Chemosphere 325 (2023) 138360 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Hazard assessment of different-sized polystyrene nanoplastics in hematopoietic human cell lines Alireza Tavakolpournegari a, 1, Balasubramanyam Annangi a, 1, Aliro Villacorta a, b, Gooya Banaei a, Joan Martin a, Susana Pastor a, Ricard Marcos a, *, 1, Alba Hernández a, 1, ** a Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Cerdanyola Del Vallès, Barcelona, Spain b Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Iquique, Chile H I G H L I G H T S G R A P H I C A L A B S T R A C T • Polystyrene nanoplastics were evaluated in Raji-B, THP-1, and TK6 cells. • TEM, confocal microscopy, and flow cytometry demonstrated cell internalization. • Cell uptake was negatively associated with the size. • Mitochondrial membrane potential loss was induced in Raji-B and THP-1 cells. • Size, biological endpoint, and cell type modulate the toxicological profile of MNPLs. A R T I C L E I N F O A B S T R A C T Handling Editor: Michael Bank The environmental presence of micro/nanoplastics (MNPLs) is an environmental and human health concern. Such MNPLs can result from the physicochemical/biological degradation of plastic goods (secondary MNPLs) or Keywords: can result from industrial production at that size, for different commercial purposes (primary MNPLs). Inde­ Polystyrene nanoplastic size pendently of their origin, the toxicological profile of MNPLs can be modulated by their size, as well as by the Raji-B/THP-1/TK6 cells ability of cells/organisms to internalize them. To get more information on these topics we have determined the Uptake ability of three different sizes of polystyrene MNPLs (50, 200, and 500 nm) to produce different biological effects Oxidative stress Mitochondrial membrane potential in three different human hematopoietic cell lines (Raji-B, THP-1, and TK6). Results show that none of the three sizes was able to induce toxicity (growth ability) in any of the tested cell types. Although transmission electron microscopy and confocal images showed cell internalization in all the cases, their quantification by flow cytometry demonstrated an important uptake by Raji-B and THP-1 cells, in comparison with TK6 cells. For the first ones, the uptake was negatively associated with the size. Interestingly, when the loss of mitochondrial membrane potential was determined, dose-related effects were observed for Raji-B and THP-1 cells, but not for TK6 cells. These effects were observed for the three different sizes. Finally, when oxidative stress induction was evaluated, no clear effects were observed for the different tested combinations. Our conclusion is that size, biological endpoint, and cell type are aspects modulating the toxicological profile of MNPLs. * Corresponding author. Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Campus of Bellaterra, 08193, Cerdanyola Del Vallès, Spain. ** Corresponding author. E-mail addresses: ricard.marcos@uab.cat (R. Marcos), alba.hernandez@uab.cat (A. Hernández). 1 Both authors contribute equally. https://doi.org/10.1016/j.chemosphere.2023.138360 Received 14 January 2023; Received in revised form 25 February 2023; Accepted 8 March 2023 Available online 10 March 2023 0045-6535/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 1. Introduction 2. Materials and methods Environmental plastic waste constitutes a serious ecological problem 2.1. Characterization of the used PSNPLs since they degrade slowly and remain in the environment for hundreds of years (Kik et al., 2020). In addition, these environmental plastics Three different sizes of pristine polystyrene nanoplastics were pur­ further degrade into micro/nanoplastics (MNPLs). Fragmented or chased from Spherotech Inc. (Chicago, USA). Namely, PSNPLs 50 nm otherwise miniaturized plastic materials in the form of MNPLs are of (PS-50; PP-008-10; with nominal sizes of 0.05–0.1 μM), PSNPLs 200 nm nagging environmental concern. Since the ubiquitous presence of (PS-200; PP-015-10; with nominal sizes of 0.1–0.2 μM), and PSNPLs 500 MNPLs in the environment is inevitable, they can be internalized by any nm (PS-500; PP-05-10; with nominal sizes of 0.4–0.6 μM. All used kind of organism, including humans. Thus, the assessment of MNPLs as PSNPLs were supplied as water dispersions. potential health risk factors for humans demands an urgent response For their characterization, PSNPLs dispersions were diluted to the (Yong et al., 2020; Rubio et al., 2020a; Mamun et al., 2023). concentration of 100 μg/mL in distilled water, or in culture medium Environmental plastic waste differs in its chemical composition. (supplemented RPMI-1640, Biowest Inc.) supplemented with 10% fetal Approximately 90% of the total amount of plastics consists of high- bovine serum (FBS), 1% glutamine, 2.5 μg/mL Plasmocin, and then density polyethylene, low-density polyethylene, polyvinyl chloride, analyzed by transmission electron microscopy (TEM) with a JEOL JEM- polystyrene, polypropylene, and polyethylene terephthalate (Tursi 1400 instrument (JEOL LTD, Tokyo, Japan). In addition, the hydrody­ et al., 2022). Consequently, it is assumed that environmental pollutants namic size and the Z-potential parameters were assessed by dynamic MNPLs are representative of these proportions. One of the main plastics light scattering (DLS) and laser Doppler velocimetry (LDV) methodolo­ used for packaging, commercial, and construction purposes is poly­ gies, using a Malvern Zetasizer Nano ZS zen3600 device (Malvern, UK). styrene (PS) (Kik et al., 2020). In addition, pristine PSMNPL beads are also used in the production of cosmetics (Xu et al., 2020). Environmental PSMNPLs, either because of plastic degradation goods (secondary 2.2. Labeling and detection of PSNPLs MNPLs) or as produced at that size for different industrial purposes (primary MNPLs), enter the exposed organism inducing a wide range of To visualize PS-50, PS-200, and PS-500 by confocal microscopy, the hazardous effects (Qiao et al., 2022). Ingestion is considered the main particles were labeled with the commercially available textile dye iDye way of entry of PSMNPLs into the human body. Using in vitro models of Poly Pink, from now iDye. The procedure was adapted from previously the intestinal barriers PSNPLs have shown their ability to translocate the published protocols for labeling micro and nanoscale polymers (Karak­ barrier and affect lymphocytic cells placed after the intestinal barrier olis et al., 2019; Nguyen and Tufenkji, 2022). Briefly, the steps were (Domenech et al., 2020). This supposes that once internalized, PSNPLs performed as follows. Suspensions of 1 mL of de different PSNPLs, at a can interact with blood cell components (as a first instance), later final concentration of 5 mg/mL were prepared and transferred to 1.5 mL reaching other organs and tissues. Consequently, hematopoietic cells are tubes, containing 0.01 g of iDye. The mixture was vigorously vortexed considered a target of PSNPLs exposure. and transferred to a 10 mL glass tube and incubated for 2 h at 70 ◦ C, and The potential toxic effects of PSNPLs may change by various factors, then cooled at room temperature. Nine mL of Milli-Q water was then including size and surface charge. In general, it is assumed that small added to the tube, and the suspension was centrifuged at 4000 rcf on an size and positive charge favor cell internalization and hazardous effects Amicon® Ultra-15 centrifugal Ultracel®-100 K filter 1 × 10 5 MWCO for (Halimu et al., 2022). Nevertheless, the effect of surface charge is 15 min. This step was repeated twice to remove the excess of iDye. The ambiguous, with negatively charged PSNPLs inducing a higher stained particles were stored at 4 ◦ C protected from light until needed. agglomeration modulating the toxic effects in ascidians (Eliso et al., To determine the labeling and fluorescence spectra, particles were 2020). Furthermore, positively charged PSMNPLs were more toxic on examined by confocal microscopy. To such end, working suspensions at human nasal epithelial cells than negatively charged ones (Huang et al., the concentration of 400 μg/mL were prepared. Two drops (20 μL) were 2022). Regarding the size effect, it is assumed that smaller sizes can placed on previously ethanol-washed glass slides and covered with a easily be internalized by cells and cross biological barriers (Ramsperger thin glass coverslip. The whole procedure was carried out under a et al., 2022). Nevertheless, in vivo results are contradictory. Thus, in the laminar flow cabinet. Aggregates of particles were then analyzed using a white-leg shrimp, although small PS sizes were more bioavailable and Leica TCS SP5 confocal microscope. The excitation wavelength was set exhibited greater damage in the guts and gills, larger sizes increased the at 561 nm, and the emission spectra were collected between 585 and biomarkers of oxidative stress and altered microbiota components (Zhou 700 nm. The emission was analyzed using the Leica Application Suite X et al., 2023). In zebrafish, larvae exposed to high-density polyethylene 3.7.5.24914 (Leica Microsystems CMS GmbH Wetzlar, Germany). Im­ showed that small size caused morphological changes in the gastroin­ ages were collected under the same conditions, as further explained in testinal cells while larger size damaged the mechanosensory receptors in section 2.9. the fish’s lateral line system (Kim et al., 2022). Especially relevant is the study carried out in mice with two different-sized polyethylene MPLs testing different parameters detecting intestinal function (gene expres­ 2.3. Cell culture sion related to epithelial, permeability, and inflammatory biomarkers) where the most marked deleterious effects were found after co-exposure Human hematopoietic cell lines TK6, THP-1, and Raji-B were used with the two sizes (Djouina et al., 2022). for this study. All the cells were purchased from Sigma Aldrich (MO, Aiming to determine the role of MNPL size on their potentially USA). TK6 as a human lymphoblastoid cell line is one of the standard hazardous effects, we have assessed the toxic effects of different sizes of mammalian cell lines used for in-vitro genotoxicity/mutagenicity tests. PSNPLs (50, 200, and 500 nm). Since the blood compartment is THP-1 is a human leukemia monocytic cell line, which is extensively considered the first target, once MNPLs have crossed the biological used to study monocyte/macrophage functions, toxicity mechanisms, barriers, blood cells were selected. Thus, three different human he­ signal-transduction pathways, and nutrient and drug transport. matopoietic cell types such as Raji-B (as a B-lymphocyte model), THP-1 Furthermore, Raji-B is the first continuous human B lymphocyte cell line (as a macrophage model), and TK6 (as a lymphoblastoid model) were of hematopoietic origin. All the mentioned cell lines were maintained in used. Cell viability, cellular uptake, intracellular ROS generation, and supplemented RPMI-1640 (Biowest Inc) medium supplemented with mitochondrial dysfunction were the evaluated effects. 10% FBS, 1% glutamine, and 2.5 μg/mL Plasmocin. All cell lines were cultured at 37 ◦ C and 5% CO2 and, to maintain the cells, the medium was changed with fresh medium every 2–3 days. 2 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 2.4. Treatment of cells fluorescence inside the cells was measured by flow cytometry (CytoFlex, Beckman Coulter, USA) with excitation/emission spectra of 561/630 nm To study the potentially toxic effects of PSNPLs, cells were exposed to respectively. A total number of 10,000 cells were scored for all the the selected concentrations of PSNPLs (PS-50, PS-200, and PS-500) conditions, and the data were analyzed using the Cytexpert software. diluted in RMPI 1640 supplemented medium, as described in the pre­ vious section. To proceed, the density of 5 × 105 cells/mL in the volume of 200 μL were seeded on 96 well plates (1 × 105 cells/well) and exposed 2.9. Intracellular ROS production and analysis to the different concentrations of PSNPLs and grown for 3 h, 24 h, and 48 h, according to the experimental endpoints. The production of intracellular ROS was measured by the dihy­ droethidium (DHE) method. The three cell lines were exposed to PSNPLs 2.5. Cell viability assay with different concentrations ranging from 0 to 50 μg/mL for 3, 24, and 48 h. After the end of the exposure time, the cells were centrifuged, The viability of the three cell lines after exposure was measured pelleted, and incubated with 10 μM of DHE in PBS for 30 min at 37 ◦ C. using the Beckman counter method. After seeding cells on 96 well plates After DHE exposure, cells were analyzed by flow cytometry (CytoFlex, with a concentration of 5 × 105 cells/mL, they were exposed to the Beckman Coulter, USA). As a positive control, cells were treated with 80 different concentrations of PSNPLs (0, 50, 100, 150, and 200 μg/mL) for μM of antimycin-A and incubated for 45 min at 37 ◦ C. The fluorescent 24 h to assess cell toxicity. After exposure, cells were diluted 1:100 to intensity was measured at 488–605 nm (excitation-emission spectrum) ISOTON and counted with a ZTM Series Coulter counter (Beckman and 10,000 cells per sample were scored and analyzed with the Cytex­ Coulter Inc., CA). The obtained percentage values were calculated as the pert software. average number of the cells counted for each treatment, compared to the untreated control culture values. 2.10. Mitochondrial membrane potential assay 2.6. Cellular uptake assessment with transmission electron microscopy To determine potential mitochondrial damage caused by PSNPLs in (TEM) Raji-B, THP-1, and TK6 cells, the mitochondrial membrane potential assay was utilized (MitoProb TMRM assay kit, Thermofisher Scientific, Among the different tools to assess PSNPLs internalization, TEM was USA). The cells were seeded in 96 well plates with a concentration of 5 used. To such end, cells were grown in T25 cell culture flasks to reach × 105 cells/mL and exposed to all sizes of PSNPLs with concentrations the density of 1.7 × 106 cells/mL. Then, each flask was treated with 3 mL (0, 50, and 100 μg/mL) lasting for 24 h. After exposure, the cells were of a concentration of 50 μg/mL of each PSNPL, previously diluted in washed twice with PBS and then they were centrifuged and the pellet RMPI 1640, for 24 h. After exposure, cells were centrifuged and the obtained was resuspended in PBS. The fluorescence intensity was pellet was fixed in 2.5% (v/v) glutaraldehyde (EM grade, Merck, measured by flow cytometry (CytoFlex, Beckman Coulter, USA) with the Darmstadt, Germany) and 2% (w/v) paraformaldehyde (Hatfield, PA, excitation/emission of 561/585 nm, respectively. For each concentra­ UK) in 0.1 M cacodylate buffer (Sigma-Aldrich, Steinheim, Germany), tion, the total number of 10.000 cells was scored and data was analyzed pH 7.4. Cells were processed following conventional procedures, as by the Cytexpert software. previously described (Annangi et al., 2015). Briefly, samples were first post-fixed with osmium trioxide, dehydrated in acetone, later embedded in Eponate 12™ resin (Ted Pella Inc., Redding, CA), and finally poly­ 2.11. Statistical analysis merized at 60 ◦ C and cut with an ultramicrotome. Ultrathin sections were placed in copper grids and contrasted with uranyl acetate and All data resulted from the average of three independent experiments, Reynolds lead citrate solutions and observed using a JEOL 1400 (JEOL including duplicates of each one of them. Data were analyzed using LTD, Tokyo, Japan) transmission electron microscope equipped with an GraphPad Prism 7 software, and the statistical analysis was performed Erlangshen (CCD GATAN ES1000W) camera. using the two-way ANOVA with Tukey’s multiple comparison test unless stated otherwise. Statistical significance was defined as a *P ≤ 0.05, **P 2.7. Cellular uptake and localization of PSNPLs analysis with confocal ≤ 0.01, ***P ≤ 0.001. microscopy 3. Results and discussion The iDye-labeled PSNPLs (iDye-PSNPLs) were used to determine PSNPLs’ internalization by confocal microscopy in the three selected cell 3.1. Characterization and dispersion of PSNPLs lines. To this end, 80,000 cells were seeded in glass bottom microwell dishes (MatTek, Ashland, OR, USA) and exposed to iDye-PSNPLs (100 The present study determined the average size and shape of the μg/mL) for 24 h. The samples were washed with PBS, and nuclei and cell supplied pristine PSNPLs, with nominal sizes of 100–200, 400–600, and membranes were stained with 1:500 Hoechst 33,342 (ThermoFisher 400–900 nm by using TEM. The obtained average sizes for PS-50, PS- Scientific, Carlsbad, CA, USA) and 1:500 Cellmask™ Deep Red plasma 200, and PS-500 wetted in distilled H2O, or in supplemented RPMI-1640 (ThermoFisher Scientific, Carlsbad, CA, USA) respectively, for 15 min at medium, are indicated in Fig. 1. In general, they are in accordance with room temperature. The iDye-PSNPLs of different sizes were visualized the manufactured specified range of sizes. Furthermore, all the shapes of intracellularly at emission wavelengths of 585 nm, under a Leica TCS the used PSNPLs were spherical, both in distilled H2O and in supple­ SP5 confocal microscope. Several fields were selected randomly per mented RPMI-1640. Table 1 shows the hydrodynamic radius (or mean sample, and the images were processed using ImageJ software having size diameter) determined with a Zetasizer Nano ZS device, both in Fiji extension. distilled H2O and in supplemented RPMI-1640. It is important to point out the protein corona generated when dispersed in the culture medium, 2.8. PSNPLs internalization assessment with flow cytometry and the tendency to agglomerate when dispersed in supplemented RPMI-1640 over water dispersion. Moreover, the measurement of the Z- Aiming to quantify the cellular uptake of PSNPLs, flow cytometry potential in supplemented RPMI-1640 revealed less stability, as re­ was assessed. All three cell lines were treated for 24 h with 100 μg/mL of flected by the lower Zeta values when compared to PSNPLs dispersed in labeled iDye-PSNPLs. After exposure, cells were washed, collected, H2O. Finally, the polydispersity index (PDI) values were close to zero centrifuged, and resuspended to 7.5 × 105 cells/mL in PBS. The suggesting they were monodispersed. 3 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Fig. 1. The graphs indicate the size distribution (mean ± SD) and polydispersity index in water and in supplemented RPMI-1640, of PS-50 (a, c); PS-200 (e, g); and PS-500 (i, k). The TEM images show round shapes in both water and in supplemented RPMI-1640 for PS-50 (b, d), PS-200 (f, h), and PS-500 (j, l). Table 1 Characteristics of the selected PSNPLs when dispersed in water or in supplemented RPMI-1640 cell culture medium. The hydrodynamic radius or average size in diameter (Z-average), the zeta potential, and the polydispersity index (PDI) of PS-50, PS-200, and PS-500 nm are shown, Data are represented as (mean ± SD). Milli-Q H2O SUPPLEMENTED RPMI-1640 PS-50 PS-200 PS-500 PS-50 PS-200 PS-500 Z-average 94.00 ± 0.86 188.00 ± 2.98 488.00 ± 7.09 94.50 ± 0.39 185.00 ± 2.10 588.70 ± 5.37 PDI 0.03 ± 0.02 0.04 ± 0.03 0.03 ± 0.02 0.09 ± 0.01 0.05 ± 0.03 0.26 ± 0.13 Z-potential − 44.90 ± 0.49 − 45.00 ± 0.24 − 50.40 ± 0.94 − 11.70 ± 3.05 − 10.10 ± 1.86 − 16.00 ± 0.81 3.2. Visualization and spectral analysis 3.3. Cell viability PSNPLs were successfully labeled with iDye, using the previously To assess the cytotoxic potential of the three different PSNPLs’ sizes, reported method (Nguyen and Tufenkji, 2022). The colored particles TK6, THP-1, and Raji-B cells were exposed to a range of concentrations were easily visualized by confocal microscopy in all three cases (Fig. 2a (0–200 μg/mL). The obtained results are indicated in Fig. 3. The data and b, c) and the obtained emission spectra from 585 to 700 nm are revealed no significant decrease in cell viability, independently of the depicted at the right of each image. The peak of emission was consis­ PSNPLs size and the concentration used, after exposures lasting for 24 h. tently between 610 and 620 nm. On the other hand, no fluorescence was Since none of the concentrations of PSNPLs in any of the cell lines was detectable for any of the not labeled PSNPLs. Interestingly, no back­ cytotoxic, the study further utilized and tested the sub-toxic doses of ground fluorescence was observed indicating a lack of leaching. PSNPLs (≤100 μg/mL) for the rest of the experiments. In agreement, the exposure of PS-50 to the same cell types (Raji-B, THP-1, and TK6) as well as in human colorectal adenocarcinoma (Caco-2) cells did not result in a Fig. 2. Visualization of PS-50, PS-200, and PS-500 labeled with iDye Poly Pink (a, b, and c, respectively). At the right, their respective spectra are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Fig. 3. Cell viability effects of different sizes and concentrations of PSNPLs in various hematopoietic cell lines. (A) The percentage of cell viability in Raji-B cells. (B) The percentage of cell viability in THP-1 cells. (C) The percentage of cell viability in TK6 cells. Exposures last for 24 h. Data are represented as (mean ± SEM). decrease in cell viability in any of them and at various concentrations another and overcome the limitations among them to provide robust and time points (Cortés et al., 2020; Rubio et al., 2020b). Besides, information on the cellular internalization of PSNPLs. PSNPLs (100 nm and 1 μm) exposure in mouse hippocampal neuronal (HT22) cells yielded a mild cytotoxic response only with the smaller 3.5. Transmission electron microscopy (TEM) sized PS-100 but not PSNPLs of 1 μm indicating a possible size effect (Liu et. al 2022). In addition, PSNPLs (20 and 70 nm) were able to reduce cell The first approach to evaluate the cellular uptake of non-fluorescent viability when used to expose human alveolar epithelial cells (A549) at pristine PSNPLs in the selected cell types was TEM. This technique helps different concentrations (Xu et al., 2019). to locate and visualize PSNPLs in the cell ultrastructure. The obtained TEM images demonstrated the significant internalization of the different 3.4. Cellular internalization of PSNPLs using different analytical tools sizes of PSNPLs (50 μg/mL, 24 h) in all the selected cell lines (Fig. 4). According to the observed micrographs, PSNPLs were found localized in The assessment of cellular internalization or uptake of PSNPLs was or near different cellular compartments based on their size, apart from undertaken by utilizing various analytical tools like TEM, confocal mi­ being present in the cytoplasm of the cells. TEM images showed PSNPLs croscopy, and flow cytometry considering they could complement one incorporation in different cell organelles like mitochondria of Raji-B Fig. 4. Representative TEM images showing the cellular internalization and localization of the three different sizes of pristine PSNPLs in Raji-B, THP-1, and TK6 cells after treatments lasting for 24 h. Arrows in red indicate PSNPLs in mitochondria, yellow in cytoplasmic regions, green in the nucleus, and blue in the endoplasmic reticulum. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 5 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 cells and endoplasmic reticulum of THP-1 cells. Furthermore, they were and PS-500 when studied in TEM, confocal microscopy, and flow able to reach the nuclei, as observed in TK6 cells. cytometry. Moreover, we found there was a cell type-specific cellular internalization considering Raji-B was more prone to the uptake of 3.5.1. Confocal microscopy PSNPLs followed by THP-1, and the lowest internalization by TK6 cells. Due to the success in staining PSNPLs (iDye-PSNPLs) they could be In support, the cellular internalization of PS-50 was also observed in the used in confocal microscopy. The confocal images of the treated cells same cell types (Raji-B, THP-1, and TK6) studied by flow cytometry revealed the significant internalization of PS-50, PS-200, and PS-500. (Rubio et al., 2020b). In agreement with our study, there was a They were mostly present in the cytoplasm and surrounding the nuclei size-dependent cellular internalization of PSNPLs in Caco-2 and A549 in TK6, THP-1, and Raji-B cells after exposures lasting for 24 h (Fig. 5). cell lines wherein PS-70 were highly internalized as compared to bigger Moreover, there was a size-dependent cellular internalization in all the sizes (PS-200 and PS-500), analyzed by both confocal microscopy and cell lines, PS-50 being the most internalized. We assume that this flow cytometry, suggesting the smaller sizes were readily accessible by elevated entry of PS-50 was due to their smaller size. the A549 and Caco-2 cells (Zhang et al., 2022). In addition, Banerjee et al. (2022) indicated that the smaller size of PSNPLs (i.e., 50 nm) was significantly internalized in HepG2 cells as compared to PS-200 and 3.6. Flow cytometry PS-500 nm, when visualized by confocal microscopy. Furthermore, it may be pointed out that the smaller size tested (i.e., PS-70 nm) would Aiming to quantify the cellular internalization of the different sizes enter via clathrin- and caveolae-mediumted endocytosis as well as of labeled PSNPLs flow cytometry was used. The intracellular presence phagocytosis, while the larger particles like PS-200 and PS-500 nm of iDye-PSNPLs (100 μg/mL) in the treated cells was determined ac­ could enter the A549 and Caco-2 cells only through phagocytosis and cording to the fluorescence intensity, as a measure of the percentage of this could explain the higher internalization of the smallest NPLs (Zhang internalized cells. The obtained results are indicated in Fig. 6. Results et al., 2022). indicate a cell-dependent internalization capacity according to the The usage of different analytical tools for cellular internalization in ranking Raji-B > THP-1 > TK6. For Raji-B and THP-1 cells, PS-50 were the present study was justified as no one tool could completely capture those showing the highest internalization, which would agree with their the NPL’s internalization thoroughly due to various factors. For small size. TK6 cells showed the lowest internalization, although the instance, the low atomic number of polymers in MNPLs could render detected uptake was statistically significant. This data confirms two them to scatter electrons weakly resulting in poor contrast under TEM things, such as a) cell internalization levels depend on the cell type, and (Sawyer et al., 2008). In addition, it warrants laborious sample prepa­ b) the uptake is size dependent, showing greater internalization with the ration and time consumption which possibly make it irrelevant to smaller size. analyze the high number of particles needed for representative studies In the current study, the data revealed there was a size-dependent (Ivleva, 2021). Moreover, MNPLs labeled with fluorophores could cellular internalization, PS-50 being highly internalized over PS-200 Fig. 5. Confocal microscopy images showing the cellular internalization of different sizes of iDye-PSNPLs (100 μg/mL) in Raji-B, THP-1, and TK6 cells after treatments lasting 24 h. Fluorescent iDye labeled PSNPLs are red, the cell membranes are green (cell mask), and the nuclei of the cells are blue (Hoechst 33,342). Magnification 63x. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 6 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Fig. 6. Histograms showing the cell internalization of the different sizes of PSNPLs in the selected hematopoietic cells by using flow cytometry. (A) The percentage of cellular internalization in Raji-B cells, (B) in THP-1 cells, and (C) in TK6 cells. Exposures last for 24 h. Data are represented as (mean ± SEM). *P < 0.05, ***P < 0.001 (2-way ANOVA and Student’s t-test). possibly shed the fluorophores in cell culture medium thereby the Similarly, amino-modified PS-20, unmodified PS-20, and PS-50 nm observation of fluorescence intracellularly might be due to free fluo­ caused loss of MMP, enhanced proton leak from mitochondria, as well as rophores (Tenuta et al., 2011). Also, the potential leaching effect of elevated adenosine triphosphate (ATP) generation, and mitochondrial fluorophores from the labeled PSNPLs was reported (Catarino et al., oxidative phosphorylation in a dose-dependent way in A549 cells 2019). Fluorescent-labeled PSNPLs (500–1000 nm) were not able to (Halimu et al., 2022). cross the epithelial barrier of zebrafish larvae rather fluorophores alone were detected in the internal tissues. Apart, the internalization of 3.8. Induction of intracellular reactive oxygen species (iROS) PSNPLs could be read in flow cytometry due to an increase in the cell complexity; however, the tendency of PSNPLs to adhere to the cell To understand the adverse health effects of PSNPLs from a biological surface may also reflect the increase in cell volume thereby affecting the point of view, different biomarkers could be analyzed in human cells. actual estimation of their internalization in that technique (Lesniak One of the most important biomarkers detecting cell toxicity and cellular et al., 2013; Zhang et al., 2022). stress could be iROS. The role of nascent oxygen species in cell signaling under normal conditions was well established; however, their imbalance 3.7. Loss of mitochondrial membrane potential due to oxidative stress could trigger DNA damage or cellular death (Bidooki et al., 2022). Furthermore, the imbalance in the oxygen free Considering the previous data showing PSNPLs’ interaction with radicals could be implicated in many debilitating diseases such as mitochondria, potential damage to these organelles was evaluated. cancer-causing, cardiovascular, and neurological disorders, among Thus, the loss of mitochondrial membrane potential (MMP) was chosen others (Frijhoff et al., 2015). The assessment of the generation of iROS, as a biomarker of effect. Flow cytometry was used to assess the loss of by PSNPLs exposure in the different cell types, showed that there were MMP, using the TMRM assay, showing that entering of PSNPLs into the significant size- and time-dependent increases in the percentage of iROS cells induced significant effects on the mitochondrial function, by in all the tested cell lines. Interestingly, significantly elevated levels of decreasing the MMP values. Results show that all PSNPLs sizes were able iROS were found in Raji-B cells treated with larger-sized PSNPLs (e.g., to cause the loss or decrease in MMP values at the tested concentrations PS-500; 25 μg/mL) compared to PS-50 and PS-200 nm (Fig. 8). On the (50 and 100 μg/mL) in Raji-B and THP-1, cells when compared to their other hand, PS-200 treated THP-1 (25 μg/mL) and TK6 (5–50 μg/mL) respective controls after 24 h (P < 0.05) (Fig. 7). On the other hand, TK6 cells showed a significantly increased production of iROS (according to cells did not show any change in MMP levels at the tested concentra­ the results shown in Fig. 8), in comparison to PS-50 and PS-500 nm after tions, as compared to their corresponding untreated controls after exposures lasting for 3 h (P < 0.05). exposure to all sizes of PSNPLs for 24 h. This lack of effect could possibly Furthermore, when the exposure time was increased to 24 h, resulted be attributed to the non-interaction of PSNPLs with the mitochondria of in significant increases in iROS in PS-500 treated THP-1 only (10–50 μg/ the TK6 cells, as it was observed by TEM, where no presence of PSNPLs mL) (P < 0.05) (see Supplementary Fig. S1), indicating the possible time in or near the mitochondria of TK6 cells could be detected. Like our data and cell-dependent transient effect. In addition, the obtained data did on the size-dependent loss of MMP in Raji-B cells, a previous study also not indicate any increase in iROS with an increase in time up to 48 h for demonstrated the size-dependent MMP effects due to exposure of Caco- all sizes of PSNPLs and for all the hematopoietic cell types (see Sup­ 2 cells to PSMPLs sized 0.1 and 5 μm. The cells treated with 5 μm plementary Fig. S2). PSMPLs had a greater loss of MMP as compared to 0.1 μm sized PS (Wu The potential of PSNPLs to induce iROS was reported in several et al., 2019). On the other hand, the smallest size of PSNPLs had a sig­ studies as well. Like our study, Rubio et al. (2020b) assessed the gen­ nificant effect on the mitochondrial membrane potential in THP-1, eration of iROS in the same cell types (Raji-B, THP-1, and TK6) after possibly indicating a cell type-specific effect. Besides, there is no spe­ treating them with PS-50 at different concentrations and time points. cific study comparing the size effect of PSNPLs on the mitochondrial The study demonstrated that there was a cell type-dependent induction dysfunction in THP-1 cells, our data proved relevant since the exposed of iROS since PS-50 (at 50 μg/mL) treated TK6 and THP-1 cells showed THP-1 cells were more responsive to different sizes of PSNPLs, in com­ significant increases of iROS after 3 h, albeit there were more elevated in parison to the assessed cells like TK6. Moreover, amino-modified TK6 even after 24 h post-treatment. On the contrary, THP-1 cells were PSNPLs (50 nm) showed a profound effect on mitochondrial metabolic not responsive to PS-50 nm induced iROS at any of the doses and time activity and in the loss of MMP in a time and dose-dependent manner, as points. Furthermore, a recent study also showed that PSNPLs could observed in the exposed alveolar macrophages (Deville et al., 2020). trigger iROS in a size-dependent manner in Caco-2 cells wherein the 7 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Fig. 7. The graphs indicate the loss of mitochondrial membrane potential after exposure to different sizes of PSNPLs viz. PS-50, PS-200, and PS-500 nm in Raji-B (A), THP-1 (B), and TK6 cells (C) after 24 h of treatments. Data are represented as (mean ± SEM). *P < 0.05, ***P < 0.001 (2-way ANOVA and Student’s t-test). smaller-sized PS-300 were able to induce higher iROS, when compared overexpression of the immunoresponsive gene 1 (irg1) protein was to PS-500 nm and micron-sized PSNPLs (Wang et al., 2020). Similarly, involved in the increased generation of ROS (as well of other inflam­ PSNPLs of 40 nm were able to alter the oxidative status in the exposed matory factors). Interestingly, and as we have observed in our study, the human bronchiolar (BEAS-2B) and in alveolar (HPAEpiC) epithelial induction of ROS by PSNPLs has been associated with the presence of cells, as there were significantly elevated levels of iROS in them after 24 mitochondrial damage in the mouse macrophages RAW 264.7 (Chen h, which could possibly be implicated in lung injury (Yang et al., 2021). et al., 2023). This agrees with our recent data exposing primary human Moreover, the surface functionalized as well as non-functionalized nasal epithelial cells to PSNPLs, where the induction of ROS and the loss PSNPLS with varying sizes (amino-functionalized PS-20, non-­ of mitochondrial membrane potential were associated with the modu­ functionalized PS-20, and PS-50 nm) triggered a dose-dependent sig­ lation of the autophagy pathway in the form of the accumulation of nificant increase in iROS, as well as the role of increased expression of LC3-II and p62, as models of autophagy markers (Annangi et al., 2023). NADPH oxidase 4 in PSNPLs induced epithelial to mesenchymal tran­ This link between increased intracellular ROS and reduced mitochon­ sition in A549 cells (Halimu et al., 2022). Similarly, the ability of ami­ drial membrane potential (and its consequent mitochondrial damage) nated PSNPLs to induce ROS has been recently detected in murine has been extensively reviewed by Shadel and Horvath (2015). alveolar macrophage (MH-S) cells (Wu et al., 2023). Looking for the In summary, it was evident that PSNPLs had the potential to induce mechanism of production of ROS and the resulting oxidative stress, and iROS due to several contributing factors like the nature of cell type, their using a proteomic approach, the authors detected that the different sizes, doses, and surface characteristics. 8 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Fig. 8. The histograms show the percentage of intracellular reactive oxygen species due to exposure of different sizes of PSNPLs viz., PS-50, PS-200, and PS-500 in Raji-B (A), THP-1 (B) and TK6 cells (C) after 3 h of treatments. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (2-way ANOVA and Student’s t-test). 4. Conclusions Author contribution statement The constant weathering or leaching of polymer plastics into sec­ RM and AH planned the experiments. AT, BA, AV, GB, JM, and SP ondary MNPLs due to various environmental factors is a major cause of carried out the experimental part. AT and BA analyzed the data, carried concern for potential human health effects. In context, this study may out the statistical analysis, prepared tables/figures, and wrote the draft provide necessary inputs for the hazard and risk assessment of MNPLs manuscript. BA, RM, and AH wrote the final manuscript. like PSNPLs when they encounter important human blood immune cell types through various routes of exposure. The release of MNPLs into the Declaration of competing interest environment in various sizes and forms may need to be considered while assessing their risk. As evidenced, PSNPLs in various sizes could trigger The authors declare that they have no known competing financial biological responses such as cellular internalization, loss of MMP, and interests or personal relationships that could have appeared to influence generation of iROS in exposed cells in a time-dependent manner. These the work reported in this paper. biological responses may have cascading effects in altering or modu­ lating the cellular functions and survival mechanisms, although they Data availability may not necessarily lead to cell death. Moreover, it is also important to understand the chronic toxic effects of PSNPLs with different sizes since Data will be made available on request. there may exist a possibility of constant or continuous environmental exposure to humans. Acknowledgments AT and JM hold Ph.D. FI fellowships form the Generalitat de Cata­ lunya. AV was supported by a Ph.D. fellowship from the National 9 A. Tavakolpournegari et al. Chemosphere 325 (2023) 138360 Agency for Research and Development (ANID), CONICYT PFCHA/ Kik, K., Bukowska, B., Sicińska, P., 2020. Polystyrene nanoparticles: sources, occurrence in the environment, distribution in tissues, accumulation, and toxicity to various DOCTORADO BECAS CHILE/2020–72210237. AH was granted an organisms. Environ. Pollut. 262. https://doi.org/10.1016/j.envpol.2020.114297. ICREA ACADEMIA award. Kim, S.A., Kim, L., Kim, T.H., An, Y.J., 2022. Assessing the size-dependent effects of This project (Plasticheal) has received funding from the European microplastics on zebrafish larvae through fish lateral line system and gut damage. Union’s Horizon 2020 research and innovation programme under grant Mar. Pollut. Bull. 185 (Pt A), 114279 https://doi.org/10.1016/j. marpolbul.2022.114279. agreement No 965196. 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