7e815d9cae11679ca16d62a3ae08ccbf Biomolecules_14_1442_2021_.pdf 7fd9aa7510e8c3e91cbaff50e4fb2e396f21d878 Biomolecules_14_1442_2021_.pdf 2d44849d0b2a840dd8a62687271849dba547dbd33d62829692d98498dd3bed75 Biomolecules_14_1442_2021_.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: Fri Oct 29 16:28:02 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: 2906211 bytes Optimized: yes PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- TQVNMO+URWPalladioL-Ital Type 1 Custom yes yes no 488 0 UAVXDJ+URWPalladioL-Bold Type 1 Custom yes yes no 491 0 LBPEFO+URWPalladioL-Roma Type 1 Custom yes yes no 494 0 GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes no 498 0 KORLVE+VnURWPalladioL Type 1 Custom yes yes no 501 0 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Differences: true Font: BaseFont: UAVXDJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: UAVXDJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LBPEFO+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: LBPEFO+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true XMP: application/pdf Josefa Domenech, Mariana de Britto, Antonia Velázquez, Susana Pastor, Alba Hernández, Ricard Marcos and Constanza Cortés 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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