Environmental Toxicology and Pharmacology 100 (2023) 104140 Contents lists available at ScienceDirect Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap Effects of true-to-life PET nanoplastics using primary human nasal epithelial cells Balasubramanyam Annangi a, Aliro Villacorta a, b, Lourdes Vela a, c, Alireza Tavakolpournegari a, Ricard Marcos a, *, Alba Hernández a, * a Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain b Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Iquique, Chile c Faculty of Health Sciences Eugenio Espejo, Universidad UTE, Quito, Ecuador A R T I C L E I N F O A B S T R A C T Edited by Dr. M.D. Coleman Since inhalation is a relevant exposure route, studies using appropriate micro/nanoplastic (MNPLs) models, representative targeted cells, and relevant biomarkers of effect are required. We have used lab-made poly­ Keywords: ethylene terephthalate (PET)NPLs obtained from PET plastic water bottles. Human primary nasal epithelial cells Nanoplastics (HNEpCs) were used as a model of the first barrier of the respiratory system. Cell internalization and intracellular PET reactive oxygen species (iROS) induction, as well as the effects on mitochondria functionality and in the mod­ HNEpCs ulation of the autophagy pathway, were evaluated. The data indicated significant cellular uptake and increased Uptake Oxidative stress levels of iROS. Furthermore, a loss of mitochondrial membrane potential was observed in the exposed cells. Mitochondrial membrane potential Regarding the effects on the autophagy pathway, PETNPLs exposure significantly increases LC3-II protein Autophagy expression levels. PETNPLs exposure also induced significant increases in the expression of p62. This is the first study showing that true-to-life PETNPLs can alter the autophagy pathway in HNEpCs. 1. Introduction stained counterparts (Xu et al., 2022a). Nevertheless, they cannot be considered representative secondary MNPLs of those polluting our The presence of microplastics and nanoplastics (MNPLs) in the environment. In such context, the degradation in the laboratory of different environmental compartments is of environmental/human plastic goods, to obtain what is denominated true-to-life MNPLs, is health concern (Chang et al., 2022). Most of the environmental MNPLs. gaining interest. Among the different polymeric materials constituting These secondary MNPLs, resulting from the physical/chemical/ bio­ plastic goods, polyethylene terephthalate (PET) stands up. The semi­ logical degradation of plastic goods, pose doubts about their potential crystalline and consistent transparency properties of plastic PETs have health hazards. Consequently, and due to the limited amount of evi­ extended their use for stretch-blown bottles, packaging, and dence, they are considered emergent environmental pollutants (Rubio manufacturing fibers for textile products (Ghasemi et al., 2021). These et al., 2020a; Gigault et al., 2021; Wu et al., 2022). Accordingly, further relevant PET characteristics have extended their use and, inevitably, studies are required to decipher the real risk associated with their most of these PET goods are wasted in landfills or other environmental environmental exposure. To such end, and to design sound experiments compartments. These are the reason why most of the proposed methods to reach such a goal, three main components must be considered in these to degrade plastic goods to obtain secondary MNPLs use PET goods as a studies: i) representative MNPLs models, ii) representative cell target source. Among the different experimental proposals, it should be noted models, and iii) an enough wide set of effect biomarkers. those using UV-laser ablation to obtain nanoplastics from PET bottles The hazard assessment of MNPLs faces a major challenge such as the (Magrì et al., 2018, 2021), the mechanical grinding of PET water bottles obtention of representative materials acting as MNPL models. During the (Rodríguez-Hernández et al., 2019; Roursgaard et al., 2022), or the last few years, polystyrene MNPLs have been used as a reference ma­ mechanical milling of PET pellets (Pignattelli et al., 2021; Lionetto et al., terial mainly because they are commercially available under different 2021). Moved that topic, a protocol to produce PETNPLs from water sizes, different surface modifications, and presenting fluorescently bottles by sanding their surface has been recently published (Villacorta * Corresponding authors. E-mail addresses: ricard.marcos@uab.cat (R. Marcos), alba.hernandez@uab.cat (A. Hernández). https://doi.org/10.1016/j.etap.2023.104140 Received 2 February 2023; Received in revised form 14 March 2023; Accepted 29 April 2023 Available online 1 May 2023 1382-6689/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 et al., 2022). Using the such protocol, we have obtained PETNPL sam­ mm mesh. Four grams of the material were placed on 40 mL of pre­ ples (sized around 100 nm) to be used in the current study. At this point, heated to 50 ◦ C 90 % trifluoroacetic acid (TFA) for 2 h under constant it must be emphasized that the environmental presence of PET waste stirring at 200 rpm on a stir heating plate (Heidolph Instruments GmbH both from direct PET goods and from polyester/polyamide/acryl fibers & Co. KG. Schwabach, Germany), and kept under agitation overnight at was calculated to be approximately 61 Mt, which relates to 20 % of the room temperature. After removing big-size agglomerates, the rest of the total plastic waste (Geyer et al., 2017). volume was distributed in equal volumes into glass tubes that were The light density of MNPLs is associated with their airborne pollution centrifuged for 1 h at 2500 RFC. Pellet was resuspended in 400 mL of and with their widespread to remote areas such as the Arctic regions 0.50 % sodium dodecyl sulfate (SDS), sonicated, and transferred to a (Evangeliou et al., 2020). Thus, MNPLs’ airborne pollution is an envi­ 250 mL graduated cylinder and the bigger fraction was left to settle for 1 ronmental problem of increasing concern (Liu et al., 2022a). To deter­ h. The top 100 mL of each cylinder was recovered and washed twice mine the risk associated with such exposure, the use of potential with water and pure ethanol. The obtained pellet was then resuspended respiratory tract target cells is required. In that context, the use of in Milli-Q water at desired concentrations and sonicated before storage human primary nasal epithelium cells (HNEpCs) look very attractive at − 80 ◦ C as depicted in Fig. 1a. For biological application particles because the nasal epithelium is the first barrier of the respiratory tract were prepared using the Nanogenotox protocol (Nanogenotox, 2011) and consequently contacts airborne pollutants, including MNPLs. (Fig. 1b). Interestingly, these types of cells have been recently used to determine the harmful effects of polystyrene nanoplastics (PSNPLs), detecting cell 2.1.2. Labeling apoptosis/necrosis, depending on their surface functionalization (Huang PETNPLs were labeled following an adaptation of previously pub­ et al., 2022). In the same study, the authors observed that in rats exposed lished protocols for different types of microplastics (Karakolis et al., via intranasal, PSNPLs thinned out the nasal mucosa enhancing the 2019; Nguyen and Tufenkji, 2022). Briefly, a working solution of 1 mL expression of transient receptor potential cation channel members. In a of PETNPLs (5 mg/mL) was placed on a 1.5 mL tube containing 0.01 g of recent paper, cells from different parts of the respiratory tract were iDye Poly Pink, iDye from now on. After vortexing and incubation at 70 compared to identify their susceptibility to the harmful effects of flame ◦ C, the mixture was cooled at room temperature and 9 mL of Milli-Q retardants; interestingly, HNEpCs were the ones showing the highest water was added. The 10 mL were centrifuged at 4000 rpm and susceptibility (Chen et al., 2022). It should remember that flame re­ filtered to remove the excess of iDye. Particles were re-resuspended on tardants are additives widely used in the plastic industry (Onoja et al., 10 mL of Milli-Q water, centrifuged twice, and finally resuspended on 2022); consequently, the use of HNEpCs seems appropriate to evaluate 1 mL of Milli-Q water and stored protected from light at 4 ◦ C (Fig. 1c). the hazard of PETNPLs, as potential airborne pollutants. MNPL exposure is considered a source of hazardous health effects in 2.1.3. Characterization humans (Rubio et al., 2020a). Multiple biomarkers of effect including Particles were characterized by confocal microscopy, transmission cytotoxicity, oxidative stress, inflammatory response, genotoxicity, and electron microscopy (TEM), scanning electron microscopy (SEM), even carcinogenicity have been reported to be affected by MNPL expo­ energy-dispersive X-ray spectroscopy (EDS), Fourier-transform infrared sures. Specifically, mitochondria were reported to be a target of MNPLs spectroscopy (FTIR), dynamic light scattering (DLS), and multiangle inducing structural changes (Cortés et al., 2020), as well as alterations in dynamic light scattering (MADLS). the mitochondrial membrane potential (Zhang et al., 2022). Since For confocal microscopy, labeled particles were diluted from the mitochondria play important roles in cell function and survival, their stock solution to a final concentration of 400 µg/mL. 20 µL drops were dysfunction may have dramatic consequences that extend beyond the placed on glass slides, previously washed with absolute ethanol, and a cell (Reddam et al., 2022). Cellular homeostasis is maintained by coverslip was placed over the samples. Drops were then let to dry and autophagy, as an evolutionarily conserved cellular mechanism protect­ analyzed using a Leica TCS SP5 confocal microscope and a Lambda scan. ing cells from intra/extracellular stressors. The lysosomal-autophagic Emission was analyzed using a Leica Application Suite X 3.7.5.24914 system is a common target of environmental pollutants that can Leica Microsystems CMS GmbH Wetzlar, Germany. Images were modulate autophagic flux by increasing it as a protective response, or by analyzed using the same software as described and ImageJ processing blocking its protective role inducing cell death (Martínez-García and and analysis software version 265 1.8.0_172. Mariño, 2020). Interestingly, this mechanism was affected in several For TEM, a carbon-covered grid was dipped in the working solution intestinal epithelial cells after exposure to PSNPLs, resulting in an of 1 mL (200 µg/mL) of PETNPLs. For SEM, a 15 µL drop of the same impaired autophagic flux and the induction of an autophagic response concentration was placed on a silica holder. Samples were let to dry (Xu et al., 2022b). Similar effects have been reported on human um­ overnight and examined using a JEOL JEM 1400 instrument (JEOL LTD, bilical vein endothelial cells where induced autophagy initiation and Tokyo, Japan) operated at 120 kV and a Zeiss Merlin (Zeiss, Oberko­ autophagosome formation were observed related to the PSNPL size (Lu chen, Germany) coupled with an X-Max 20 mm EDS system (Oxford et al., 2022). This information remarks on the usefulness of the auto­ Instruments, Oxford, UK) for TEM and SEM-EDX, respectively. The INCA phagia pathway as a biomarker for identifying the potentially harmful Energy software (INCA, Grinnell, IA, USA) was used to analyze EDS effects of MNPL exposures. signals. According to the above indicated, this study aims to determine the For FTIR, PET films and PETNPLs suspensions were examined using hazardous effects of true-to-life MNPLs on HNEpCs focusing mainly on an ATR Tensor 27 and a Vertex 80 (Bruker Corporation, Billerica, the effects on mitochondria functionality and autophagy pathways. Massachusetts, USA), respectively. Film pieces (3 ×3 cm) from PET bottles were analyzed directly while for the PETNPLs stock solution 2. Materials and methods (10 mg/mL), a drop was placed on a gold mirror and let to dry inside a Petri dish, for one week. The gold mirror was used as a control. 2.1. Preparation, labeling, and characterization of PET To determine the hydrodynamic behavior of PETNPLs, 5 mL (100 µg/mL) was prepared using an airway epithelial cell growth me­ 2.1.1. Preparation dium with a supplemental mix. From the suspension, 1.5 mL was placed Lab-made PETNPLs were prepared following the procedure detailed on a DTS0012 cuvette, and the hydrodynamic size was determined. in our recent work (Villacorta et al., 2022), following the initial idea Additionally, 1 mL was transferred to a DTS1070 cuvette, and the z- reported by Rodríguez-Hernández et al. (2019). Briefly, commercially potential was measured. All evaluations were carried out on a Zeta­ available PET plastic water bottles were sanded using a diamond rotary sizer® Ultra device from Malvern Panalytical (Cambridge, United burr. The powder obtained from this procedure was sieved using a 0.20 Kingdom). 2 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 Fig. 1. (a) Schematic representation of PETNPLs production from the environmentally available water bottles to obtain the initial water-dispersed stock. (b) Preparation of PETNPLs for biologic applications using the Nanogenotox dispersion protocol. (c) Samples from (b) were stored at − 80 ◦ C, as ready-to-use aliquots (RTU) or labeled using iDye PolyPink. 2.2. Cell culture changes in the iROS levels were induced. Exposed cells were analyzed in a CytoFlex flow cytometer (Beckman Coulter, USA) and 20,000 cells Cell culture was performed as recently published (Annangi et al., were evaluated using the CytoFlex software. 2023). The main components such as cryopreserved primary human nasal epithelial cells (HNEpC, catalog no. C-12620), airway epithelial 2.6. Mitochondrial membrane potential assay cell growth media (AEGM) (catalog no. C-21160), and DetachKit (cat­ alog number C-41200) were purchased from PromoCell (GmbH, Ger­ The determination of the mitochondrial membrane potential (MMP) many). HNEpC were selected because the nasal epithelium is considered in HNEpC was performed as recently published (Annangi et al., 2023) the first barrier in contact with environmental air pollutants, including using the MitoProbe™ TMRM kit for flow cytometry (Invitrogen, catalog MNPLs. Moreover, the nasal epithelium is involved in the mucociliary no. M20036). As above indicated, only the highest concentration clearance of foreign particles trapped in the nasal cavity (Ramezanpour (100 µg/mL) was tested once no toxic effects were observed. Exposed et al., 2018; Scherzad et al., 2019). cells were analyzed in a CytoFlex flow cytometer (Beckman Coulter, USA). As a positive control, 1 µL of 5 mM CCCP (carbonyl cyanide 2.3. Cell viability 3-chlorophenylhydrazone) was used. A total of 20,000 single cells per sample were analyzed using the CytoFlex software. Cell viability was performed as recently published (Annangi et al., 2023). Viability was determined by measuring the number of cells after exposure. The number of cells was determined with a Beckman counter 2.7. Total protein extraction and Western blot analysis (Beckman Coulter, Brea, CA). Cells were seeded in 6-well plates and treated with concentrations ranging from 0 to 100 µg/mL of PETNPLs Protein extraction and Western blot were performed as recently for 24 h. After exposure, cell viability was calculated by averaging three published (Annangi et al., 2023). The expression of autophagy-related independent viability experiments compared to the untreated control proteins (LC3-II and p62, for induction and blockage, respectively), (blank control, untreated cells grown in AGEM with the supplemental was assessed by Western blot. From the obtained protein lysate, 50 µg of mix). protein was run on SDS-PAGE gel and transferred to PVDF membranes. After the transfer of proteins, the membranes were blocked, incubated 2.4. Intracellular uptake of iDye labeled PETNPLs by confocal microscopy with primary antibodies against LC3-II, p62, and GAPDH, washed thrice with TBST, and incubated with HRP-conjugated secondary antibody. Cell uptake was determined as recently published (Annangi et al., The membranes were further developed using an enhanced chem­ 2023). Identifying cell uptake is essential for the relevance of the rest of iluminescence system (ECL, Cell Signaling Technologies) and the the biomarkers. In our case cell uptake was determined by confocal quantification of protein expression was determined by ImageJ microscopy. PETNPLs were stained with the textile dye iDye Poly Pink software. (iDye) (Karakolis et al., 2019). Cells were treated with the stained PETNPLs (iDyePETNPLs, 100 µg/mL) for a period of 24 h, and the 2.8. Immunocytochemistry iDyePETNPLs were visualized intracellularly under a Leica TCS SP5 confocal microscope. For image processing, the Imaris 9.5 software was For the cell localization of LC3II and p62, the procedure recently used. published was used (Annangi et al., 2023). Treated cells were fixed, blocked (2 % BSA), and incubated with anti-LC3II and anti-p62 anti­ 2.5. Estimation of iROS by the DCFH-DA assay bodies overnight. The next day, cells were labeled with the secondary antibodies Alexa Fluor 488 goat anti-rabbit and goat anti-mouse for Intracellular ROS detection was performed as recently published anti-LC3II and anti-p62, respectively. The fluorescence images for (Annangi et al., 2023), and based on previous studies (Rubio et al., intracellular LC3II, p62, and iDyePETNPLs, were captured in a Leica TCS 2020b), by using the dichlorodihydrofluorescein diacetate assay SP5 confocal microscope and processed with the Imaris 9.5 software. (DCFH-DA). Since no toxic effects were observed after PETNPLs expo­ Chloroquine was used as a positive control (Cuomo et al., 2019). The sure, only the highest concentration of 100 µg/mL was tested to detect if ImageJ software was used to quantify the fluorescence. 3 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 2.9. Statistical analysis However, the particle size distribution by numbers shows a high per­ centage under 100 nm, with the main peak close to 93 nm. The average The Kolmogorov-Smirnov & Shapiro-Wilk test and Levene’s test of the three independent measurements, as well as the measurement were used to determine the normality and the homogeneity of the data. correlation values, are presented in Fig. 2c and d, respectively. To The Student t-test and the two-way ANOVA with Tukey’s multiple confirm the chemical identity, the SEM/EDS spectra showed that only comparison tests were used for the data showing normal distribution carbon (C), oxygen (O), and silica (Si) were dominant, confirming the and equal variance. Data are represented as mean ± SEM and the sta­ lack of contaminants that could interfere with future hazard assessment tistical significance was defined as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. experiments (Fig. 2e). FTIR spectra confirm that the starting material shares the same peaks reported in the literature (Chen et al., 2013; 3. Results and discussion Johnson et al., 2021). One of the main issues to overcome when true-life MNPLs are used is effectively tracking them at the cellular level. To Electronic microscopy was used to investigate the shape and size demonstrate the suitability of the used iDye Poly Pink confocal images distribution of the obtained material. The data showed the shape of are the first and most reliable evidence. As indicated in Fig. 2g, the particles as irregular (Fig. 2a), as a model representation of what might maximum emission was obtained between the 590–620 nm region and be found in the environment, due to the different weathering conditions the lambda scan reveals maximum fluorescence peaks at 600 and to which polymers would be subjected. It is important to remark on the 615 nm (red line in Fig. 2h). No fluorescence signals were detectable for relevance of using true-to-life MNPLs as the PETNPLs reported here, unlabeled PETNPLs (green in the same figure). This would confirm mainly due to the lack of information on the weathering environmental previous labeling results reported at the micro (Karakolis et al., 2019) or conditions affecting this polymer, despite being one of the most nano (Nguyen and Tufenkji, 2022) scale for plastic polymers. frequently utilized one-use plastics (Geyer et al., 2017). In this way, our experimental approach helps to shed light on the characteristics of po­ 3.1. Cell viability tential environmental MNPLs. The average size distribution of the ob­ tained PETNPLs is presented in Fig. 2b. We found an average particle Cell viability decrease due to PETNPLs exposure was determined, as size of 62.38 nm ± 3.51 (S.E.M). As expected, the calculated poly­ a measure of cytotoxic response (Fig. 3A). More than focusing on po­ dispersity index was high reaching 0.73. Considering the inherent tential alterations in the cell membrane functionality, we have just polydisperse nature of the obtained PETNPLs, DLS measurements were determined the ability of cell growth as a cytotoxicity parameter. The carried out on airway epithelial cell growth media with the supple­ data indicated that PETNPLs were not able to cause any significant mental mix, determining size distribution by intensity and number, as decrease in cell viability at all the tested concentrations, as compared to suggested by the EU guidelines (European Commission, 2011). The untreated control cells. This suggested the exposure of HNEpCs with z-potential of the samples was − 16.10 and the z-average was, as ex­ PETNPLs did not result in cytotoxicity. This lack of decrease in cell pected, higher than the average size measured by TEM, which may be viability after PETNPLs (1–100 µg/mL) exposure agrees with an initial mediated by the formation of the bio-corona on the PETNPLs. These paper reported by Villacorta et al. (2022) using two human lympho­ measurements are represented on the size distribution by intensity blastic cell lines, and it was also observed in different human cell lines where the particles contribute to the signal according to their size. such as colorectal adenocarcinoma (Caco-2), liver cancer line HepG2 Fig. 2. (a) TEM image of PETNPLs. (b) Size distribution on percentage, with an average size of 62.38 nm, a standard error of 3.51, and a calculated polydispersity index of 0.73. (c) DLS size distribution by number (light blue) and intensity (orange). (d) The indicated correlations correspond to (c) measurements. (e) EDS profile obtained from SEM coupled with EDS showing the dominant presence of only carbon (purple line) and oxygen (blue line) with the silicon signal from particle holder (red line). (f) FTIR profile from PET film from bottles (light blue) and from PETNPLS suspension (orange) showing matching peaks corresponding with PET. (g) iDye labeled PETNPLs aggregates emission intensity. (h) Lambda scan of unlabeled PETNPLs (green) and iDyePETNPLs (red). 4 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 Fig. 3. Polyethylene terephthalate nanoplastics’ cytotoxic response in HNEpCs. (A) The graph represents the cell viability after PETNPLs treatment lasting for 24 h using different concentrations vs untreated controls. Data represented as mean ± SEM. (B)The confocal images highlight the cellular internalization of iDyePETNPLs in HNEpCs after exposures (100 μg/mL) lasting for 24 h. i) Untreated control. ii) localization of iDyePETNPLs (in red), possibly in endosomes of treated cells, iii) Presence of iDyePETNPLs (in red) within and surrounding nuclei (blue). Note: cell membrane stained in grey using cell mask. Magnification 63x. and immortalized hepatic (HepaRG) cells (Stock et al., 2021) where the recent past (Yang et al., 2021, Halimu et al., 2022, Shi et al., 2022). In exposure was able to cause mild toxic effects, but at high and unrealistic this study, the ability of PETNPLs to induce iROS was measured in the concentrations. Similarly, slight but significant decreases in cell viability treated cells by the DCFH-DA assay using flow cytometry. Our results were observed in the human alveolar epithelial tumor cell line (A549), revealed that there were significant increases in the percentages of iROS but only at very high (98.4 and 196.79 μg/mL) concentrations (Zhang when the cells were exposed to 100 µg/mL of PETNPLs for 24 h, in et al., 2022). It should be mentioned that the used PET was from a comparison with the observed in untreated cells (Fig. 4A-i and A-ii, commercial source, not a true-to-life PET. In another study, Tolardo P < 0.05). These data concur with the significant internalization of et al. (2022) used PET obtained from laser ablation of plastic water PETNPLs and their presence in the various intracellular regions. In bottles and found toxic effects in Caco-2 and HepG2 cells, but the au­ support of our data, PETNPLs (at 49.2 μg/mL) were able to induce sig­ thors attributed this toxicity to the high degree of surface oxidation due nificant increases in iROS after exposure to human alveolar epithelial to the laser ablation. cells (A549) (Zhang et al., 2022). Interestingly, Aguilar-Guzmán et al. Based on our data, we utilized only 100 µg/mL of PETNPLs for (2022) also showed a slight but significantly elevated iROS production further experiments since no significant decreases in cell viability were in macrophages exposed to different concentrations (7.5–60 μg/mL). detected and, consequently, this concentration could be regarded as a However, this increase did not show a concentration-dependent pattern. possible sub-toxic. In addition, we evaluated different biological re­ Contrary, PETNPLs obtained from commercial plastic food containers sponses such as cellular internalization, imbalance iROS, loss of MMP, did not induce significant iROS in Caco-2 and HepG2 cells (Roursgaard and modulation of autophagy pathway due to PETNPLs after 24 h of et al., 2022), as also obtained in human cell lines like THP-1 (monocytes) exposure only, as this may be sufficient to provide the necessary inputs and TK6 (lymphoblasts) (Villacorta et al., 2022). in relation to hazard and risk assessment of our true-to-life PETNPLs. Furthermore, the functions of mitochondria could be affected due to MNPLs exposure since mitochondria are susceptible to oxidative stress 3.2. Intracellular localization, generation of iROS, and loss of MMP by or iROS which could lead to a decrease in MMP or their injury (Zorov PETNPLs exposure et al., 2014; Jeong et al., 2016; Hu and Palić, 2020). Considering, whether PETNPLs could affect mitochondrial functions such as loss of The confirmation of the internalization of MNPLs into cells is a MMP was determined in PETNPLs exposed HNEpCs after 24 h of treat­ necessary step to be conducted when evaluating the cellular response, as ment. The generated data suggested a significant loss of MMP due to evidenced in a recent study by Liu et al. (2021). Authors demonstrated PETNPLs exposure, in comparison to untreated cells (Figs. 4B-i and B-ii, that the used polystyrene MNPLs were significantly internalized by the P < 0.05). The imbalance of iROS and loss of MMP could be related to a cells via different processes like passive membrane penetration and significant cellular internalization of PETNPLs. In support, Zhang et al. active endocytosis. In context, our study determined the internalization (2022) also demonstrated the decrease in MMP induced by oxidative and intracellular localization of PETNPLs in HNEpCs exposed to stress after exposure of A549 cells to PETNPLs; nevertheless, they did 100 µg/mL for 24 h by utilizing confocal microscopy. As shown in not cause cell apoptosis. However, the internalized PETNPLs (at Fig. 3B, iDyePETNPLs were significantly internalized by the cells and 15 μg/mL) in macrophages did not damage mitochondria although there possibly localized in endosomes (Fig. 3B-ii), as well as surrounding and was a slight and constant iROS production (Aguilar-Guzmán et al., within the nuclei of the treated cells (Fig. 3B-iii), in comparison to the 2022). observed in untreated controls. In support, a recent study also demon­ strated the presence of PETNPLs as electron-dense particles in endo­ 3.3. Defective or insufficient autophagy by PETNPLs somes as well as near nuclear regions of macrophages (RAW 264.7). Interestingly, they were able to alter the endosomes’ structure thereby Several studies did indicate that the higher incidence of iROS or affecting their integrity (Aguilar-Guzmán et al., 2022). Similarly, mitochondrial damage by environmental agents like MNPLs, as PETNPLs were mostly internalized in the endo-lysosomes of human observed in this study, was known to play a potential role in the mod­ Caco-2 intestinal epithelial cells (Magrì et al., 2018). ulation of the autophagy pathway (Hu and Palić, 2020; Wang et al., The potential of MNPLs to generate iROS in different human lung 2021; Li et al., 2022). Therefore, this work also evaluated the effects of alveolar and bronchiolar cells has been proven by several studies in the PETNPLs on this pathway by looking at the expression of important 5 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 Fig. 4. (A)The flow cytometry data indicate the generation of intracellular reactive oxygen species (iROS) by PETNPLs in HNEpCs at 24 h post-treatment. i) The dot plots represent the percentage of iROS in PETNPLs exposed cells (far right quadrant) over untreated control⋅H2O2 acted as a positive control. ii) The graph represents the significant increase in the percentage of iROS in PETNPLs treated cells vs untreated control cells. (B) The flow cytometry data suggest the loss of mitochondrial membrane potential (MMP) in PETNPLs exposed HNEpCs for 24 h. i) The dot plots represent the decrease in fluorescence intensity as an indicator of loss of MMP due to the treatment of PETNPLs to HNEpCs (far left quadrant) versus untreated control. CCCP was used as a positive control. ii) The histogram depicts the significant loss of MMP in PETNPLs treated cells vs untreated control cells. Data represented as mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001. autophagy-related protein biomarkers viz., the microtubule-associated bronchiolar epithelial (BEAS-2B) cells. Such an autophagic effect was protein 1 light chain 3 (LC3-II) and the ubiquitin-binding protein attributed to the induction of endoplasmic reticulum stress (Lim et al., (p62), by using western blotting and immunocytochemistry. The for­ 2019). Likewise, PSNPLs (100 and 500 nm) exposure was able to cause a mation or turnover rate of LC3-II, an autophagosome marker, would significant size- and dose-dependent accumulation of autophagosomes indicate the autophagic activity and its upregulation could signal the and reduction in autolysosomes in human umbilical vein endothelial increase in intracellular autophagosomes (Wei et al., 2022). The current cells (HUVECs), as measured by the significant increases in the levels of data suggested that the cells treated with PETNPLs in a combination LC3-II, mCherry-GFP-LC3 puncta, and Beclin 1 autophagic markers (Lu with chloroquine, which acts as an inhibitor of the autophagosome and et al., 2022). Also, varying sizes of PSMPLs (0.5, 1, and 5 μM) raised the lysosomal fusion, significantly increased the expression levels of the levels of expression of LC3-II in exposed HUVECs, indicating their role in LC3-II protein in comparison to untreated cells; nevertheless, the cells the induction of autophagy and cell death (Lee et al., 2021). A similar treated with PETNPLs alone could not result in a significant increase in effect was observed with PSMPLs (2 μM) which elevated the protein the expression of the LC3-II marker as measured by immunoblotting expression levels of LC3-II and Beclin 1 in human kidney proximal (Fig. 5A, P < 0.05). In addition, the cells treated with iDyePETNPLs tubular epithelial cells (HK-2 cells) (Wang et al., 2021). However, showed a significant accumulation of LC3-II, as compared to untreated PSMPLs with 1 µM size were reported as unable to affect the expression cells, by using confocal microscopy, corroborating the potential of levels of LC3-II and p62 in exposed HUVECs (Lu et al., 2023). Like our PETNPLs in modulating the autophagy pathway. Interestingly, the data, a co-localization was observed under confocal microscopy, be­ concurrent presence or co-localization of LC3-II and iDyePETNPLs was tween PSNPLs (100 nm) and LC3-II puncta in exposed mice embryonic also observed in confocal microscopy (Fig. 5B-i, and B-ii, P < 0.05). This fibroblast cells, suggesting they could trigger the formation of auto­ signifies the usage of multiple methods to assess autophagy, a highly phagosomes (Han et al., 2021). dynamic process, using biomarkers like LC3-II as there is no one The p62 would get degraded in the process of clearance of cell debris stand-alone assay that can monitor autophagy (Zhang et al., 2016). in a normal complete autophagy pathway. Nonetheless, impediments in Moreover, there were also no significant increases in the expression the degradation of this protein would indicate the loss of autophagy flux levels of LC3-II in cells treated with rapamycin (positive control), when (Yang and Wang, 2022). Hence, our study demonstrated that the cells analyzed by western blotting. treated with PETNPLs alone, resulted in significant increases in the In support, exposures to other MNPLs like polystyrene nanoplastics expression levels of p62, when compared to the untreated control (PSNPLs) resulted in dose-dependent increases in the accumulation of (Fig. 6A, P < 0.05). A similar effect was also observed under confocal LC3-II, associated with the formation of autophagosomes, in human microscopy wherein there were significant increases in the 6 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 Fig. 5. (A) The protein expression of important autophagy pathway marker i.e., LC3-II after exposure of HNEpCs to PETNPLs, alone or in combination with chloroquine for 24 h. The graph represents the quantitative changes in levels of LC3-II expression in PET-NPLs treated and untreated cells (top) and the respective western blot for LC3-II (bottom). (B) The confocal images represent the accumulation of LC3-II after exposure of HNEpCs to 100 μg/mL of iDyePETNPLs for 24 h. i) The cells with intracellular localization of PETNPLs (red), expression of LC3-II (green), and close association or co-localization of PETNPLs (red) and LC3-II (green) (merge: yellow, shown in inset) compared to untreated control cells. Chloroquine as a positive control for LC3-II (green). Nuclei stained in blue with Hoechst dye. ii) The histogram depicts the measurement of the relative average area of fluorescence (green/LC3-II puncta, A.U.) due to iDyePETNPLs versus untreated control. Magnification 63x. Data represented as mean ± SEM. ***P ≤ 0.001. Note: Rapa and CQ in graphs and blots represent rapamycin and chloroquine, respectively. Fig. 6. (A) The graph suggests the quantitative changes in levels of p62 expression in PETNPLs treated and untreated cells (top) and respective western blot for p62 (bottom). (B) The confocal images represent the accumulation of p62 after exposure of HNEpCs to 100 μg/mL of iDyePETNPLs for 24 h. i) The cells with intracellular localization of PETNPLs (red), expression of p62 (green), and cytoplasmic presence and co-localization of PETNPLs (red) and p62 (green) (merge: yellow, shown in inset) compared to untreated control cells. Chloroquine as a positive control for p62 (green). Nuclei stained in blue with Hoechst dye. ii) The graph suggests the measurement of the relative average area of fluorescence (green/p62 puncta, A.U.) due to iDyePETNPLs versus untreated control. Magnification 63x. Data repre­ sented as mean ± SEM. ***P ≤ 0.001. Note: Rapa and CQ in graphs and blots represent rapamycin and chloroquine, respectively. accumulation of p62 puncta in PETNPLs exposed cells, as compared to the loss of autophagy flux (Fig. 6B-I, and B-ii, P < 0.05). This kind of the untreated control. Moreover, the data revealed a clear effect was also necessitated by widely studied nanoplastics like PSNPLs co-localization or close association of p62 puncta and PETNPLs in the since their exposure to macrophages (RAW 264.7) revealed an increased treated cells, indicating their potential role in the blockage or causing expression of p62, suggesting an impairment in the degradation capacity 7 B. Annangi et al. Environmental Toxicology and Pharmacology 100 (2023) 104140 of autophagy (Tan et al., 2020). Moreover, the exposure of PSNPLs to a Ph.D. FI fellowship from the Generalitat de Catalunya. A. Hernández human colon carcinoma cell line (RKO) and to mice primary intestinal was granted an ICREA ACADEMIA Award. epithelial cells inhibited the autophagy as evidenced by increases in the This project (Plasticheal) has received funding from the European p62 protein levels, as well as its aggregation in PSNPLs treated RKO cells Union’s Horizon 2020 Research and Innovation Programme under grant (Xu et al., 2022b). However, PSMPLs (2 μM) did not increase the p62 agreement No 965196. This work was partially supported by the Spanish protein expression levels in the treated HK-2 cells (Wang et al., 2021). Ministry of Science and Innovation [PID2020-116789, RB-C43] and the The relationship between the excessive production of iROS and loss Generalitat de Catalunya (2021-SGR-00731). of mitochondrial membrane potential or mitochondrial dysfunction due to various stress conditions in the induction of autophagy has been well References established (Filomeni et al., 2015; Fang et al., 2017). 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