Long-Term Exposure to Real-Life Polyethylene Terephthalate Nanoplastics Induces Carcinogenesis In Vitro

Abstract

Micro/nanoplastics (MNPLs) are environmental contaminants originating mainly from plastic waste degradation that pose potential health risks. Inhalation is a major exposure route, as evidenced by their detection in human lungs, with polyethylene terephthalate (PET) among the most abundant particles in respiratory airways. However, the harmful effects of particle bioaccumulation remain unclear, as chronic effects are understudied. To assess long-term effects, specifically carcinogenic effects, BEAS-2B cells were exposed to PET-NPLs for 30 weeks. Genotoxicity, carcinogenic phenotypic hallmarks, and a panel of genes and pathways associated with cell transformation and lung cancer were examined and compared across three exposure durations. No significant effects were observed after 24 h or 15 weeks of exposure. However, a 30-week exposure led to increased genotoxic damage, anchorage-independent growth, and invasive potential. Transcriptomic analysis showed the upregulation of several oncogenes and lung cancer-associated genes at the end of the exposure. Further analysis revealed an increase in differentially expressed genes over time and a temporal gradient of lung cancer-related genes. Altogether, the data suggest PET-NPLs' potential carcinogenicity after extended exposure, highlighting serious long-term health risks of MNPLs. Assessing their carcinogenic risks under chronic scenarios of exposure is crucial to addressing knowledge gaps and eventually developing preventive policies.

Keywords: carcinogenicity; chronic exposure; human health risk; nanoplastics (NPLs); new approach methodologies (NAMs); polyethylene terephthalate (PET); respiratory toxicity.

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Interaction of PET-NPLs with BEAS-2B cells. (A) Cytotoxicity assessment of PET-NPLs in BEAS-2B cells. (B) Flow cytometry analysis showing fluorescence intensity (PE-A) in the cells as a measure of cellular interaction. The gray curve represents the negative control (NT), while the blue curve corresponds to PET-NPL-treated cells. (C–F) Confocal microscopy images: (C) BEAS-2B cells without treatment and (c1) the corresponding IMARIS 3D reconstruction; (D) BEAS-2B cells exposed to PET-NPLs for 24 h and (d1) the corresponding IMARIS 3D reconstruction. (E, F) Orthogonal views illustrating localization of PET-NPLs inside the cells.

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Long-term phenotypical effects of PET-NPLs in BEAS-2B cells. (A) Genotoxic damage, (B) anchorage-independent growth ability, and (C) invasive potential. Passage-paired negative control (NT) and methylmethanesulfonate (MMS, positive control).

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BEAS-2B transcriptomic state after 30 weeks of PET-NPLs exposure. (A) Number of DEGs (downregulated, upregulated, and total) and mean-difference plot showing the distribution of DEGs’ expression. (B) Percentage of enriched terms from DO collection corresponding to cancer-related or other terms. (C) Fold change for the genes related to the most common lung cancer mutations and the top 10 genes co-mentioned with lung cancer expressed as log2-fold-change (L2FC) values.

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Transcriptomic progression during chronic exposure to PET-NPLs. (A) Number of DEGs after 24 h,15 weeks, and 30 weeks of exposure from darker to lighter color, respectively. Significance is not represented. (B) Venn diagram illustrating shared DEGs between 15 (yellow) and 30 weeks (blue) of exposure (C) Lung-carcinogenesis-related enriched terms found after 15 and 30 weeks of exposure. Intensity is correlated with the significance level.

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Mapping of transcriptomic data to the nonsmall cell lung cancer pathway. The figure displays fold changes in gene expression levels for annotated genes in the hsa05223 pathway from the KEGG database. Gene boxes are split in two parts: the left side corresponds to the fold-change value at 15 weeks of exposure, and the right side corresponds to the fold-change value at 30 weeks of exposure.

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Gene expression values for genes following a trend in their expression levels during chronic exposure. Gene expression values are represented as log fold-change values (L2FC). (A1) Genes mapping EMT hallmark collection classified by their cascade position (receptor, regulator, or effector). (A2) Genes mapping the top 100 genes comentioned with lung cancer term according to Enrichr, along with manually annotated genes. (B) Histone panel according to Histone DB 2.0. (C) Putative biomarker genes that might act as early detectors of the transformation phenotype.