkers, however, have shown that microplastics
affect the innate and adaptive immune systems, and cause cellular and tissue damage.
Nevertheless, the toxicity mechanisms and defense against microplastics are still unclear.

42

Human exposure to microplastics is much more complex than the scenario simulated herein.
Further research using different experimental designs are needed to better understand the
microplastic effects on human health, such as long-term exposure, different microplastic
shapes, sizes and chemical composition, and presence of contaminants or microorganisms
adsorbed on the microplastic surface. Therefore, the study described in manuscript 2
compared the health effects on female and male mice exposed to microplastics for a longer
period of time.

43

3-

(In prep) Sex-based and time-dependent effects of microplastic

exposure in mice

3.1

Chapter summary

Plastic pollution has been identified by the United Nations as the second most ominous threat
to the global environment after climate change. Once in nature, plastics can be fragmented
into smaller pieces giving rise to another problem: microplastics. Despite substantial evidence
suggesting that humans are exposed to microplastics mainly through diet and inhalation, little
is known about the fate and effects of these particles on mammals. Here we investigated the
effects of polyethylene microplastics on mammalian health, using a mouse model. To do this,
female and male mice were fed food containing microplastics for four and sixteen weeks and
a third group was allowed an additional four-week recovery period to assess the reversibility
of the potential effects. Microplastics were able to translocate from the intestinal lumen into
different organs of both female and male mice and remained embedded in the tissues even
after the recovery period, confirming plastic biopersistence. Although no histopathological
signs were observed in the tissues, female mice showed altered mRNA levels of genes related
to inflammation and tissue injury repair. Overall, longer exposure to microplastics caused
more pronounced effects than short exposure, and some of these effects were reversed after
the recovery period. The present study shed light on the often-overlooked sex differences in
the response to microplastic exposure. Further research is warranted to better understand the
effects of exposure times, particularly long-term impacts, and the use of female and male
models is imperative to translate experimental findings to human populations.

44

3.2

Introduction

Plastics are ubiquitous in both terrestrial and aquatic ecosystems, with an estimated 250
million metric tons entering the oceans by 2025 (Wright and Kelly, 2017). Microplastics
come from a variety of sources, including manufactured small plastic particles designed for
commercial use (primary microplastics) and larger plastic debris that degrades into smaller
pieces due to natural processes (secondary microplastics). Although a modern wastewater
treatment plant can remove up to 98% of the microplastics in the effluent, it is estimated that
65 million microplastic particles are still released into the receiving water every day (Murphy
et al., 2016). In addition, microplastics are concentrated in the sludge subsequently used as
fertilizer thus contaminating the soil (Hirt and Body-Malapel, 2020).
Emerging evidence suggests that human exposure to microplastics occurs mainly through diet
and inhalation. Although the effects on human health are uncertain, recent studies have found
microplastics in human tissues ( Ibrahim et al., 2021; Ragusa et al., 2021; Schwabl et al.,
2019). Mouse models have been used to elucidate the microplastic potential risk to
mammalian health. Researches have shown that microplastics can translocate across the gut
barrier into different organs (Araújo and Malafaia, 2021; Deng et al., 2018, 2017; Jin et al.,
2018a) and cause intestinal homeostasis disturbance (Jin et al., 2018a; Li et al., 2020; Lu et
al., 2018; Luo et al., 2019a), neurotoxic effects (Deng et al., 2017), metabolism disorder
(Deng et al., 2017; Jin et al., 2018a; Lu et al., 2018) and reproductive impairment (B. Hou et
al., 2021; Jin et al., 2021; Xie et al., 2020).
Recent researches have highlighted the possible adverse effects of microplastic exposure on
immune responses. It is thought that microplastics greater than 150µm are not able to cross
the intestinal barrier, however they remain in the mucus layer and might lead to local
intestinal inflammation and microbiota disturbance (Hirt and Body-Malapel, 2020). Particles
45

smaller than 150µm may translocate from the gut cavity to the lymph and circulatory system,
causing systemic exposure (Barboza et al., 2018). Microplastic oral exposure caused gut
inflammatory cell infiltration (Deng et al., 2020; Qiao et al., 2019b), altered immune cell
counts in the blood and spleen (Park et al., 2020), and elevated levels of immunoglobulins
and pro-inflammatory cytokines (B. Hou et al., 2021; Jin et al., 2018b; Li et al., 2020; Park et
al., 2020; Xie et al., 2020). Moreover, microplastics can exacerbate inflammation in a preexisting inflammatory condition, as demonstrated in dextran sodium sulfate (DSS)-induced
colitis mice exposed to PS microplastics (Zheng et al., 2021). However, these studies have
not considered the sex-based differences in immune response against microplastics.
Immune responses against both foreign and self-antigens depend on sex. Generally, females
have stronger innate and adaptive immune responses compared with males in a variety of
species, including human and mouse (Klein and Flanagan, 2016). The sex differences in
immune responses are caused by genetic, hormonal and environmental factors (Costa et al.,
2018; Klein and Flanagan, 2016). Many signaling proteins are encoded on the X
chromosome, such as pattern recognition receptors (toll-like receptor (TLR) 7 and TRL8),
cytokine receptors (IL2RG and IL13RA2) and transcriptional regulators (FOXP3) (Klein and
Flanagan, 2016). Males are more vulnerable to X-linked diseases as they have a single X
chromosome, while the cellular mosaicism associated with X inactivation in females provides
a more extensive repertoire of proteins (Fish, 2008). Additionally, these X genes are found in
higher copy number in females, as approximately 15% of X genes in humans and 3% in mice
escape X inactivation (Klein and Flanagan, 2016). Moreover, some immune cells, such as
natural killer cells, macrophages, dendritic cells, neutrophils, B- and T-cells, have sex-steroid
hormone receptors and respond directly to the absence, presence or change of hormone
levels. Environmental factors, such as microbiome and diet, also have differential effects on
the immune response. Nutritional supplementation caused different epigenetic changes of
46

immune-related genes in female and male fetuses. Similarly, vitamin supplementation
differently affected immune cell numbers and cytokine production in female and male infants
(Klein and Flanagan, 2016).
Complement pathway activation and complement levels in the serum differ between females
and males in both humans (Costa et al., 2018) and mice (Kotimaa et al., 2016). Although
female and male mice show similar complement functional activity at the level of C3
activation in all three initiating pathways, females have lower terminal functionality, i.e. C6 –
C9 activation (Kotimaa et al., 2016). Generally, testosterone and progesterone have a
suppressive effect on immune function, while estradiol can be pro-inflammatory at low
concentrations or anti-inflammatory at high concentrations (Klein and Flanagan, 2016).
Estrogens and progesterone tend to favor immune responses towards T-helper (TH) 2 while
supressing TH1 and TH17 differentiation. Testosterone supresses TH2 and TH17-induced
adaptive immune responses while the effect on TH1 differentiation is unclear (Roved et al.,
2017). Estrogens increase the size of regulatory T (TREG)-cell population and decrease the
negative selection of naïve B-cells, which leads to survival of autoreactive B-cells and higher
antibody production (Fish, 2008). Testosterone decreases B- and T-cell proliferation,
antibody and cytokine production and supresses TLR4 expression in monocytes and
macrophages (Fish, 2008).
The immunocompetence-handicap hypothesis is a model that explains the interactions
between endocrine system, secondary sexual traits, immune system and parasite burden
(Folstad and Karter, 1992). Briefly, testosterone is required for the development of many
secondary sexual traits and dominance in males, therefore relating to reproductive success.
However, testosterone supresses the immune system and increases the susceptibility and
pathogenicity of parasitic infections. Hormonal self-regulation and plasticity of sexual trait
47

expression play an important role balancing the costs and benefits of each component
(Folstad and Karter, 1992).
Sex-based differences in immune response play a major role in disease incidence in females
and males. In general, men are more susceptible to viral, bacterial, fungal and parasitic
infections while the prevalence of autoimmune diseases is higher in women (Fish, 2008).
Sex-dependent factors influence susceptibility and progression of diseases, as well as efficacy
and side-effects of drugs. These findings made the National Institutes of Health (NIH) require
that all NIH-funded clinical trials include women as subjects. Similarly, the Natural Sciences
and Engineer Research Council of Canada (NSERC) asks the grant applicants to consider sex
and gender in the project’s research design.
The present study aimed to determine the microplastic effects on mammalian health using a
mouse model. We analyzed microplastic tissue distribution and accumulation, histopathology
and expression levels of genes related to inflammation (complement factor B - Cfb, Creactive protein - Crp, serum amyloid P-component - Apcs) and tissue injury repair (cellular
communication network factor 1 - Ccn1). We compared sex-specific responses to
microplastics as well as the effects of different exposure times.

48

3.3

Methods

Care of animals
Five to six-week old BALB/c mice (n=18 females, n=18 males) were ordered from Charles
River Laboratories (Pointe-Claire, Quebec, Canada). The mice weighed approximately 20
grams. The animals were housed in the Animal Care Facility, Brandon University, Brandon,
Canada, on a 12-hour light/dark period with food and water provided ad libitum. Each cage
housed three mice. Temperature and relative humidity were maintained between 20°C - 25°C
and 50% - 70%, respectively. The cages used were clear polycarbonate with stainless steel
lids. The composition of the bedding used for nesting was corncob and natural paper. Paper
rolls and apple tree sticks were used for environmental enrichment. Upon arrival, the mice
were acclimated for two weeks before beginning the experiment to ensure there were no signs
of stress or illness. The male and female mice were randomly assigned to one control group
(n=9) and one microplastic-treated group (n=9). The cages were cleaned once a week, when
the feces were collected and the mice were weighed. Mice were anesthetized with 5%
isoflurane until unconscious and then immediately euthanized with CO2 at a flow rate of 20%
chamber air displacement per minute. The animal care protocol was approved by the Brandon
University Animal Care Committee (BUACC) in accordance with guidelines of the Canadian
Council on Animal Care (BUACC Certificate #2019R04-1).

Microplastic exposure and sampling
MB-containing food was prepared as described in manuscript 1. Please refer to page 21.
The experimental procedure was conducted as outlined in the Figure 3-1. The animals were
randomly divided into two groups: nine were assigned to the control group and nine were
49

assigned to the MB-treated group. Upon arrival, the mice were fed microplastic-free food and
the experiment began after two weeks of acclimation. After four weeks of exposure, three
mice from each group were sacrificed and samples collected. To sacrifice the animals, they
were first sedated and anaesthetized with isoflurane gas, followed by carbon dioxide
euthanasia. Kidney, liver, spleen, ovaries, testes, small and large intestines were harvested
and weighed. Another three mice from each group were sacrificed after sixteen weeks of
exposure. At this point, all the remaining mice were fed microplastic-free food and were
sacrificed after four weeks (recovery period).
Exposure end

Exposure start

2 weeks

4 weeks

12 weeks
Sacrifices:
3 Control mice
3 MB-treated mice

4 weeks
Sacrifices:
3 Control mice
3 MB-treated mice

Sacrifices:
3 Control mice
3 MB-treated mice

Figure 3-1: Experimental design
Microplastic exposure began after two weeks of acclimation. The sacrifices occurred after
four weeks and sixteen weeks of exposure. The exposure was then terminated and all
remaining mice were fed microplastic-free food and were sacrificed after four weeks.

Microplastic intake and egestion
Microplastic intake and egestion analyses were performed as described in manuscript 1.
Please refer to page 24.

Food consumption, body weight and relative organ weight
Food consumption and body weight were measured as described in manuscript 1. Please refer
to page 24. Liver, spleen, kidneys, ovaries, testes, small and large intestines were harvested
and weighed. Small and large intestines were opened longitudinally, and the intestinal content
50

was carefully removed with forceps before weighing the tissues. To calculate the relative
organ weight the organ weight was divided by the body weight measured immediately after
euthanasia.

Histological analysis and tissue accumulation
Histological analysis was performed as described in manuscript 1. Please refer to page 25.

Gene expression analysis
Liver, spleen and large intestine were harvested for gene expression analysis. The protocol
was described in manuscript 1. Please refer to page 26.

Statistical analysis
Food consumption and body weight data were tested using two-way repeated measures
ANOVA (one factor repetition) and multiple comparisons versus control group (Holm-Sidak
method) was used to isolate the group that differs from the others. Relative organ weight and
gene expression data were tested using unpaired t-Test. All tests were performed using
SigmaPlot 11.0 at a significance level of p ≤ 0.05 after passing normality and equal variance
tests.

51

3.4

Results

Food consumption and body weight
Food consumption was measured by cage daily – each cage housed three mice. The results of
the two-way repeated measures ANOVA revealed that there was no significant main effect of
microplastic exposure on food consumption of female mice (df=1, p=0.169). However, there
was a significant interaction between treatment (control, microplastic) and time (days 1 to
140) (df=139, p=0.041), i.e. the effect of microplastic exposure on food consumption depends
on the day. Multiple comparisons versus control group (Holm-Sidak method) showed that
significant difference was observed on days 6, 7, 70, 83, 96, 99, 109, 111, 113 and 123, with
decreased food consumption on all these days, except for day 7 when the consumption was
increased (marked with * - see Figure 3-2a). For male mice, however, statistical analysis
showed no statistically significant effect of microplastic exposure on food consumption
(df=1, p=0.846) and no statistically significant interaction between treatment and time
(df=139, p=0.281) (Figure 3-3a).
Individual mice were weighed every week. The results of the two-way repeated measures
ANOVA revealed that there was no statistically significant effect of microplastic exposure on
body weight of female and male mice (Figure 3-2b, Figure 3-3b). There was a statistically
significant effect of time on body weight of female (df=19, p<0.001) and male mice (df=19,
p<0.001) however it was due to the mouse expected growth during puberty phase. There was
no statistically significant effect of microplastic exposure on body weight of female (df=1,
p=0.802) and male mice (df=1, p=0.477) and there was no statistically significant interaction
between treatment (control, microplastic) and time (weeks 1 to 20) in female (df=19,
p=0.429) and male mice (df=19, p=0.978).

52

Microplastic intake and egestion
Food consumption was measured by cage daily - each cage housed three mice. On average,
PE microplastic-treated cages consumed 9.03g/day, i.e. each mouse consumed 3g/day,
assuming equal food consumption. As the microplastic concentration in the mouse food was
approximately 3,960particles/Kg, each mouse was exposed to 11.88 PE particles/day for up
to sixteen weeks followed by a four-week recovery period.
Microplastic egestion was estimated counting the number of PE fluorescent microplastics
present in the feces. There was a significant decrease in the amount of microplastics in the
feces after the sixteenth week, when the exposure was terminated for both female and male
mice (df=19, p<0.001) (Figure 3-2c, Figure 3-3c).

53

a

b

c

54

Figure 3-2: Female food consumption, body weight and microplastic egestion.
a) The presented values are the average food consumption per cage. Statistically significant
difference was observed only on days 6, 7, 70, 83, 96, 99, 109, 111, 113 and 123 (marked
with *). Linear regression for female control group: y = 0.003x + 9.5519 R² = 0.0092, and
female MB group: y = -0.0027x + 8.9928 R² = 0.0098.
b) Individual body weight. The presented values are the mean±SEM. No statistically
significant difference was observed between control and MB-treated groups. Linear
regression for female control group: y = 0.1914x + 19.855 R² = 0.9255, and female MB
group: y = 0.1725x + 19.794 R² = 0.9363.
c) Microplastic egestion. Feces were reconstituted in DI water, filtrated and analyzed under a
fluorescence stereomicroscope. The presented values are the mean±SEM of the amount of
microplastics found in 1g of feces. There was a sharp drop after the sixteenth week, when the
exposure was terminated.

55

a

b

c

56

Figure 3-3: Male food consumption, body weight and microplastic egestion.
a) The presented values are the average food consumption per cage. No statistically
significant difference was observed. Linear regression for male control group: y = -0.0002x +
9.3986 R² = 4E-05, and male MB group: y = -0.0003x + 9.2572 R² = 0.0002.
b) Individual body weight. The presented values are the mean±SEM. No statistically
significant difference was observed between control and MB-treated groups. Linear
regression for male control group: y = 0.2424x + 22.956 R² = 0.9269, and male MB group: y
= 0.201x + 23.671 R² = 0.7581.
c) Microplastic egestion. Feces were reconstituted in DI water, filtrated and analyzed under a
fluorescence stereomicroscope. The presented values are the mean±SEM of the amount of
microplastics found in 1g of feces. There was a sharp drop after the sixteenth week, when the
exposure was terminated.

57

Relative organ weight
Liver, spleen, kidneys, ovaries, testes, small and large intestines were harvested and weighed
immediately after euthanasia. The relative organ weight was calculated by dividing the organ
weight by body weight (g/g). Ovaries from microplastic-treated mice showed a decreased
relative weight compared to the control after four weeks (p≤0.05) and sixteen weeks of
exposure (p≤0.001). A significant decrease was also observed for female liver after the
recovery period (p≤0.05). Spleen, kidneys, testes, small and large intestines showed no
significant change in relative weight throughout the experiment (Figure 3-4).

58

a

b

c

59

Figure 3-4: Relative organ weight.
Liver, spleen, kidneys, ovaries, testes, small and large intestines were harvested and weighed.
Control (Ctrl) and MB-treated groups after a) four weeks of exposure, b) sixteen weeks of
exposure and c) recovery period. The relative organ weight was calculated dividing the organ
weight by the body weight (g/g). The presented values are the mean±SEM. Statistically
significant differences are marked with * (p≤0.05) and ** (p≤0.001).

60

Histological analysis and tissue accumulation
Liver, spleen, kidney, ovary, small and large intestines were used for histopathological
analysis. The H&E staining showed normal cellular morphology with no signs of
inflammation in all tissues analyzed after four and sixteen weeks of exposure and four week
recovery period. There was no evidence of necrosis or neutrophil infiltration in control mice
as well as microplastic-treated mice (Figure 3-5).
No PE microplastic was found in the kidney and ovary, while 1-3 particles were found in the
liver and intestines of both female and male mice and in the spleen of female mice after four
and sixteen weeks of exposure. Some PE microplastics were observed in the liver and
intestines after the recovery period (Table 3-1 and Figure 3-6).

61

Figure 3-5: Histological analysis after microplastic exposure.
Representative photomicrographs of H&E stained liver, spleen, kidney, small and large
intestines and ovary of control and MB-treated mice. Scale bar = 100µm.

62

Table 3-1: MB accumulation in different tissues.
MB particles were observed in female and male mouse tissues after four and sixteen weeks of
exposure and recovery period.
Female
4-week
exposure
16-week
exposure
4-week
recovery

4-week
exposure
16-week
exposure
4-week
recovery

Liver

I B1
I B2
I B3
II B1
II B2
II B3
III B1
III B2
III B3

Male
I B1
I B2
I B3
II B1
II B2
II B3
III B1
III B2
III B3

Spleen

2

Kidney

Ovary

2

Small intest.

Large intest.

1
1

2
1

1
1
2

1

1

2
1

Liver
2

Spleen

Small intest.
2

Large intest.
1

2

3
2

2
1

1

1

1

63

Kidney

Figure 3-6: MB found in mouse tissues.
Fluorescent red PE MB (arrows) in the liver, spleen, small and large intestines. Right column:
20x objective (scale bar = 50 µm), middle and right columns: 40x objective (scale bar = 20
µm).

64

Gene expression analysis
The expression levels of serum amyloid P-component (Apcs), complement factor B (Cfb), Creactive protein (Crp) and cellular communication network factor 1 (Ccn1) were measured in
the liver (Figure 3-7). Cfb and Ccn1 gene expressions were also measured in the large
intestine (Figure 3-8) and spleen (Figure 3-9). Small intestine gene expression analysis was
not included in this study as the qPCR data did not show consistency and reproducibility.
In general, female and male mice showed different gene expression patterns throughout the
experiment. Microplastic-treated mice appeared to show upregulation or downregulation of
some genes compared with the control mice, however this was only statistically significant
(p≤0.05) in females for Cfb and Ccn1 after sixteen weeks of exposure and Apcs after the
recovery period in the liver, and Ccn1 after sixteen weeks of exposure in the large intestine.

65

Relative gene expression levels

a

2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

b

Relative gene expression levels

Apcs
2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Relative gene expression levels

2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Crp

*

Ccn1

*
Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

Apcs

c

Cfb

Cfb

Crp

Ccn1

*

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

Apcs

Cfb

Crp

Ccn1

Figure 3-7: Gene expression in the liver.
Apcs, Cfb, Crp and Ccn1 gene expression profiles of control (Ctrl) and MB-treated female
and male mice after a) four weeks of exposure, b) sixteen weeks of exposure, and c) recovery
period. 18s and Gapdh were used as normalizer genes. Mean ± SEM (n=3). Statistically
significant differences are marked with * (p≤0.05).
66

Relative gene expression levels

a

3.00
2.80
2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

b

Relative gene expression levels

Cfb

Ccn1

3.00
2.80
2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

*

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

c

Relative gene expression levels

Cfb

Ccn1

3.00
2.80
2.60
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

Cfb

Ccn1

Figure 3-8: Gene expression in the large intestine.
Cfb and Crp gene expression of control (Ctrl) and MB-treated female and male mice after a)
four weeks of exposure, b) sixteen weeks of exposure, and c) recovery period. 18s and Gapdh
were used as normalizer genes. Mean ± SEM (n=3). Statistically significant differences are
marked with * (p≤0.05).
67

Relative gene expression levels

a

2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

b

Relative gene expression levels

Cfb

Ccn1

2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

c

Relative gene expression levels

Cfb

Ccn1

2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Ctrl - Female
MB - Female
Ctrl - Male
MB - Male

Cfb

Ccn1

Figure 3-9: Gene expression in the spleen.
Cfb and Crp gene expression of control (Ctrl) and MB-treated female and male mice after a)
four weeks of exposure, b) sixteen weeks of exposure, and c) recovery period. 18s and Gapdh
were used as normalizer genes. Mean ± SEM (n=3).
68

3.5

Discussion

Microplastics have been reported worldwide and a growing body of evidence suggests that
humans are exposed to these environmental pollutants. Although the effects on human health
are unknown, microplastic potential to cause toxic effects has been documented in both
invertebrate and vertebrate species (Lei et al., 2018b), and in in vitro as well as in in vivo
studies (Brown et al., 2001). The estimated daily consumption of microplastics for a North
American diet ranges from 106 to 142 particles depending on age and sex. Inhalation
contributes an additional 97 to 170 particles and these numbers escalate even more if the
source of water is exclusively bottled water (Cox et al., 2019). In the present study, mice
were exposed to approximately 11.88 PE microplastic particles/day for up to sixteen weeks
followed by a four-week recovery period. According to Cox et al. (2019) this value represents
a realistic, but low concentration that humans might be exposed to via diet.
In the present study, PE microplastic exposure did not affect food consumption of male mice
while only minor changes were observed for female mice. No significant difference was
observed in body weight of female and male mice after four weeks and sixteen weeks of
exposure and recovery period. In contrast, a significant loss of body weight was observed in
male mice after PS and PE microplastic exposure as early as three weeks of oral exposure
(Jin et al., 2021; Lu et al., 2018; Park et al., 2020; Xie et al., 2020). Other researchers have
reported no effect on body weight and food consumption after microplastic exposure in male
rats (Rafiee et al., 2018; Xu et al., 2004) and male mice (Araújo and Malafaia, 2021; Deng et
al., 2017; B. Hou et al., 2021; Stock et al., 2019). These contradictory results could be
explained by differences in experimental design, such as microplastic characteristics and
administration method. Most of the studies that reported a significant reduction in body
weight used PS microplastics and an extremely high dose administered by oral gavage.
69

Both female and male mice were able to egest microplastics in feces within the first week of
exposure. A drastic reduction was observed after the sixteenth week, when the exposure was
terminated. Nonetheless, a small number of microplastics was observed in the recovery
period. This finding suggests that PE microbeads might be retained in the gastrointestinal
tract; this could be explained by the microbead properties, such as small size and spherical
shape. Similarly, plastic microspheres were quickly egested by Xenopus tadpoles (Hu et al.,
2016), sea bass larvae (Mazurais et al., 2015) and Daphnia (Rosenkranz et al., 2009) and
were almost completely eliminated after 6 days, 2 days and 4 hours, respectively. Under
visual inspection, the microplastics showed no signs of deterioration or fragmentation, which
confirms that microplastics are bioresistant, i.e. refractory to digestion and dissolution.
In the present study, no significant change was observed in the relative weight of kidney,
spleen, testes and small and large intestines of female and male mice throughout the
experiment. Likewise, no significant change was reported in relative weight of kidney,
spleen, liver, heart, lung, and testes after oral exposure to PS microparticles (Stock et al.,
2019). Although no difference was observed in the liver weight of mouse dams exposed to PS
microplastics during gestation and lactation, their offspring showed an increased liver weight
along with hepatic lipid accumulation and altered metabolite levels, suggesting a
transgenerational effect (Luo et al., 2019a).
The liver’s main function includes the production of blood plasma proteins and bile,
endocytosis-based filtering of the blood and detoxification of substances. Approximately
25% of the incoming blood supply comes from the aorta via the hepatic artery, while 75%
comes from the lower gastrointestinal tract through the portal vein. The arterial and venous
blood flows into the sinusoids, passing along and over the basolateral membrane of rows of
hepatocytes and drains into the hepatic vein and ultimately the inferior vena cava. This
70

organization ensures that hepatocytes are the first cells exposed to everything ingested and
absorbed, either nutrients or toxic substances. Hepatocytes excrete lipids, salts and degraded
proteins from their apical membrane into small channels (canaliculi) that feed bile contents
through the intrahepatic biliary tree. Bile is drained from the liver into the gall bladder for
storage and released into the intestinal lumen during feeding to promote emulsification,
digestion and adsorption of dietary fats as well as the removal of foreign substances and
endogenous waste products (Schulze et al., 2019). The liver also produces a variety of
proteins that play an important role in the innate immunity, such as complement proteins,
opsonins, chemokines and fibrinogen. As some of these proteins rapidly increase in the blood
upon inflammatory stimulation they are called acute-phase reactants (APR) (Zhou et al.,
2016).
As would be expected for a highly vascularized organ that receives blood directly from the
gut and has a pivotal role in the immune system, the liver is thought to be affected by
microplastic exposure. In the present study, 1-2 microplastic particles were found in the liver
of female and male mice after four and sixteen weeks of exposure, as well as after the
recovery period. Interestingly, a significant relative weight decrease was observed in female
mouse liver after the recovery period, whereas no difference was found during the
microplastic exposure period. Increased liver weight in diabetic mice has been associated
with increased triglyceride accumulation and influx of fatty acid into the liver (Zafar and
Naqvi, 2010). However, mice exposed to PS microplastics showed hepatic lipid droplets and
decreased liver relative weight (Deng et al., 2017). Similarly, a significant loss of liver and
epididymal fat relative weight has been reported in mice after PS microplastic exposure (Lu
et al., 2018). Exposure to microplastics has been reported to cause hepatic metabolism
disorder, as indicated by altered metabolomic profile and biomarkers related to amino acid,
bile acid, glucose and lipid metabolism (Deng et al., 2017; Jin et al., 2018a; Lu et al., 2018;
71

Luo et al., 2019a, 2019b; Zheng et al., 2021). In addition, PE microplastic exposure caused
hepatotoxicity in tadpoles, promoting immune cell infiltration, blood vessel dilation, sinus
congestion, hydropic degeneration, steatosis, hypertrophy and hyperplasia along with
hepatocyte nuclear abnormalities (Araújo et al., 2020b). In the present study, however, no
histological difference was observed between control and PE microplastic treated group
throughout the experiment.
Contrary to expectations, the reduction of liver relative weight as well as upregulation of
Apcs gene expression in the liver occurred in female mice only after the recovery period; no
significant difference was observed after four and sixteen weeks of exposure. Although the
mice were allowed a four-week period without exposure, microplastics remained embedded
in the liver and probably exerted a delayed toxic effect. The reason why these effects were
not observed during the exposure period is unknown and intriguing, since SAP is a major
acute-phase reactant in mice. CRP is a major acute-phase reactant in humans, while in mice it
is a trace plasma component and only a minor acute-phase reactant. In contrast, SAP is highly
inducible in mice but only slightly in humans (Roy et al., 2017), showing a 50-fold increase
in mouse serum within 24-48 hours of injection of LPS and returning to the basal level after
32 days (Pepys et al., 1979).
CRP and SAP belong to the pentraxin superfamily, characterized by a cyclic pentameric
structure, sequence homology and calcium-dependent interactions with the ligands (Mold et
al., 2001). CRP and SAP are pattern recognition molecules produced by the liver and secreted
into the blood to interact with pathogens and cell debris and promote their clearance by
binding to IgG receptors on phagocytic cells (Mold et al., 2001). In addition, SAP can
activate the classical complement cascade and interact with monocytes, neutrophils and
macrophages to modulate the immune response and wound healing (Cox et al., 2014; Pilling
72

and Gomer, 2018). SAP influences the migration and spreading of neutrophils, inhibits
monocyte differentiation into fibrocytes and promotes macrophage polarization toward an
anti-inflammatory/anti-fibrotic phenotype (Cox et al., 2014). SAP has been demonstrated to
bind to a variety of microbial polysaccharides and toxins as well as chromatin and DNA in
circulation, enhancing the clearance of autoantigens to prevent autoimmunity (Mold et al.,
2001).
Hepatocytes constitutively produce most of the complement proteins and further elevate their
production after inflammatory stimuli (Zhou et al., 2016). Transcriptome analysis showed
that up to 51 genes were differentially expressed in larval zebrafish injected with PS
microplastics, most notably the alternative complement pathway genes (eg.: cfhl3, cfhl4, cfb
and c9), suggesting that microplastics are integrated in immunological recognition processes
(Veneman et al., 2017). In the present study, female mice showed an increased Cfb gene
expression after sixteen weeks of exposure to PE microplastics. Although microplastics do
not have any corresponding receptors, they need to be tagged by complement proteins and
cleared from the body by phagocytic cells. The alternative complement pathway seems likely
to be affected by microplastic exposure since its activation does not depend on the presence
of antibodies (Veneman et al., 2017). CFB is unique to the alternative complement pathway
and undergoes cleavage when bound to spontaneously occurring C3 (H2O), forming the C3
convertase (C3bBb). During infection, C3b on the microbial surface recruits more CFB to
amplify the concentration of C3 convertase and generate large numbers of C3b. Regardless of
the initiating pathway, up to 90% of the C3b molecules are generated via activation of the
alternative complement pathway (Owen et al., 2018).
Considering that microplastics can potentially cause tissue damage, it is expected that injury
repair mechanisms to be engaged after microplastic exposure. CCN1 has been related to
73

inflammation, injury repair and wound healing (Choi et al., 2015; Kim et al., 2018). CCN1 is
a secreted matricellular protein that is involved in all tissue repair phases: inflammation,
proliferation and maturation. CCN1 binds to specific integrin receptors on distinct cell types
to promote clearance of apoptotic neutrophils by macrophages, induce cell proliferation and
differentiation through Jag1/Notch signaling, and promote angiogenesis and matrix
remodeling. CCN1 is essential to prevent fibrosis during wound healing, as it induces
fibrogenic myofibroblast senescence (Kim et al., 2018). In the present study, the expression
level of Ccn1 gene was measured. After sixteen weeks of exposure to PE microplastics,
female mice showed upregulation of Ccn1in the liver and in the large intestine.
As the intestinal epithelium is constantly exposed to microbes and ingested toxins, the
intestinal cells are prone to damage (Kim et al., 2018). CCN1 plays an essential role in
intestinal tissue repair by promoting proliferation of colonic epithelial cells. Administration
of CCN1 accelerated mucosal restitution of DSS-induced colitis in mice mostly due to
macrophage and fibroblast IL-6 secretion (Choi et al., 2015). In the present study, however,
histological analysis did not reveal any signs of intestinal damage in PE microplastic-treated
female and male mice despite the presence of 1-3 particles in the small and large intestine
after four and sixteen weeks of exposure and recovery period. A reasonable explanation is
that early stages of tissue damage may not be detected in histological analysis.
The spleen is the largest secondary lymphoid organ in the body and is responsible for
filtering the blood of foreign materials and old or damaged erythrocytes and for initiating an
immune response against blood-borne pathogens (Cesta, 2006). The spleen is functionally
and morphologically divided into red pulp and white pulp. The white pulp is essentially made
of lymphatic tissue while the red pulp consists of venous sinuses and splenic cords that filter
the blood (Cesta, 2006). Particles larger than 100-200nm are incapable of crossing the
74

endothelial slit of splenic sinuses and are retained in the red pulp, where they are internalized
by macrophages and slowly destroyed (Cataldi et al., 2017).
In the present study, 1-2 microplastic particles were found in the splenic red pulp and white
pulp of female mice after four weeks and sixteen weeks of exposure, while no particle was
observed after the recovery period. In a recent study, abnormal nuclear and mitochondrial
morphology was observed in female mouse spleens after PE microplastic oral exposure,
along with inhibited dendritic cell maturation and increased helper T cell to cytotoxic T cell
(CD4+/CD8+) ratio (Park et al., 2020). After intraperitoneal injection of PS microplastics in
mice, no microplastics were found in the spleen, nonetheless, the red pulp and the marginal
zone of the white pulp seemed to be activated (Tomazic-Jezic et al., 2001). In the present
study, however, no histopathological changes were observed in female and male mouse
spleen after exposure to PE microplastics.
Histological analysis revealed normal tissue morphology without noticeable pathological
signs in the liver, kidney, spleen, ovary and intestines of female and male mice after four and
sixteen weeks of exposure to PE microplastics and after the recovery period. Similarly
exposure to PS nano/microplastics showed no histological alteration in kidney, liver, spleen,
lung, testis and intestine of mice (Stock et al., 2019) and rats (Walczak et al., 2015). No
histological changes were observed in zebrafish gill, liver, brain, kidney and intestines after
exposure to PE micro fragments (Karami et al., 2017) and PS microbeads (Lei et al., 2018b).
On the other hand, histopathological changes have been reported in other studies. Zebrafish
showed intestinal damage, such as villi and epithelial damage, thinning of the gut wall and
congestive inflammation (Qiao et al., 2019b) as well as cracking of villi and splitting of
enterocytes (Lei et al., 2018b). Mice exposed to PS microplastics showed hepatic ballooning
degeneration (Luo et al., 2019a) and lipid droplets and inflammation in the liver (Deng et al.,
75

2017). Edema, loose glands, small vessels proliferation and inflammatory cell infiltration has
been reported in mouse intestine after PE microplastic exposure (Li et al., 2020).
Histopathological changes were also reported in mouse testes after exposure to PS
microplastics, as shown by disordered spermatogenic cells, abscission and multinucleated
gonocytes (B. Hou et al., 2021; Jin et al., 2021; Xie et al., 2020).
Histological sections were used to investigate microplastic distribution and accumulation in
the liver, spleen, kidney, ovary and small and large intestines. No PE microplastic was found
in the kidney or ovary, while 1-3 particles were found in the liver and intestines of both
female and male mice and in the spleen of female mice after four and sixteen weeks of
exposure. Studies have shown that particles smaller than 150µm may translocate from the gut
to the lymph and circulatory system, however the absorption of these particles is limited
(<0.3%) and only particles smaller than 20µm is expected to penetrate deeply into organs
(Barboza et al., 2018). In the present study, some PE microplastics remained in the liver and
intestines even after the recovery period, confirming plastic bioresistance and biopersistence.
A recent study reported only a few particles in the gut wall and no particles in the liver,
kidney and spleen of mice exposed to PS microplastics (Stock et al., 2019), whereas other
studies showed microplastic accumulation in the mouse gut, liver, kidney and testes (Araújo
and Malafaia, 2021; Deng et al., 2018, 2017; Jin et al., 2021, 2018a).
Studies have shown that microplastic adverse effects might be caused by plastic additives, as
they are not chemically bound to the polymeric chain and can be easily released into the
tissues (Campanale et al., 2020). Plasticizers are well known endocrine disrupting chemicals
and mainly affect female fertility in a range of species, including humans (Rattan et al.,
2017). Phthalates and bisphenol A are used to confer flexibility to plastic products but due to
their toxic effects, they have been replaced by alternative plasticizers. However, due to their
76

similar structure, the alternatives are thought to cause equivalent toxicological effects (Rattan
et al., 2017). In the present study, a significant decrease in ovarian relative weight was
observed after four and sixteen weeks of exposure to PE microplastics. Due to the supplier’s
trade secret, it was not possible to associate this result with microplastic chemical
composition; however it is reasonable to speculate that some plasticizer might have
contributed to ovarian weight decrease. It is worth mentioning that the inherent toxic effects
of microplastics might augment the toxicity of phthalates, as indicated by the aggravated
toxic effects on mice co-exposed to DEHP and PE microplastics compared with exposure to
DEHP or microplastics alone (Deng et al., 2020). Recently, microplastic reproductive
toxicology has been reported in male mice, as indicated by testicular histopathological
changes, inflammation and oxidative stress, low-quality sperm cells and decreased serum
testosterone levels (B. Hou et al., 2021; Jin et al., 2021; Xie et al., 2020).
As might be expected, no significant difference was observed in ovarian weight between PE
microplastic-treated mice and control mice after the recovery period, suggesting that the
effect was reversed. The reversibility of the toxic effects depends on which follicle stage was
targeted. For example, targeting of primordial follicles for an extended period of time leads to
irreversible toxic effects, as the follicular reserve is non-renewable. By contrast, the effects
on more mature follicles, such as antral follicles, can be reversed if the toxicant is removed,
as the early stages of folliculogenesis are unaffected (Hannon and Flaws, 2015). However, in
the present study no difference was observed in ovarian histology between control and
treatment groups after exposure to microplastics. In addition, no PE microplastics were found
in the ovaries, suggesting that the toxic effect is not related to the physical damage caused by
microplastics. Further research is warranted to investigate how microplastics affect ovarian
function and the potential to cause reproductive disorders in females.

77

It is especially important to notice the sex-based differences in the response to microplastic
exposure. Females and males have distinct innate and adaptive immune responses as well as
different immunological responses to self and foreign antigens (Klein and Flanagan, 2016).
Research has shown that female and male mice have similar complement activity until the C3
activation level, while females have lower terminal pathway activation (C6-C9) (Kotimaa et
al., 2016). In the present study, both male and female mice accumulated PE microplastics in
the tissues, but only females appeared to have triggered immune response and tissue repair
mechanisms, as demonstrated by the upregulation of Cfb, Apcs and Ccn1 genes. These results
suggest that the alternative complement pathway and the pattern recognition molecule SAP
are likely to be involved in the immune response against microplastics in females, while it
was not possible to determine the defense mechanisms induced in males.

78

3.6

Conclusions

Emerging evidence shows that humans are exposed to microplastics, yet the effects on human
health have not been clarified. The present study aimed to contribute to the understanding of
the microplastic effects on mammalian health using an original approach. Our experimental
design included a four-week recovery period to assess the reversibility of the adverse effects
and we used both female and male mouse models to compare sex-based differences in the
response to microplastic exposure.
Microplastics were able to translocate across the intestinal epithelial barrier and accumulated
in the liver, spleen and intestinal wall and remained in the tissues even after the recovery
period, confirming microplastic biopersistence and bioresistance. Although tissue
accumulation was reported in both female and male mice, females seemed to be more
affected. Females showed increased expression levels of genes related to inflammation (Cfb,
Apcs) and tissue injury repair (Ccn1) as well as reduction in ovarian weight. Some of these
effects appeared to be reversible, as the ovarian weight and Cfb and Ccn1 gene expression
returned to normal levels after the recovery period, while the effect on Apcs gene expression
seemed to be delayed and noticeable only in the recovery period.
Overall, longer exposure to microplastics caused more pronounced effects than short
exposure, and some of these effects were reversed after the depuration period. Microplastics
affected the expression of genes that coordinate a broad range of biological functions, such as
immune system activation, wound healing and tissue repair. These results suggest that
microplastics might have a systemic impact and the severity of effects depends on the
exposure time. Further research is warranted to better understand the potential long-term
health hazards and the sex-based differences in the biological response against microplastics.

79

4-

Conclusions

Microplastics have become one of the most concerning environmental problems in recent
years. Although a growing body of evidence suggest that humans are exposed, the fate and
effects of microplastics on human body are controversial and unclear. Many studies have
used invertebrate and vertebrate models to evaluate the potential adverse effects on wildlife
and humans.
The present research project confirmed that only a small fraction of microplastics cross the
gut barrier and accumulate in the tissues; the majority of them are more likely to be
eliminated via feces. Either microbeads or microfibers had a minor effect on food
consumption and no effect on body weight. At environmental concentrations, microplastics
do not seem to pose a serious health risk during short-term exposure, however longer
exposure seems to be more harmful. Microplastics affected more females than males, and the
effects were time-dependent. Some effects worsened with longer exposure (e.g. ovarian
weight), others reversed after a depuration period (e.g. Cfb and Ccn1 gene expression,
ovarian weight) and others have a delayed manifestation (e.g. Apcs gene expression, liver
weight). Only a few microplastics were found in the tissues, nevertheless they remained there
even after the recovery period, confirming plastic bioresistance and biopersistence. The genes
analyzed in this study are involved in a variety of biological functions, such as immune
system activation, wound healing and tissue repair, which suggests that microplastics might
have a systemic impact.
The microplastic problem has just started; even if plastic production were completely
stopped, the existing plastic in the landfills and environment would continue to degrade into
microplastics and nanoplastics and human exposure to these particles would keep increasing.
We acknowledge that the mouse exposure to microplastics performed herein does not
80

completely represent the human exposure, as it did not take into consideration the
nano/microplastic diversity and the cumulative effects of a lifetime of exposure. The present
study, however, described for the first time the sex-based differences in the mouse response
to microplastics, showing that females are more affected than males. Furthermore, exposure
time plays an important role on the outcome, as some effects were time-dependent, with
longer exposure causing worse effects, others reversed after a period without exposure, and
others showed a delayed manifestation.

81

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Appendices
Appendix 1: Histological preparations
1.

Tissues were fixed in 10% neutral buffered formalin for at least 24 hours at room

temperature.
Reagent
Quantity
Formaldehyde 37-40%
100mL
Distilled water
900mL
Sodium phosphate monobasic 4g
Sodium phosphate dibasic
6.5g
Adjust the pH to 6.8, if necessary.

2.

After trimming, tissues were dehydrated as follows:

70% ethanol: 1 hour
80% ethanol: 1 hour
95% ethanol: 1 hour
100% ethanol I: 1 hour
100% ethanol II: 1 hour
Xylene I: 1 hour
Xylene II: 1 hour
3.

Tissues were infiltrated with paraffin wax (Fisher Scientific TissuePrep®) in stainless

steel base molds on a hot plate at 95°C. The tissues were infiltrated with paraffin twice, for 1
hour each. After infiltration, samples were embedded in paraffin and immediately placed on
ice to for fast cooling. Embedded samples were kept at room temperature until sectioning.

92

4.

Samples were sectioned at 5µm thickness using a Shandon AS325 microtome.

Sections were 10-60µm apart in order to sample different depths of the tissue as follows:
Tissue
Spleen
Ovary
Liver
Kidney
Small intestine
Large intestine

5.

µm apart
10
10
50
60
50
25

The sections were floated in waterbath at 42°C (Fisher TissuePrepTM flotation bath,

model 134) and placed on glass slides.
6.

The slides were air dried and placed on a hot plate for 30 minutes at 60°C to allow the

sections to adhere to the slide.
7.

Slides were dewaxed and hydrated as follows:

Xylene I: 10 minutes
Xylene II: 10 minutes
100% Ethanol I: 5 minutes
100% Ethanol II: 5 minutes
95% Ethanol: 2 minutes
70% Ethanol: 2 minutes
Washing in running tap water: 2 minutes
8.

Slides were stained as follows:

Harris hematoxylin: 2 minutes
93

Washing in running tap water: 3 minutes
Acid-alcohol solution: 10 dips
Washing in running tap water: 1 minute
95% ethanol: 30 seconds
Eosin Y: 30 seconds
95% ethanol: 2 minutes
100% ethanol I: 1 minute
100% ethanol II: 1 minute
Xylene: 5 minutes
Cover slipping using Permount® mounting media.

94

Appendix 2: Total RNA extraction protocol (TRIzol method)
The protocol was obtained from Invitrogen User Guide.
1.

1 mL of TRIzol® Reagent (Ambion, Life Technologies, USA) was added per 50–100

mg of tissue and the sample was homogenized using a tissue homogenizer (Tissue-Tearor,
Biospec Products). The sample volume should not exceed 10% of the volume of TRIzol®
Reagent used for lysis.
2.

The homogenized sample was incubated for 5 minutes at room temperature to permit

complete dissociation of the nucleoproteins complex.
3.

0.2 mL of chloroform (Sigma-Aldrich) was added per 1 mL of TRIzol® Reagent used

for lysis and sample was incubated for 2-3 minutes at room temperature.
4.

Sample was centrifuged for 15 minutes at 12,000 x g at 4°C (Eppendorf refrigerated

centrifuge 5417R). The mixture separated into a lower red phenol-chloroform, and
interphase, and a colorless upper aqueous phase.
5.

The aqueous phase containing the RNA was transferred to a new tube by angling the

tube at 45° and pipetting the solution out carefully to avoid transferring any of the interphase
or organic layer into the pipette.
6.

0.5 mL of isopropanol (Fisher Scientific) was added to the aqueous phase, per 1 mL

of TRIzol® Reagent used for lysis and sample was incubated for 10 minutes at room
temperature.
7.

Sample was centrifuged for 10 minutes at 12,000 x g at 4°C.

8.

Total RNA precipitate formed a white gel-like pellet at the bottom of the tube. The

supernatant was discarded.
95

9.

The pellet was resuspended in 1 mL of 75% ethanol (BDH®) per 1 mL of TRIzol®

Reagent used for lysis. The sample was vortexed briefly.
10.

Sample was centrifuged for 5 minutes at 7500 x g at 4°C and the supernatant was

discarded.
11.

The RNA pellet was air dried for 5–10 minutes at room temperature.

12.

The pellet was resuspended in 20 - 100 µL of nuclease-free water (Invitrogen) by

pipetting up and down.
13.

Sample was incubated in a water bath at 55–60°C for 10–15 minutes.

14.

The RNA yield was measured using a nanophotometer (NP80, Implen) using

nuclease-free water as a blank. Samples with a 260/280 ratio close to 2.0 and concentration
above 500ng/µL were considered acceptable for downstream applications.
RNA samples were stored at -80°C until reverse transcription.

96

Appendix 3: Reverse transcription (cDNA synthesis)

The reverse transcription protocol was obtained from Thermo Fisher Scientific user guide
with addition of a pre-treatment with nuclease digest. All reagents were thawed on ice and
homogenized through vortexing and spinning. Reverse transcription was performed using the
High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher
Scientific, Lithuania).

1.

RNA samples were pre-treated with nuclease digest as follows:
Reagent

Volume (µL)

RNase free water

5.0*

RNA sample (2µg AKA 2000ng)

3.0*

10x DNase I buffer

1.0

DNase I

1.0

Total volume

10

*Note: The volume of RNA sample was calculated to correspond to 2µg depending on the
extraction concentration. Water was added to complete the total volume (10µL).

2.

Samples were incubated for 15 minutes at room temperature.

3.

1µL of 25mM EDTA was added to each sample.

4.

After homogenization, samples were incubated for 10 minutes at 65°C using a thermal

cycler (Techne Progene).
5.

The 2X Reverse Transcription master mix was prepared as follows:

97

Reagent

Volume (µL)

10x RT buffer

2.0

25x dNTP mix

0.8

10x random primer

2.0

Multiscribe reverse transcriptase

1.0

RNase inhibitor

1.0

Nuclease-free water

3.2

Total

10.0

6.

10 µL of 2X RT master mix was pipetted into each 0.2mL PCR tube.

7.

10 µL of RNA sample from the nuclease digest was added to each tube, pipetting up

and down two times to mix.
8.

Samples were vortexed and centrifuged to spin down the contents and to eliminate

any air bubbles.
9.

Samples were loaded into the thermal cycler programmed for the following

conditions:

Temperature
Time

10.

Step 1
25°C
10 min

Step 2
37°C
120 min

Step 3
85°C
5 min

Step 4
4°C
hold

The cDNA yield was measured using a NP80 NanoPhotometer (Implen, Munich,

Germany) using a solution of nuclease-free water (15µL) and 10x RT buffer (2µL) as a
blank. Samples with a 260/280 ratio between 1.6-1.8 and concentration above 500ng/µL were
considered acceptable for downstream application.
11.

cDNA samples were diluted (1:80), aliquoted and stored at -20°C until qPCR.

98

Appendix 4: TaqMan RT-PCR
This protocol was adapted from Thermo Fisher Scientific and Corbett Rotor-Gene 6000 user
guides. All test samples (expression assay), no template control (NTC), and normalizer genes
(18S ribosomal RNA and GADPH) were run in duplicate. Statistical analysis was performed
using the average from 3 independent runs.

1.

The efficiency of the reaction was determined for both target and reference genes

before running the test samples. cDNA from treated and control samples were pooled
together to make a 2-fold serial dilution across 5 points. This serial dilution was used to
stablish a standard curve (concentration plotted against the Ct values). The efficiency was
calculated as follows:
Efficiency = 10 (-1/slope) – 1
Efficiency between 90 – 105% and R2 > 0.985 were considered acceptable.

2.

All reagents were thawed on ice. The reagents were resuspended by vortexing then

briefly centrifuged. The reaction mix was prepared as follows:
Reagent
Volume (µL)
20x Taqman Expression Assay
1.0
2x Taqman Gene Expression Master Mix 10.0
cDNA template*
4.0
RNase free water
5.0
Total volume
20.0
*Note: The volume of cDNA template was calculated to correspond to approximately 100ng,
not exceeding 20% of the PCR reaction mix. Water was added to complete the total volume
(20µL).
3.

Reactions were prepared in 0.1mL strip tubes placed into a precooled loading block.
99

4.

Corbett Rotor-Gene 6000 was set as follows:
Stage
Temperature Time
Hold
95°C
10 minutes
Cycle (x50) 95°C
15 seconds
58 - 60°C
60 seconds

Channel set up:
Name = green
Source = 470nm
Detection = 510nm
Gain = 10

The “Perform Optimization Before 1st Acquisition” function was selected.
5.

The Ct values were calculated by importing the appropriate standard curve and

following slope correction by the Rotor-Gene software.
6.

Relative quantification of mRNA levels was performed according to 2-△△Ct method

(Livak and Schmittgen, 2001). Briefly, △Ct was calculated by subtracting the geometric
mean Ct of the normalizer genes (18s and Gapdh) from the target gene Ct. Then, △△Ct was
calculated by subtracting the control group average △Ct from each individual △Ct. The fold
change was calculated by the formula 2-△△Ct for each mouse.

100