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Plastic pollution constitutes an escalating global crisis and poses serious threats to ecosystems, human and animal health, food, water, and economies. An expanding body of evidence indicates that micro- and nanoplastics cannot be controlled by biological tissue barriers; they can circulate through tissues, reach distant organs, and readily accumulate within various biological systems [1-6]. Among the many types of plastics, polyethylene terephthalate (PET) is one of the most common polymers exposed in daily human and animal life. Notably, PET fragments into micro- and nanoparticles that have sparked significant interest in potential health implications upon ingestion. In the present study, we investigated the effects of PET microplastics on human intestinal cell cultures subjected to a range of concentrations (~200; 1000; 10,000; 20,000; and 72,000 particles/mL) of PET particles (quantified size range ~6–8 μm, containing a polydisperse mixture of micro- and nanoplastics). The chemical fingerprint and ultrastructural characteristics of the PET microplastics were confirmed by Raman spectroscopy and scanning electron microscopy (SEM) analysis (Figure S1). Integrated SEM micrographs (Figure S1C–D) revealed irregularly shaped micro- to nanoscale PET particles with measured dimensions around ~213–217 nm, while Raman spectral analysis (Figure S1F,G) displayed distinct characteristic peaks across the 600–1700 cm−1 region (Figure S1G), together confirming both the nanoscale morphology and molecular identity of the detected particles and providing direct structural–chemical validation of their presence in the samples. Our findings demonstrate a toxicological cascade in which PET microplastics compromise intestinal barrier function and perturb cellular homeostasis, with oxidative stress, endoplasmic reticulum (ER) stress, and cell death emerging as principal intermediaries in the affected tissue disruptions. In our model of exposure, namely gut-on-a-chip, a pivotal metric in our investigation was the transepithelial electrical resistance (TEER) assay, which quantitatively assesses the integrity of the epithelial barrier. Remarkably, increasing PET microplastic concentrations resulted in a clear, dose- and time-dependent reduction in TEER (Figure 1A). Concurrently, we employed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate metabolic activity as an indicator of overall cellular viability (Figure 1B). While minimal toxicity was observed at lower concentrations, the higher doses (20,000 particles/mL and above) induced considerable cell death on day four. Notably, TEER reduction occurred at lower doses (1000 p/mL) prior to significant cytotoxicity, suggesting specific barrier disruption distinct from cell death. With the aim of elucidating the underlying mechanisms, we first measured intracellular reactive oxygen species (ROS) generation. A marked increase in ROS production paralleled increasing concentrations of PET microplastics, even in the first hour of treatment (Figure 1C), and this effect persisted for 24 h (Figures 1D and S1B). Beyond these intracellular stress markers, we sought to determine whether this internal state prompted a functional response, as reflected in secreted proteins. By analyzing culture supernatants with proximity extension assay-targeted proteomics, we profiled the cellular secretome and identified profound, dose-dependent alterations. A heatmap of the proteomic data shows a distinct clustering pattern, separating the high-dose exposure groups from the control and low-dose groups (Figure 1E). Relatively low doses of micro- and nanoplastics, such as 200 particles/mL, which are relevant to daily exposure [2, 3], show significant effects on levels of many proteins (Figures 1F and S2). This protein-level data shows that the antioxidant enzyme peroxiredoxin (PRDX1) and the junctional protein coxsackievirus and adenovirus receptor (CXADR) are significantly downregulated, while the danger signal extracellular newly identified RAGE-binding protein (EN-RAGE) and key inflammatory mediators IL-1α, C-X-C motif chemokine ligand 1 (CXCL1), and triggering receptor expressed on myeloid cells 1 (TREM1) are significantly upregulated in a dose-dependent manner (Figure 1F). The dose-dependent physiological and proteomic changes were demonstrated by an intense transcriptional change, as observed in RNA sequencing of the exposed epithelial cells. Evaluations of global gene expression, including principal component analysis (PCA) (Figure 2A) and a comprehensive heatmap of differentially expressed genes (Figure 2B), revealed distinct clustering patterns that reflected the PET microplastic dose. To investigate the functional consequences of these gene expression alterations, gene ontology (GO) enrichment analysis revealed that upregulated genes were significantly enriched in pathways related to apoptosis, endoplasmic reticulum (ER) stress, and cellular responses to oxidative stress (Figure 2C). Detailed heatmaps for these key pathways illustrate a clear particle-number-dependent upregulation of genes involved in oxidative stress (Figure 2D), chemical stress (Figure 2E), ER stress (Figure 2F), and the apoptotic signaling pathway (Figure 2G). Low-dose PET microplastic exposure elicited minimal transcriptional perturbations, consistent with adaptive homeostatic responses, whereas high-dose exposure triggered widespread pathway dysregulation indicative of oxidative stress and metabolic reprogramming (Figure S3). This widespread transcriptional reprogramming towards stress and apoptosis provides a mechanistic basis for the ultimate decline in cellular function and health. In this context, environmental factors, including micro- and nanoplastics, contribute to the disruption of the epithelial barrier, which plays a critical role in the pathogenesis of various diseases [7]. This study clearly demonstrates, for the first time, that the micro- and nanoplastic burden of PET at daily exposure levels compromises both barrier integrity and core metabolic capacity, which could impair nutrient absorption and immune defenses, particularly under chronic or high-level exposure scenarios. Given the pervasive presence of PET in the everyday environments of both humans and animals, the findings highlight the potentially serious consequences of PET exposure and emphasize the urgency of further investigation into its regulatory and health impacts [2, 3]. Overall, our results delineate the multifaceted nature of microplastic-induced cellular damage, represented by diminished epithelial barrier function, decreased cell viability, and widespread transcriptional shifts converging on oxidative stress, ER stress, and programmed cell death pathways. These in vitro observations contribute to the body of evidence concerning the potential molecular mechanisms of microplastic ingestion on human health. The findings support the continued evaluation and scrutiny of permissible microplastic thresholds in food and water, particularly in the context of increasing global plastic production. Additional in vivo and dose–response studies will be critical for extrapolating these findings and refining our understanding of how microplastics manifest toxicity within the human gastrointestinal tract. Ultimately, our findings illustrate the sensitivity of the intestinal epithelium as a primary target for ingested pollutants and toxic substances. S.A., M.A., K.C.N., and C.A.A. conceptualized and designed the study. S.A. conducted the primary investigation with experimental support from H.B., L.C., O.A., C.Z., Y.P., D.Y., X.L., and E.I.B. S.A. and I.O. were responsible for data analysis, visualization, and interpretation of the results, with technical support from C.B. and B.R. C.A.A. and M.A. supervised the project. H.I.A. was responsible for the Raman spectroscopy analysis. N.B.M. and M.E.S. conducted the SEM analysis. S.A., H.B. and C.A.A. wrote the manuscript with critical input from M.A., K.C.N., and R.D. All authors critically reviewed the content and approved the final version for submission. Open access publishing facilitated by Universitat Zurich, as part of the Wiley - Universitat Zurich agreement via the Consortium Of Swiss Academic Libraries. The authors have nothing to report. R.D. is a non-executive Board Director at Seed Health Inc. I.O. is chair of the EAACI Epithelial Cell Biology Working Group. K.C.N. currently reports Grants from National Institute of Allergy and Infectious Diseases (NIAID), National Heart, Lung, and Blood Institute (NHLBI), National Institute of Environmental Health Sciences (NIEHS); Stock options from Phylaxis and IgGenix; Co-founder of Latitude, Alladapt, BeforeBrands, and IgGenix; Consultant for Excellergy; National Scientific Committee member at Immune Tolerance Network (ITN). Patents include Granulocyte-based methods for detecting and monitoring immune system disorders, Methods and Assays for Detecting and Quantifying Pure Subpopulations of White Blood Cells in Immune System Disorders, Biological sample preparation device and methods of using the same, Immune cell activation device and method of using the same, Mixed allergen composition and methods for using the same, Methods of Isolating Allergen Specific Antibodies, Microfluidic devise and diagnostic methods for Basophil Activation, and Methods for detecting heavy metals in biological samples. M.A. has received research grants from the Swiss National Science Foundation, Bern; research grant from Stanford University; Leading House for the Latin American Region, Seed Money Grant. She is the Scientific Advisory Board member of Stanford University Sean Parker Asthma Allergy Center, CA; Advisory Board member of LEO Foundation Skin Immunology Research Center, Copenhagen; and Scientific Co-Chair of World Allergy Congress (WAC) Istanbul, 2022, Scientific Programme Committee Chair, EAACI. C.A.A. has received research grants from the Swiss National Science Foundation, European Union (EU CURE, EU Syn-Air-G), Novartis Research Institutes (Basel, Switzerland), Stanford University (Redwood City, Calif), Seed Health (Boston, USA) and SciBase (Stockholm, Sweden); is the Co-Chair for EAACI Guidelines on Environmental Science in Allergic diseases and Asthma; is on the Advisory Boards of Sanofi/Regeneron (Bern, Switzerland, New York, USA), Stanford University Sean Parker Asthma Allergy Center (CA, USA), Novartis (Basel, Switzerland), Glaxo Smith Kline (Zurich, Switzerland), Bristol-Myers Squibb (New York, USA), Seed Health (Boston, USA), and SciBase (Stockholm, Sweden); and is the Editor-in-Chief of Allergy. Other authors declare that they have no relevant conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1: (A) Representative brightfield (top) and fluorescence microscopy images (bottom) of the PET particles used in the study. The particles were stained with Nile Red to confirm their polymeric nature. Scale bars are 275 μm for 10× magnification and 150 μm for 20× magnification. (B) Time-course analysis of intracellular Reactive Oxygen Species (ROS) generation in intestinal epithelial cells exposed to various concentrations of PET particles over 24 h. ROS levels are expressed relative to the control group at the start of the experiment. Data points represent the mean ± standard deviation. (C) Scanning electron microscopy (SEM) images of PET microplastics at low and intermediate magnifications (400×, 1000×, and 5000×). The micrographs reveal irregular, fragmented particle morphology with rough and heterogeneous surface topography, consistent with mechanically degraded PET microplastics. Scale bars: 100 μm and 10 μm, as indicated. (D) High-resolution SEM images (50,000×, 100,000×, and 200,000× magnification) demonstrate the presence of nanoscale fragments associated with the parent PET microplastics. Discrete nanoparticulate structures with diameters of approximately 213.5 nm and 217.3 nm are indicated, providing direct evidence of co-existing nanoplastic fractions. Scale bars: 1 μm and 300 nm. (E) Optical microscopy image of PET microplastics dispersed on a glass slide, illustrating particle size heterogeneity and irregular shape distribution. The image shows the presence of mixed-size plastic fragments, which supports the environmentally relevant exposure model used in this study. Scale bar: 200 μm. (F) Raman intensity map at 1614 cm−1 overlaid on the optical image, where brighter regions reflect the particles' 3D morphology. (G) Raman spectrum of the particle showing characteristic PET bands, confirming its identity as polyethylene terephthalate. Figure S2: Violin plots show the relative abundance (Normalized Protein eXpression, NPX) of individual proteins secreted into the culture medium after 48 h of exposure to increasing concentrations of PET microplastics. Data were analyzed by a one-way ANOVA followed by Tukey's Honest Significant Difference (HSD) post hoc test. Asterisks indicate a significant difference compared to the control group (p.adj < 0.05, *p.adj < 0.01, **p.adj < 0.001). Figure S3: Dose-dependent transcriptomic responses following PET microplastic exposure. (A) Bar plot showing the number of differentially expressed genes (DEGs) across increasing exposure doses, stratified by upregulated and downregulated transcripts. A progressive increase in DEG counts is observed with rising particle concentrations. (B) UpSet plot illustrating the intersection of DEGs across dose groups, demonstrating minimal overlap between low- and high-dose exposures and highlighting dose-specific transcriptional signatures. (C) Gene set enrichment analysis (GSEA) of significantly dysregulated pathways, including mitochondrial translation, embryonic organ development, actin filament bundle organization, cell–cell adhesion via plasma membrane adhesion molecules, and negative regulation of sprouting angiogenesis. Dot size indicates gene count, and color represents adjusted p-values. (D) Spearman correlation analysis between exposure dose and pathway activity scores, identifying both positive and negatively correlated biological processes, including ferroptosis, signal transduction by p53 class mediators, vitamin transport, and cellular response to heat stress. (E–F) Representative dose–response correlations for selected pathways, including positive chemotaxis, endosome-to-lysosome transport, ferroptosis, and glycoprotein metabolic processes, demonstrating graded activation or suppression with increasing exposure levels. (G–H) Expression profiles of oxidative stress- and xenobiotic-response genes (CYP1A1, NQO1, SLC7A11, TXNRD1, HMOX1, SQSTM1, RELB, and GADD45A) showing strong positive correlations with dose. (I) Negative dose-dependent expression trends of metabolic and lipid-related genes (ACSS1, SLC2A5, VIL1, and DGAT2), indicating suppression of anabolic pathways at higher exposure concentrations. Correlation coefficients (ρ) and false discovery rates (FDR) are indicated within each panel. Exposure doses are presented as log10 (Dose +1). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.