Abstract

   Microplastics are a widespread environmental hazard and their impact on
   human health has become a growing concern in last years. Recently, the
   potential role of microplastics in the development of various diseases,
   including diabetes, has been highlighted. Therefore, the aim of this
   study was to investigate the effects of PET microplastics on the
   pancreas using immature pigs as a model organism. We analyzed the
   global transcriptomic profile of the pancreas by RNA-Seq in piglets
   treated with either a low (0.1 g/day) or a high dose (1 g/day) of PET
   microplastics for 4 weeks. The analysis revealed a dose-dependent
   effect of PET microplastics on gene expression. A low dose changed the
   expression of one gene, while a high dose affected the expression of 86
   genes. The differentially expressed genes, including immune cell
   markers, cytokines and chemokines, may activate the immune system in
   the pancreas in a way that is characteristic of the pathogenesis of
   diabetes. In addition, PET microplastics induced oxidative stress in
   the pancreas. These above imply that oral exposure to PET microplastics
   could be a new risk factor for the development of diabetes.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12864-025-11760-1.

   Keywords: Diabetes, Pig, Pancreas, Environmental hazard, Cytokines,
   Chemokines, Risk factor

Introduction

   Microplastics (MPs) are plastic particles smaller than 5 mm, which can
   be either produced intentionally (primary) or generated by the
   degradation of larger plastic parts (secondary) [[34]1]. Their
   ubiquitous presence in water, food, and air has raised great concern
   about their impact on human health in recent years. Recent studies have
   confirmed the presence of MPs in human tissues including the placenta,
   testicles, liver, brain and blood [[35]2–[36]4]. Furthermore,
   investigations have demonstrated that microplastic particles can
   influence various physiological processes, such as activation of the
   immune response, triggering inflammation and inducing oxidative stress
   [[37]5–[38]7]. There is evidence that MPs may contribute to metabolic
   diseases associated with insulin resistance by dysregulating the gut
   microbiota [[39]8] transferring harmful microorganisms and metabolites
   to the liver via the gut-liver axis [[40]9] or affecting blood glucose
   levels [[41]10]. In addition, we have previously shown that PET
   microplastics alter the expression of miRNA in serum-derived
   extracellular vesicles (EVs), which are associated with insulin
   resistance and the development of type II diabetes [[42]11].

   Diabetes mellitus (DM) is a long-term metabolic disease characterized
   by constantly elevated blood glucose levels, either due to insufficient
   insulin production or the body’s inability to effectively use the
   insulin it produces [[43]12]. This disease affects people of all ages,
   genders, and regions, making it one of the leading causes of mortality
   and morbidity worldwide. Therefore, diabetes is considered one of the
   fastest growing health challenges of the twenty-first century and
   represents a significant financial burden for healthcare systems
   worldwide. An estimated 10.5% of the world’s population currently live
   with this disease [[44]13]. Based on their pathogenesis, two main types
   of diabetes can be distinguished: type 1 diabetes (T1D) and type 2
   diabetes (T2D) [[45]14].

   Dysregulation of insulin synthesis is closely related to the
   pathophysiology of both T1D and T2D [[46]15, [47]16]. Insulin is known
   to be a central player in the regulation of metabolism, particularly in
   the maintenance of glucose homeostasis [[48]17]. There is also evidence
   that dysregulation of insulin signaling is linked to the development of
   cognitive impairment and dementia [[49]18]. Studies in animal models
   have shown that insulin resistance and impaired insulin signaling
   exacerbate neurodegenerative processes in Alzheimer’s disease by
   affecting amyloid-beta accumulation and neuronal deficits [[50]19,
   [51]20]. Therefore, understanding the mechanisms that impair insulin
   synthesis is essential to describe the processes underlying many
   diseases.

   In light of our previous findings and the existing literature, we
   conducted this study to determine the impact of PET microplastics on
   the global transcriptomic profile and oxidative status of the pancreas,
   using immature piglets as a model organism. The pig is considered a
   valuable model for the study of diabetes due to its physiological
   similarities to humans, particularly in terms of pancreatic structure,
   metabolic function, and pathophysiological responses [[52]21]. These
   similarities make pigs particularly relevant for investigating complex
   diseases such as diabetes and metabolic syndrome, which involve insulin
   resistance. Moreover, the metabolic pathways involved in insulin and
   glucose regulation in pigs are comparable to those in humans. In this
   study, we used two doses of PET microplastics – a low dose of 0.1
   g/day/animal and a high dose of 1 g/day/animal. The doses were selected
   on the basis of estimates that humans consume an average of 0.1 to 5 g
   of microplastics per week [[53]22]. However, determining the precise
   intake of microplastics is a challenge as exposure levels varies
   depending on individual lifestyle and environmental factors.
   Nevertheless, the doses used in this study – approximately 0.7 and 7 g
   per week – appear to reflect a realistic level of human exposure based
   on average intake estimates. Furthermore, it is important to emphasize
   that the average exposure of infants to PET is significantly higher
   than that of adults [[54]23], and the main objective of our study was
   to investigate the effects on a young organism.

Results

The effect of PET microplastics on differential gene expression in pancreas

   Treatment of piglets with a low dose of PET microplastics revealed only
   one differentially expressed gene (DEG) in the pancreas – actin gamma 2
   (ACTG2), which was downregulated (Fig. [55]1A). In contrast, treatment
   with a high dose of PET microplastics resulted in the identification of
   86 DEGs, with 7 genes being downregulated and 79 upregulated (Figs.
   [56]1B, [57]2A). Gene Ontology (GO) annotation of biological processes
   (BP) included 533 terms, while 174 terms were classified under
   molecular functions (MF) and 94 under cellular components (CC).
   According to GO analysis, the DEGs involved in the pancreatic response
   to PET microplastics were associated with processes such as immune
   response (C–C motif chemokine receptor 7 (CCR7), C–C motif chemokine
   ligand 17 (CCL17), tumor necrosis factor superfamily member 14
   (TNFSF14), lymphocyte activation family member X1 (LAX1), interferon
   regulatory factor 8 (IRF8), C–C motif chemokine ligand 22 (CCL22),
   C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand 10
   (CXCL10), lymphotoxin beta (LTB), complement C3 (C3),
   ENSSSCG00000056654, ENSSSCG00000036445)), chemotaxis (CCR7, CCL17,
   CCL22, CXCL9, CXCL10, ENSSSCG00000036445, dedicator of cytokinesis 2
   (DOCK2)), cytokine activity (interleukin 12B (IL12B), CCL17, CCL22,
   LTB, CXCL9, CXCL10, ENSSSCG00000036445), and B cell differentiation
   (recombination activating 1 (RAG1), caspase recruitment domain family
   member 11 (CARD11), interleukin 2 receptor subunit gamma (IL2RG),
   membrane spanning 4-domains A1 (MS4 A1), lymphoblastic leukemia
   associated hematopoiesis regulator 1 (LYL1)) (Fig. [58]2B). KEGG
   pathway analysis indicated that the identified DEGs were involved in
   cytokine − cytokine receptor interaction (IL12B, CCR7, CCL17, TNFSF14,
   tumor necrosis factor receptor superfamily member 18 (TNFRSF18), CCL22,
   LTB, CXCL9, CXCL10, ENSSSCG00000036445, IL2RG), Th1 and Th2 cell
   differentiation (IL12B, runt-related transcription factor 3 (RUNX3),
   CD3 delta subunit (CD3D), IL2RG, Janus kinase 3 (JAK3)), T cell
   receptor signaling pathway (CARD11, phosphoinositide-3-kinase catalytic
   subunit delta (PIK3 CD), CD3D, lymphocyte cytosolic protein 2 (LCP2)),
   Toll-like receptor signaling pathway (IL12B, PIK3 CD, CXCL9, CXCL10),
   NF-kappa B signaling pathway (TNFSF14, CARD11, LTB, TNF receptor
   associated factor 1 (TRAF1)), and the JAK-STAT signaling pathway
   (IL12B, PIK3 CD, IL2RG, JAK3) (Fig. [59]2C). The list of the top 20
   DEGs, along with detailed results of the DEGs, GO and KEGG analyses can
   be found in Tables S1, S2, S3, S4.

Fig. 1.

   [60]Fig. 1
   [61]Open in a new tab

   Volcano plots illustrating changes in gene expression profiles in
   porcine pancreas under the influence of PET microplastics. Plot A shows
   gene expression changes following a low dose of PET microplastics
   treatment, plot B shows changes after a high dose, and plot C compares
   gene expression between high and low doses of PET microplastics. The
   X-axis displays the logarithmic fold changes in expression (log2 FC),
   while the Y-axis represents the -log10 transformed adjusted p-values
   (q-values). A horizontal dashed line marks the significance threshold
   for the adjusted p-value (0.05), and vertical dashed lines denote the
   fold change cut-off (absolute value of log2 FC > 1.5). Each dot
   represents a gene’s expression level, with red dots indicating
   significantly overexpressed genes and green dots indicating
   significantly underexpressed genes (both with an adjusted p-value
   < 0.05). Grey dots represent genes with non-significant changes in
   expression

Fig. 2.

   [62]Fig. 2
   [63]Open in a new tab

   Gene expression analysis in porcine pancreas after exposure to high
   dose of PET microplastics. A Heatmap showing the expression levels of
   differentially expressed genes in control and high dose PET
   microplastics-treated groups. Rows represent individual genes, and
   columns represent samples from each treatment group (control vs. high
   dose). Colors indicate the level of gene expression, with red
   representing upregulated genes, green representing downregulated genes,
   and black indicating no significant change in expression. The
   dendrogram on the left clusters genes based on similarity in expression
   patterns. B Sankey diagram illustrating enriched biological processes
   and pathways associated with the differentially expressed genes in the
   high dose of PET microplastics-treated group. This diagram shows how
   specific genes (on the right) are linked to various biological
   processes (on the left). Each pathway is represented by a different
   color, and lines connect genes to the biological processes in which
   they are involved. C Bubble plot showing pathway enrichment analysis
   for the genes affected by high dose of PET microplastics. Each pathway
   is listed on the Y-axis, and the X-axis displays the rich factor, which
   represents the ratio of differentially expressed genes associated with
   each pathway to the total number of genes in that pathway. The color of
   each bubble indicates the statistical significance (p-value) of the
   enrichment, with a gradient from red (less significant) to blue (more
   significant). The size of the bubbles represents the number of genes
   associated with each pathway

Comparison between the low and high doses of PET microplastics

   The analysis identified 105 differentially expressed genes (DEGs) in
   the pancreas of gilts administered a high dose of PET microplastics
   (MPs), compared to those given a low dose. Of these, 7 genes were
   downregulated and 98 were upregulated (Figs. [64]1C, [65]3A). The Gene
   Ontology (GO) annotation of biological processes (BP) included 663
   terms, while 195 terms were classified under molecular functions (MF)
   and 105 under cellular components (CC). Most of the processes were
   associated with immune system activation, including immune response
   (CCL17, C–C motif chemokine receptor 2 (CCR2), TNFSF14, IRF8, membrane
   associated ring-CH-type finger 1 (MARCHF1), swine leukocyte
   antigen-DQB1 (SLA-DQB1), CXCL10, C–C motif chemokine ligand 19 (CCL19),
   complement C7 (C7), C3), follicular B cell differentiation (IRF8, SPI-1
   proto-oncogene (SPI1)), inflammatory response (tetraspanin 2 (TSPAN2),
   CCL17, CCR2, complement C4 A (C4 A), CXCL10, CCL19, C3), and T cell
   costimulation (TNFSF14, CARD11, CD5 molecule (CD5)) (Fig. [66]3B). KEGG
   pathway analysis revealed that the DEGs were involved in the chemokine
   signaling pathway (FGR proto-oncogene, SRC family tyrosine kinase
   (FGR), phospholipase C beta 2 (PLCB2), CCL17, CCR2, PIK3 CD, CXCL10,
   CCL19, phosphatidylinositol-3,4,5-trisphosphate-dependent RAC exchange
   factor 1 (PREX1), JAK3), cytokine − cytokine receptor interaction
   (CCL17, CCR2, TNFSF14, CXCL10, interleukin 10 receptor subunit alpha
   (IL10RA), CCL19, IL2RG, ENSSSCG00000009633, colony stimulating factor 1
   (CSF1)), Th1 and Th2 cell differentiation (RUNX3, SLA-DQB1, CD3D,
   IL2RG, JAK3), and the PI3 K − Akt signaling pathway (PIK3 CD, tenascin
   C (TNC), integrin subunit alpha 8 (ITGA8), IL2RG, integrin subunit
   alpha 4 (ITGA4), JAK3, CSF1, integrin subunit beta 7 (ITGB7)) (Fig.
   [67]3C). The list of the top 20 DEGs, along with detailed results of
   the DEGs, GO and KEGG analyses can be found in Tables S1, S2, S3, S4.

Fig. 3.

   [68]Fig. 3
   [69]Open in a new tab

   Gene expression analysis in porcine pancreas comparing the effects of
   high and low doses of PET microplastics. A Heatmap displaying the
   expression levels of differentially expressed genes in the pancreas for
   both high dose and low dose PET microplastics treatment groups. Rows
   represent individual genes, and columns represent samples from each
   treatment group (low dose vs. high dose). Colors indicate the level of
   gene expression, with red representing upregulated genes, green
   indicating downregulated genes, and black showing genes with no
   significant change in expression. The dendrogram on the left clusters
   genes based on similarity in their expression patterns across both
   doses. B Sankey diagram illustrating enriched biological processes and
   pathways linked to the differentially expressed genes when comparing
   high and low doses of PET microplastics. This diagram shows how
   specific genes (on the right) connect to biological processes (on the
   left), such. Each pathway is represented by a different color, with
   lines connecting genes to the biological processes they influence,
   highlighting differences in pathway involvement between the two doses.
   C Bubble plot representing pathway enrichment analysis for genes with
   differential expression between high dose and low dose PET
   microplastics exposure. Pathways are listed on the Y-axis, while the
   X-axis shows the "rich factor, "which reflects the ratio of
   differentially expressed genes in each pathway to the total number of
   genes in that pathway. The color gradient of each bubble indicates the
   statistical significance (p-value) of the enrichment, with a range from
   red (less significant) to blue (more significant). The size of the
   bubbles represents the number of genes associated with each pathway

Real-time PCR

   The analysis revealed that the mRNA abundance of CCL22, CXCL10, CXCL9,
   IL12B, IL2R, TNFSF14, IRF8, JAK3, was upregulated in the pancreas of
   piglets treated with a high dose of PET microplastics. Notably, the
   expression patterns observed for these DEGs were consisted with results
   obtained from RNA-Seq analysis (Supplemental Fig. [70]1).

Oxidative stress markers

   Exposure of piglets to a low dose of PET microplastics led to an
   increase in pancreatic malondialdehyde (MDA) levels, an indicator of
   lipid peroxidation. Moreover, the low dose of PET microplastics reduced
   the activity of the following antioxidants in the pancreas: catalase
   (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST),
   while it increased glutathione peroxidase (GPx) activity. In contrast,
   significant changes following treatment with a high dose of PET
   microplastics were observed only in SOD activity, which was decreased
   (Fig. [71]4).

Fig. 4.

   [72]Fig. 4
   [73]Open in a new tab

   Activity level of oxidative stress marker (MDA) and antioxidant enzymes
   (GPx, SOD, CAT, GST) in the pancreas of the experimental groups that
   received a low dose (LD) and a high dose (HD) of PET microplastics
   compared to the control (C). Significant differences between the groups
   are indicated by asterisks (*p < 0.05, **p < 0.01)

Discussion

   This study provides evidence that PET microplastics may pose a
   potential threat by altering pancreatic physiology. We demonstrated
   that PET microplastics alter the transcriptome profile and markers of
   oxidative stress in the pancreas. Notably, the differentially regulated
   genes, particularly at the high dose, are involved in the regulation of
   cytokine and chemokine activity, lymphocyte differentiation, TLR4 and
   NF-kappa B signaling pathways. Additionally, our findings indicate that
   PET microplastics increased the expression of IL-12β in the pancreas, a
   cytokine previously established as crucial role in the development and
   pathogenesis of autoimmune diseases by driving the recruitment of
   inflammatory cells [[74]24]. In non-obese diabetic (NOD) mice,
   increased expression of this cytokine was found in islet cells, in
   parallel with the destruction of β-cells [[75]25]. Another study in NOD
   mice indicated that IL-12 plays a significant role in the transition
   from non-destructive to destructive insulitis [[76]26, [77]27].
   Elevated levels of IL-12 were also associated with increased insulin
   resistance and impaired glucose tolerance [[78]28].

   Our study showed that PET microplastics upregulated the expression of
   various members of the TNF superfamily such as TNFSF3 (LTβ), TNFSF14
   (LIGHT) and TNFRSF18 (GITRL). These pro-inflammatory cytokines affect
   both the immune system and β-cell function by binding to their
   corresponding receptors (TNFRSF) [[79]29]. Evidence suggests that
   TNFSF3 receptors, which are predominantly expressed on immune cells and
   lymphoid stromal cells, contribute to the ectopic formation of tertiary
   lymphoid organs (TLOs), which trigger chronic inflammation and
   autoimmune responses [[80]30]. Such processes facilitate persistent
   immune activation and β-cell destruction, thereby promoting the
   development and progression of type 1 diabetes [[81]31, [82]32].
   Additionally, TNFSF14 has been linked to immune-mediated destruction of
   β-cells in diabetic mice [[83]33], and elevated levels of this cytokine
   have been observed in patients with type 2 diabetes [[84]34]. TNFRSF18,
   in turn, exacerbates autoimmune diabetes by selectively activating
   aggressive T cells while suppressing Tregs. It has been shown that
   blocking TNFRSF18 expression can prevent the onset of diabetes
   [[85]35]. Collectively, these findings suggest that PET microplastics
   may increase the risk of β-cell damage and contribute to the
   development of diabetes by upregulating key cytokines such as IL-12β,
   TNFSF3, TNFSF14 and TNFRSF18.

   Exposure to a high dose of PET microplastics was found to increase the
   expression of several chemokines in the pancreas, including CXCL10,
   CXCL9, CCL17 and CCL22. Chemokines are specific cytokines that mediate
   the chemotaxis of immune cells by binding to surface receptors.
   Elevated levels of CXCL10 have been found in the serum of patients with
   newly diagnosed type 1 or type 2 diabetes and in patients at high risk
   of developing the disease [[86]36–[87]39]. Further studies conducted in
   mouse models have shown that CXCL10 is overexpressed in the pancreatic
   islets even prior detectable onset of insulitis [[88]40, [89]41].
   Moreover, CXCL9, in conjunction with CXCL10, also changes the mass and
   function of β-cells, and deletion of CXCR3 in mice delays the onset of
   type 1 diabetes [[90]42]. Thus, PET microplastics may activate immune
   response in the pancreas and increase the risk of type 1 diabetes.
   Interestingly, we also observed an increased expression of CCL17 and
   CCL22, which act via the CCR4 receptor, which is predominantly
   localized on Tregs. These chemokines are known to protect pancreatic
   β-cells from autoimmune attack in type 1 diabetes. CCL17 and CCL22
   recruit Tregs to the islets, reducing autoreactive CD8 + T cells and
   mitigating β-cell destruction [[91]43]. The simultaneous upregulation
   of chemokines that promote (CXCL10, CXCL9) and protect against (CCL17,
   CCL22) type 1 diabetes suggests a complex immune response to PET
   microplastics. Initially, microplastics may promote β-cell destruction,
   but tissue injury could trigger a feedback mechanism that produces
   protective chemokines, such as CCL17 and CCL22, to limit further
   damage.

   The current research provides evidence that PET microplastics may
   modify the immune response by upregulating the expression of markers
   associated with T cell infiltration such as CD3D, CD6, RUNX3 and LCP2
   [[92]44, [93]45]. Furthermore, PET microplastics upregulated the
   expression of JAK3, a key enzyme in the JAK-STAT signaling pathway
   involved in regulating immune cell activity. JAK3, in combination with
   the IL-2 receptor, which was also upregulated in this study, plays a
   critical role in T cell development and the progression of type 1
   diabetes [[94]46]. Inhibition of JAK3 has been shown to protect against
   diabetes onset in mouse models, highlighting its importance in diabetes
   pathogenesis [[95]47]. Though T cells are largely responsible for
   β-cell destruction in type 1 diabetes, there is growing evidence that B
   cells also play a role in the pathogenesis of the disease. Notably,
   there is evidence that depleting B cells delays the progression of type
   1 diabetes in newly diagnosed patients [[96]48]. In our study, PET
   microplastics increased the expression of markers important for B cell
   activation [[97]49] such as CD20 (MS4 A1), PAX5 and BLK, what suggest
   that PET microplastics may accelerate autoimmune processes leading to
   β-cell dysfunction. The upregulation of both T cell and B cell markers
   following exposure to PET microplastics implicates these particles in
   immune dysregulation associated with type 1 diabetes.

   Consistent with these immune-related changes, the present study showed
   an increased expression of interferon regulatory factors (IRFs),
   particularly IRF4 and IRF8. It is worth noting that both factors play a
   crucial role in the pathogenesis of type 1 diabetes by modulating
   interactions between β-cells and immune cells [[98]32]. Overactivation
   of IRF4 has been associated with increased secretion of diabetogenic
   cytokines by CD4 + T cells, promoting the development of type 1
   diabetes [[99]50, [100]51]. IRF8, in turn, is essential for the
   maturation of dendritic cells (DCs) and the production of type I
   interferons, which may exacerbate autoimmune attacks on β-cells
   [[101]32, [102]52].

   In addition to transcriptomic effects, the colorimetric analysis showed
   that a low dose of PET microplastics had a stronger effect on the
   oxidative status of the pancreas than a high dose. Oxidative stress can
   be assessed by measuring the level of antioxidants and the
   concentration of substances derived from the action of oxygen free
   radicals on biological molecules, such as MDA. Our study demonstrated
   that a low dose of PET microplastics increased the level of MDA,
   indicating heightened oxidative stress. Concurrently, we observed a
   decrease in the activity of key antioxidant enzymes– CAT, SOD, GST. The
   reduced capacity of the antioxidant defense system plays an important
   role in the development of pancreatic β-cell dysfunction and is
   characteristic of early acute pancreatitis [[103]53, [104]54]. In
   addition, the elevated MDA level indicates increased lipid
   peroxidation, reflecting oxidative damage to pancreatic cells by oxygen
   free radicals [[105]55]. The fact that the low dose had only a minimal
   effect on the transcriptomic profile of the pancreas, while it had a
   significant effect on oxidative status, which suggests that both low
   and high doses of PET microplastics can affect pancreatic function,
   though different mechanisms. While the high dose may induce more
   pronounced transcriptional changes detectable by RNA-Seq, the low dose
   appears to induce oxidative stress through mechanisms that do not
   require extensive changes in gene expression or there are already
   changes at other molecular levels. Furthermore, the lack of correlation
   between gene expression and enzyme activity might be due to many
   factors, including differences in mRNA and protein stability or
   transcriptional and post-transcriptional regulation [[106]56, [107]57].
   In addition, functional feedback loops may also play a role, in which
   high concentrations of antioxidant enzymes suppress further gene
   expression or high mRNA levels do not necessarily lead to increased
   protein activity due to regulatory mechanisms [[108]58].

   In conclusion, our study shows that orally ingested PET microplastics
   disrupt pancreatic gene expression and oxidative balance in a
   dose-dependent manner. The upregulation of immune-related genes and the
   impairment of antioxidant defenses may indicate inflammation-induced
   damage to β-cells and a possible role in the development of diabetes.
   These results emphasize the biological effects of PET microplastics and
   the urgent need for further research into their contribution to
   metabolic diseases.

Materials and methods

Animals

   All experimental protocols were approved by the Local Ethics Committee
   of the University of Warmia and Mazury in Olsztyn (Decision No.
   10/2020, dated February 26, 2020). All methods were carried out in
   accordance with Polish law, which defines the conditions and methods of
   conducting experiments on animals, and the European Community Directive
   (EU Directive 2010/63/EU) on the ethical use of experimental animals.
   All methods were performed in accordance with ARRIVE guidelines. The
   animals, sourced from a farm in Lubawa, Poland, were kept under
   standard laboratory conditions with unrestricted access to fresh water
   (ad libitum) and a nutritionally appropriate diet. The housing
   environment was maintained at a temperature of 20–22 °C, with humidity
   levels controlled between 55–60%. To eliminate potential plastic
   contamination, all plastic materials were removed from the
   surroundings, and stainless steel was used for feeding and watering
   equipment. Prior to the experiment, the farm infrastructure where the
   gilts were housed also consisted of stainless steel. Bedding materials,
   including wood, were provided to ensure high animal welfare standards.

   The study lasted four weeks and involved 15 eight-week-old gilts
   (Pietrain × Duroc) with an average body weight of approximately 20 kg.
   The animals were randomly divided into three groups: control group
   (CTR, n = 5) – received empty gelatin capsules orally, low-dose group
   (LD, n = 5) – administered a daily oral dose of 0.1 g/pig of PET
   microplastics (MPs) encapsulated in gelatin, and high-dose group (HD,
   n = 5) – given 1 g/pig of PET MPs per day in gelatin capsules. Capsules
   were provided one hour before the morning feeding. The PET
   microplastics used in the experiment were in the form of
   semi-crystalline polyethylene terephthalate (PET) powder, obtained from
   Goodfellow Cambridge Ltd., UK (Cat. No. [109]ES306031/1). The physical
   properties of the plastic particles have been detailed in previous
   studies. In brief, the particle sizes ranged from 7.6 to 416.9 µm, with
   a predominant diameter of 158.5 µm. The particles exhibited diverse
   shapes, including spherical, fibrous, and irregular forms, with both
   sharp and rounded edges [[110]59]. After the four-week exposure period,
   the animals were humanely euthanized. The euthanasia protocol included
   an initial intramuscular administration of atropine (0.05 mg/kg, Polfa,
   Poland), followed by xylazine (3 mg/kg, Vet-Agro, Poland) and ketamine
   (6 mg/kg, Vetoquinol Biowet, Poland). Approximately 20 min later, once
   full anesthesia was confirmed, an intravenous overdose of sodium
   pentobarbital (0.6 mL/kg, Biowet, Poland) was administered. The absence
   of vital functions was verified by the lack of a pupillary reflex,
   pulse, and respiration. Immediately after confirmation, pancreatic
   tissue samples were collected for subsequent analysis.

RNA isolation, library preparation and sequencing procedure

   RNA was isolated from 15 samples using the RNeasy Mini Kit (Qiagen,
   Germany) in accordance with the manufacturer’s guidelines. The quality
   and concentration of the extracted RNA were assessed using a Tecan
   Infinite M200 plate reader (Tecan Group Ltd., Switzerland), while RNA
   integrity was verified with an Agilent Bioanalyzer 2100 (Agilent
   Technologies, USA). The procedure for library preparation and
   sequencing has been previously detailed [[111]51]. In summary, mRNA was
   first fragmented and then converted into cDNA using the TruSeq Stranded
   mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). Unique adapters
   were attached to the double-stranded cDNA fragments, ensuring strand
   specificity. The prepared libraries were then pooled and sequenced on
   the Illumina NovaSeq 6000 platform using a paired-end (PE) 2 × 150 bp
   sequencing strategy.

Quality control and genome mapping

   The quality of raw paired-end reads was assessed using FastQC and
   Trimmomatic. Sequences were filtered based on the following criteria:
   (a) a minimum length of 120 bp, (b) a PHRED score greater than 20, and
   (c) uniform read length. High-quality trimmed reads were then aligned
   to the Sus scrofa 11.1 genome assembly using the STAR Aligner,
   referencing ENSEMBL annotation (release 98). The mapping results were
   subsequently indexed and sorted by genomic coordinates. Gene expression
   levels were obtained by processing the ballgown files and executing the
   prepDE.py script.

Differentially expressed genes

   The identification of differentially expressed genes (DEGs) was
   performed using DESeq2, applying a false discovery rate (FDR < 0.05)
   and a fold-change threshold of absolute log2 FC > 1.5. Changes in gene
   expression patterns in the porcine pancreas following in vivo exposure
   to PET microplastics were examined through high-throughput
   transcriptome sequencing. The transcriptomic effects of PET
   microplastics were evaluated across three comparisons: low-dose PET MPs
   (LD PET) versus control (CTR), high-dose PET MPs (HD PET) versus CTR,
   and HD PET versus LD PET. Additionally, fragments per kilobase of
   transcript per million mapped reads (FPKM) were computed as a
   normalized expression metric, considering sequencing depth and genomic
   feature length. Enrichment analysis of key biological processes and
   metabolic pathways among DEGs was conducted using the enrichGO and
   enrichKEGG functions from the clusterProfiler R package. The following
   parameters were used: Organism – pig, Ontology categories – CC, MF, or
   BP, p-adjusted value cut-off – 0.05, and p-adjustment method – BH.
   Visualization of expression and functional profiles was carried out
   using R Bioconductor packages such as ggplot2, circlize, and GOplot
   [[112]60, [113]61].

Real-time PCR validation

   Differentially expressed genes were randomly selected for validation by
   real-time PCR using the AriaMx real-time PCR system (Agilent
   Technology, USA) as previously described [[114]62]. Primer sequences
   (Supplemental Table S4) for the reference and target genes (CCL22,
   CXCL10, IL12B, IL2R, TNFSF14, IRF8, JAK3) were designed using Primer
   Express Software 3 (Applied Biosystems, USA). PCR reaction mixes with a
   final volume of 25 μl consisted of cDNA (4 ng), 300 μM of each primer,
   12.5 μl of Power SYBR Green PCR Master Mix (Applied Biosystems, USA)
   and RNase-free water. The abundance of the tested mRNAs was calculated
   using the comparative Pfaffl method [[115]63]. The constitutively
   expressed PPIA and RPLP4 genes were implemented as reference genes, and
   the geometric mean values of the expression levels were used for
   analysis. Real-time PCR results were analyzed using Statistica software
   (version 13.1; Statsoft Inc. Tulsa, OK, USA) with Student’s t-test and
   expressed as means ± SEM. The results were considered statistically
   significant at p ≤ 0.05.

Oxidative stress and antioxidant activity

   Pancreatic tissue homogenates were prepared to analyze the oxidative
   status. Approximately 25 mg of the pancreatic tissue samples were
   homogenized according to the manufacturer’s instructions to ensure the
   integrity of the enzymatic activities. The homogenates were then
   subjected to various assays to determine oxidative stress marker and
   antioxidants activities using commercial kits from Cayman Chemical
   (USA) – Superoxide Dismutase Assay Kit (SOD, 706,002), Catalase Assay
   Kit (CAT, 707,002), Glutathione Peroxidase Assay Kit (GPx, 703,102),
   Glutathione S-Transferase Assay Kit (GST, 703,302), and the TBARS (TCA
   Method) Assay Kit (700,870) for the measurement of malondialdehyde
   (MDA). All enzymatic activities, with the exception of MDA, were
   normalized to the protein concentration determined using the BCA
   Protein Assay Kit (ThermoFisher, USA). Absorbance measurements for the
   assays were performed using the Infinite M200 Pro Reader in conjunction
   with Tecan i-control software (Tecan, Switzerland), following the
   manufacturer’s instructions for each assay kit. The results were
   analyzed using Statistica software (version 13.1; Statsoft Inc. Tulsa,
   OK, USA) with Student’s t-test and expressed as means ± SEM. The
   results were considered statistically significant at p ≤ 0.05.

Supplementary Information

   [116]Supplementary Material 1.^ (1.4MB, zip)

Acknowledgements