Size- and polymer-dependent toxicity of amorphous environmentally relevant micro- and nanoplastics in human bronchial epithelial cells

Abstract

Background: Knowledge of the toxicological impact of micro- and nanoplastics (MNPs) on the human airway epithelium is limited and almost exclusively based on experiments applying high doses of spherical polystyrene (PS) particles. In this study, we investigated the toxicity of a broad size range of amorphous MNPs generated from different environmentally-relevant polymers.

Methods: Bronchial epithelial cells (BEAS-2B) were exposed to three different doses of polyvinylchloride (PVC), polypropylene (PP), or polyamide (PA) particles (< 1 μm-10 μm), as well as leachates from these polymers. Toxicity was evaluated by assessment of cytotoxicity, inflammation (IL-8 release and inflammatory gene expression) and oxidative stress (DCFH-DA assay and antioxidant gene expression). Furthermore, the molecular mechanism behind MNP-induced inflammation was investigated by studying activation of two well-known inflammation related transcriptional factors (NF-κB and AP-1).

Results: Only PA nanoplastics induced significant cell death, IL-8 secretion and inflammatory gene expression compared to vehicle control. PA-induced inflammation was accompanied by NF-κB, but not AP-1, transcriptional activity. PA did not increase cellular ROS levels; however, it did lead to increased expression of the antioxidant gene superoxide dismutase 2. In addition to PA, exposure to < 1 µm and 1-5 µm PP particles resulted in elevated IL-8 secretion, likely due to the presence of talc added as filler. None of the leachates affected cytotoxicity or inflammation.

Conclusion: Toxicity of MNPs to human bronchial epithelial cells was dependent on polymer type, size and dose. Nanoplastics, especially PA, were more toxic to bronchial epithelial cells than microplastics and induced cytotoxicity and an inflammatory response.

Supplementary information: The online version contains supplementary material available at 10.1186/s43591-025-00126-9.

Keywords: Inflammation; Inhalation toxicology; NF-ĸB; Polyamide; Polypropylene; Polyvinylchloride.

Fig. 1

Particle shape characterization. Representative Scanning electron microscopy (SEM) images of the different size fractions of Polyvinylchloride (PVC), Polypropylene/Talc (PP/Talc) and Polyamide (PA) micro- and nanoplastics

Fig. 2

Static Light Scattering (SLS) measurements of micro- and nanoplastics (MNPs) and talc. Representative number-based (black lines) and volume-based (red lines) distribution spectra are shown for A polyvinylchloride (PVC), B polypropylene/talc (PP/Talc), C polyamide (PA) and D talc. MNPs and talc incubated for 0 h and 24 h in exposure medium (DMEM, 0.05% BSA, 1% FBS, 1% 1-propanol)

Fig. 3

Evaluation of cytotoxicity upon exposure to polypropylene mixed with talc (PP/Talc), polyvinyl chloride (PVC), and polyamide (PA). Cells were exposed to the vehicle control (exposure medium; DMEM, 0.05% BSA, 1% FBS, 1% 1-propanol) or to MNPs of the respective polymers in exposure medium. Applied doses are displayed in the bar charts, in μg/mL. Released lactate dehydrogenase (LDH) levels were measured after 24 h exposure. Maximum LDH release was achieved by exposure to Triton X-100 (2%). Cytotoxicity data upon exposure to PP/Talc and talc (A), PVC (B), and PA (C) MNPs in size fractions of < 1 µm, 1–5 µm and 5–10 µm. Every data point represents an independent exposure experiment, each performed in technical triplicate. Data presented as mean percentage of the maximum LDH release ± SD. ****P < 0.0001 compared to vehicle control

Fig. 4

IL-8 secretion upon exposure to polypropylene mixed with talc (PP/Talc), polyvinyl chloride (PVC), and polyamide (PA) micro- and nanoplastics. Cells were exposed to the vehicle control (DMEM, 0.05% BSA, 1% FBS) or to the different size fractions of the respective polymers. Applied doses are displayed in μg/mL. IL-8 secretion upon parallel exposure to positive control TNF-α (50 ng/ml) (A), PP/Talc (B), PVC (C) and PA (D) in size fractions of < 1 µm, 1–5 µm and 5–10 µm. Talc and milled talc doses were identical to quantities of talc present in the PP/Talc particle suspensions. Every data point represents an independent exposure experiment, each performed in technical triplicate. Average absolute IL-8 levels (± SD) are compared with the vehicle control: *P < 0.05; **P < 0.01; ****P < 0.0001

Fig. 5

Pro-inflammatory gene expression and activation of NF-κB pathway upon exposure to polyamide (PA) nanoplastics. Cells were exposed to PA particles of < 1 µm (Applied doses are displayed in μg/mL), or TNF-α (50 ng/mL) in parallel experiments. A, B Gene expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) and C-X-C Motif Chemokine Ligand 1 (CXCL1) C, D Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was measured through luminescence intensity in stably transduced BEAS-2B cells with an NF-κB luciferase reporter. E, F Activator protein 1 (AP-1) activation was measured through luminescence intensity in stably transduced BEAS-2B cells with an AP-1 luciferase reporter. G IL-8 protein secretion after PA nanoplastics exposure, with different concentrations of NF- κB inhibitor (IKK-16). Every data point represents an independent exposure experiment, each performed in technical triplicate. Data are presented as mean fold change/vehicle control ± SD or absolute values (IL-8). Differences were considered statistically significant when P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6

Antioxidant gene expression and intracellular ROS formation upon exposure to polyamide (PA) nanoplastics. Cells were exposed to PA nanoplastics (< 1 µm) (Applied doses are displayed in μg/mL) for 24 h. A Intracellular ROS was measured with the DCFH-DA assay B Gene expression of superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2). C Ratio between reduced glutathione (GSH) and oxidized glutathione (GSSG). D Gene expression of SOD1 after PA nanoplastics exposure, with different concentrations of NF-κB inhibitor (IKK-16) E Gene expression of SOD2 after PA nanoplastics exposure, with different concentrations of IKK-16. F IL-8 protein secretion after PA nanoplastics exposure, with different concentrations of antioxidant quercetin. Every data-point represents an independent exposure experiment, each performed in technical triplicate. Data are displayed as mean fold change/vehicle control or absolute IL-8 protein levels ± SD. P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001