acacf4a9a112d746f28530514f7796b6 pmc_34680075.pdf 4c180d7abc962cb7af4df9327e4bba8e03fb38ef pmc_34680075.pdf 4caef46362ac59aa773f04697cfbd619640b107fe524cecb3aca649a99be3d34 pmc_34680075.pdf Title: Long-Term Effects of Polystyrene Nanoplastics in Human Intestinal Caco-2 Cells Subject: The increasing presence of micro- and nanoplastics (MNPLs) in the environment, and their consequent accumulation in trophic niches, could pose a potential health threat to humans, especially due to their chronic ingestion. In vitro studies using human cells are considered pertinent approaches to determine potential health risks to humans. Nevertheless, most of such studies have been conducted using short exposure times and high concentrations. Since human exposure to MNPLs is supposed to be chronic, there is a lack of information regarding the potential in vitro MNPLs effects under chronic exposure conditions. To this aim, we assessed the accumulation and potential outcomes of polystyrene nanoparticles (PSNPs), as a model of MNPLs, in undifferentiated Caco-2 cells (as models of cell target in ingestion exposures) under a relevant long-term exposure scenario, consisting of eight weeks of exposure to sub-toxic PSNPs concentrations. In such exposure conditions, culture-media was changed every 2–3 days to maintain constant exposure. The different analyzed endpoints were cytotoxicity, dysregulation of stress-related genes, genotoxicity, oxidative DNA damage, and intracellular ROS levels. These are endpoints that showed to be sensitive enough in different studies. The obtained results attest that PSNPs accumulate in the cells through time, inducing changes at the ultrastructural and molecular levels. Nevertheless, minor changes in the different evaluated genotoxicity-related biomarkers were observed. This would indicate that no DNA damage or oxidative stress is observed in the human intestinal Caco-2 cells after long-term exposure to PSNPs. This is the first study dealing with the long-term effects of PSNPs on human cultured cells. Keywords: nanoplastics; polystyrene nanoparticles; genotoxicity; intestinal barrier; Caco-2 cells Author: Josefa Domenech, Mariana de Britto, Antonia Velázquez, Susana Pastor, Alba Hernández, Ricard Marcos and Constanza Cortés Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 12 04:01:11 2021 CEST ModDate: Tue Oct 12 04:56:50 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2962278 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- TQVNMO+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 UAVXDJ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 LBPEFO+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes no 23 0 KORLVE+VnURWPalladioL Type 1 Custom yes yes no 28 0 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WinAnsi yes yes no 280 0 LLMJAJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 283 0 LLMJAF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 295 0 LLMJAG+PalatinoLinotype CID TrueType Identity-H yes yes yes 298 0 LLMJAH+PalatinoLinotype TrueType WinAnsi yes yes no 304 0 LLMJAJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 307 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:40:06 CET RepresentationInformation: pmc_34680075.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-10 08:31:37 CET Size: 2962278 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 499 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Nanoplastic Particles Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Short-Term Exposure to Nanopolystyrene Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Nanopolystyrene’s Cytotoxicity Assessment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Intracellular Nanopolystyrene Detection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Long-Term Exposure to Nanopolystyrene Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Nanopolystyrene Internalization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Real-Time RT PCR Gene Expression Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Genotoxic and Oxidative DNA Damage Assessment in the Comet Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Oxidative Stress Assessment with the DCFH-DA Method Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Nanoplastic Particles Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Short-Term PSNPs Cytotoxicity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Short-Term PSNPs Internalization, Cellular Localization, and Subcellular Structural Effects Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Long-Term y-PSNPs Internalization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Gene Expression Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Genotoxic and Oxidative DNA Damage Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Intracellular ROS Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Info: Title: Long-Term Effects of Polystyrene Nanoplastics in Human Intestinal Caco-2 Cells Author: Josefa Domenech, Mariana de Britto, Antonia Velázquez, Susana Pastor, Alba Hernández, Ricard Marcos and Constanza Cortés Subject: The increasing presence of micro- and nanoplastics (MNPLs) in the environment, and their consequent accumulation in trophic niches, could pose a potential health threat to humans, especially due to their chronic ingestion. In vitro studies using human cells are considered pertinent approaches to determine potential health risks to humans. Nevertheless, most of such studies have been conducted using short exposure times and high concentrations. Since human exposure to MNPLs is supposed to be chronic, there is a lack of information regarding the potential in vitro MNPLs effects under chronic exposure conditions. To this aim, we assessed the accumulation and potential outcomes of polystyrene nanoparticles (PSNPs), as a model of MNPLs, in undifferentiated Caco-2 cells (as models of cell target in ingestion exposures) under a relevant long-term exposure scenario, consisting of eight weeks of exposure to sub-toxic PSNPs concentrations. In such exposure conditions, culture-media was changed every 2 3 days to maintain constant exposure. The different analyzed endpoints were cytotoxicity, dysregulation of stress-related genes, genotoxicity, oxidative DNA damage, and intracellular ROS levels. These are endpoints that showed to be sensitive enough in different studies. The obtained results attest that PSNPs accumulate in the cells through time, inducing changes at the ultrastructural and molecular levels. Nevertheless, minor changes in the different evaluated genotoxicity-related biomarkers were observed. This would indicate that no DNA damage or oxidative stress is observed in the human intestinal Caco-2 cells after long-term exposure to PSNPs. This is the first study dealing with the long-term effects of PSNPs on human cultured cells. 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