36616c6bbace52ca72250768574f025d pmc_9953511.pdf cd490fe52d7fa631033ba81585f8ab49bf0be2d4 pmc_9953511.pdf 195c2c0b2cba9b45c055686e98cafe31738f46577085fb9704406080186f54b8 pmc_9953511.pdf Title: Hazard Assessment of Polystyrene Nanoplastics in Primary Human Nasal Epithelial Cells, Focusing on the Autophagic Effects Subject: The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among them, inhalation exposure to MNPLs is the least studied area, even though their widespread presence in the air is dramatically increasing. In this context, this study focused on the potential hazard of polystyrene nanoplastics (PSNPLs with sizes 50 and 500 nm) in human primary nasal epithelial cells (HNEpCs), with the first line of cells acting as a physical and immune barrier in the respiratory system. Primarily, cellular internalization was evaluated by utilizing laboratory-labeled fluorescence PSNPLs with iDye, a commercial, pink-colored dye, using confocal microscopy, and found PSNPLs to be significantly internalized by HNEpCs. After, various cellular effects, such as the induction of intracellular reactive oxygen species (iROS), the loss of mitochondrial membrane potential (MMP), and the modulation of the autophagy pathway in the form of the accumulation of autophagosomes (LC3-II) and p62 markers (a ubiquitin involved in the clearance of cell debris), were evaluated after cell exposure. The data demonstrated significant increases in iROS, a decrease in MMP, as well as a greater accumulation of LC3-II and p62 in the presence of PSNPLs. Notably, the autophagic effects did indicate the implications of PSNPLs in defective or insufficient autophagy. This is the first study showing the autophagy pathway as a possible target for PSNPL-induced adverse effects in HNEpCs. When taken together, this study proved the cellular effects of PSNPLs in HNEpCs and adds value to the existing studies as a part of the respiratory risk assessment of MNPLs. Keywords: nanoplastics; polystyrene; HNEpCs; uptake; oxidative stress; mitochondrial membrane potential; autophagy Author: Balasubramanyam Annangi, Aliro Villacorta, Montserrat López-Mesas, Victor Fuentes-Cebrian, Ricard Marcos and Alba Hernández Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Feb 7 02:27:53 2023 CET ModDate: Tue Feb 7 02:40:22 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3204826 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CHZVTV+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 NOMQKQ+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 PMMDJH+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 OYGWHX+VnURWPalladioL Type 1 Custom yes yes no 23 0 ENXZUH+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 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TrueType WinAnsi yes yes no 185 0 KPHAMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 188 0 KPHAMP+PalatinoLinotype TrueType WinAnsi yes yes no 194 0 KPHANB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 197 0 KPHAMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 207 0 KPHAMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 210 0 KPHAMP+PalatinoLinotype TrueType WinAnsi yes yes no 216 0 KPHANB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 219 0 KPHAMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 231 0 KPHAMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 234 0 KPHAMP+PalatinoLinotype TrueType WinAnsi yes yes no 240 0 KPHANB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 243 0 KPHAMN+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 258 0 KPHAMO+PalatinoLinotype CID TrueType Identity-H yes yes yes 261 0 KPHAMP+PalatinoLinotype TrueType WinAnsi yes yes no 267 0 KPHANB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 270 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-25 02:25:09 CEST RepresentationInformation: pmc_9953511.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-16 02:18:26 CET Size: 3204826 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 479 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Characterization of Polystyrene Nanoplastics (PSNPLs) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Cell Viability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Intracellular Uptake by Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Estimation of Intracellular ROS Levels by Using the DCFH-DA Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Mitochondrial Membrane Potential Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Total Protein Extraction and Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Immunocytochemistry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Physicochemical Characterization of the Used PSNPLs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Cell Viability Assessment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Intracellular Localization of PSNPLs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Generation of iROS by PSNPLs Exposure Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Loss of Mitochondrial Membrane Potential by PSNPLs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: PSNPLs Cause Defective Autophagy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Info: Title: Hazard Assessment of Polystyrene Nanoplastics in Primary Human Nasal Epithelial Cells, Focusing on the Autophagic Effects Author: Balasubramanyam Annangi, Aliro Villacorta, Montserrat López-Mesas, Victor Fuentes-Cebrian, Ricard Marcos and Alba Hernández Subject: The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among them, inhalation exposure to MNPLs is the least studied area, even though their widespread presence in the air is dramatically increasing. In this context, this study focused on the potential hazard of polystyrene nanoplastics (PSNPLs with sizes 50 and 500 nm) in human primary nasal epithelial cells (HNEpCs), with the first line of cells acting as a physical and immune barrier in the respiratory system. Primarily, cellular internalization was evaluated by utilizing laboratory-labeled fluorescence PSNPLs with iDye, a commercial, pink-colored dye, using confocal microscopy, and found PSNPLs to be significantly internalized by HNEpCs. After, various cellular effects, such as the induction of intracellular reactive oxygen species (iROS), the loss of mitochondrial membrane potential (MMP), and the modulation of the autophagy pathway in the form of the accumulation of autophagosomes (LC3-II) and p62 markers (a ubiquitin involved in the clearance of cell debris), were evaluated after cell exposure. The data demonstrated significant increases in iROS, a decrease in MMP, as well as a greater accumulation of LC3-II and p62 in the presence of PSNPLs. Notably, the autophagic effects did indicate the implications of PSNPLs in defective or insufficient autophagy. This is the first study showing the autophagy pathway as a possible target for PSNPL-induced adverse effects in HNEpCs. When taken together, this study proved the cellular effects of PSNPLs in HNEpCs and adds value to the existing studies as a part of the respiratory risk assessment of MNPLs. 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