Journal of Hazardous Materials 438 (2022) 129470 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat Research Paper Long-term exposure to nanoplastics alters molecular and functional traits related to the carcinogenic process Irene Barguilla a, Josefa Domenech a, Sandra Ballesteros a, Laura Rubio b, 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, Barcelona, Spain b Nanobiology Laboratory, Department of Natural and Exact Sciences, Pontificia Universidad Católica Madre y Maestra, PUCMM, Santiago de los Caballeros, Dominican Republic 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 • The carcinogenic risk of polystyrene nanoplastics (PSNPLs) was evaluated. • Long-term exposures (6 months) were used. • A sensitive cell model showing cell- transforming phenotype was used. • PSNPLs exposure exacerbated most of the evaluated hallmarks of cancer. • This data constitutes a relevant warning on the potential carcinogenic risk of MNPLs. A R T I C L E I N F O A B S T R A C T Editor: Youn-Joo An Micro/nanoplastics (MNPLs) are considered emergent pollutants widely spread over all environmental com­ partments. Although their potential biological effects are being intensively evaluated, many doubts remain about Keywords: their potential health effects in humans. One of the most underdeveloped fields is the determination of the Polystyrene nanoplastic potential tumorigenic risk of MNPLs exposure. To shed light on this topic, we have designed a wide battery of Long-term exposure different hallmarks of cancer applied to prone-to-transformed progress MEF cells exposed to polystyrene Cell transformation nanoplastics (PSNPLs) in the long term (6 months). Interestingly, most of the evaluated hallmarks of cancer are Oncogenic phenotype Carcinogenesis exacerbated after exposure, independently if they are associated with an early tumoral phenotype (changes in stress-related genes, or microRNA deregulation), advanced tumoral phenotype (growing independently of anchorage ability, and migration capacity), or an aggressive tumoral phenotype (invasion potential, changes in * Corresponding authors at: Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Cer­ danyola del Vallès, Barcelona, Spain. E-mail addresses: ricard.marcos@uab.es (R. Marcos), alba.hernandez@uab.es (A. Hernández). https://doi.org/10.1016/j.jhazmat.2022.129470 Received 25 January 2022; Received in revised form 8 June 2022; Accepted 24 June 2022 Available online 27 June 2022 0304-3894/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 pluripotency markers, and ability to grow to form tumorspheres). This set of obtained data constitutes a relevant warning on the potential carcinogenic risk associated with long-term exposures to MNPLs, specifically that induced by the PSNPLs evaluated in this study. 1. Introduction 2. Materials and methods One of the most worrisome aspects of the widespread micro- and 2.1. PSNPs and y-PSNPs characterization nanoplastic (MNPLs) pollution is the potential health effects triggered by their long-term exposure. Secondary MNPLs, generated from the Pristine PSNPLs (PP-008–10) and yellow fluorophore-conjugated environmental degradation of macroplastic waste, are present every­ PSNPLs (y-PSNPLs) (FP-00552-2) were purchased from Spherotech where and, consequently, humans are continuously exposed to them by (Chicago, USA). Pristine PSNPLs were used in all experimental work different routes including ingestion, inhalation, and dermal deposition except for the quantification of PS nanoparticles internalization by flow (Bouwmeester et al., 2015; Domenech and Marcos, 2021). Therefore, cytometry, for which the fluorescently labeled particle was required. risk assessment approaches evaluating the potential long-term effects of Nanoplastic characterization was performed using a Zetasizer device MNPLs exposure are urgently required (Brachner et al., 2020). and transmission electron microscopy (TEM). For that purpose, the Up to date, several studies have found detrimental biological effects PSNPLs dispersions were diluted to a final concentration of 100 μg/mL associated with MNPLs exposure, mostly following acute regimes. Using in distilled water or in a culture medium (DMEM). The hydrodynamic in vitro approaches different authors have described the ability of MNPLs size and Z-potential parameters were analyzed in triplicates with a to efficiently internalize in diverse cell lines, and translocate through Malvern Zetasizer Nano ZS zen3600 device (Malvern, UK), applying physiological barriers (Hesler et al., 2019; Domenech et al., 2020), dynamic light scattering (DLS) and electrophoretic light scattering (ELS) inducing reactive oxygen species (ROS) formation (Schirinzi et al., approaches. On the other hand, carbon-coated TEM grids were dipped 2017), DNA damage (Rubio et al., 2020a), inflammation (Ballesteros into the PSNPLs dispersions and visualized on a JEOL JEM-1400 in­ et al., 2020; Hu and Palić, 2020), mitochondrial dysfunction, stress, and strument (JEOL LTD, Tokyo, Japan). To determine the mean particle autophagy (Wang et al., 2021). However, very few studies explore these size, 100 randomly selected PSNPLs were measured using Image J -or other- endpoints in a chronic exposure context, which would be more software with the Fiji extension. representative of a real-life exposure scenario. Among those studies found in the literature, the work by Domenech et al. (2021) showed that Caco-2 cells exposed to low-range doses of polystyrene nanoplastic 2.2. Cell culture conditions (PSNPLs) altered the expression levels of stress-related genes such as HO1 and SOD2. However, no other pieces of evidence of ROS induction, Mouse embryonic fibroblasts, phenotypically sensitive to oxidative genotoxicity, or oxidative DNA damage were observed, despite the damage (Ogg1-/-), and initially transformed by 30 weeks of chronic so­ constant intake of PSNPLs by the cells, and the observed structural dium arsenite (AsIII) exposure (2 µM) (Bach et al., 2016), were used. changes associated with its accumulation. The impact of the sub-chronic These cells are referenced from this point forward as exposure to MNPLs has also been explored after a 48-day exposure of prone-to-transformation progress (PTP) cells. PTP cells were grown in HT-29 cells to polystyrene microplastics (PSMPLs). Thus, Visalli et al. Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Life (2021) showed that ROS and DNA damage induction by short-term ex­ Technologies, NY, USA) supplemented with 10 % fetal bovine serum posures to PSMPLs were less evident under sub-chronic exposure set­ (Biowest, France) and 2.5 µg/mL Plasmocin (InvivoGen, CA, USA) in a tings, hinting at the adaptability of the intestinal cells to PSMPLs humidified atmosphere of 5 % CO2 and 95 % air at 37 ºC. exposure. Taken together, these results prove that differential and accumulative effects are induced depending on the exposure settings. It is important to point out the critical lack of information on 2.3. PSNPLs cytotoxicity carcinogenicity-related endpoints associated with MNPLs exposure. It should be considered that carcinogenic effects are potential hazards To assess the potential cytotoxicity associated with the exposure to associated with environmental long-term exposures. Therefore, in this PSNPLs in PTP cells, viability was determined by the Beckman counter study, we aimed to propose/use a battery of in vitro carcinogenesis method with a ZTM Series Coulter-counter (Beckman Coulter Inc., CA, biomarkers, including stress-response deregulation, microRNA expres­ US). Briefly, 24 h after seeding 80,000 cells on 12-well plates, cells were sion alteration, invasive potential, and cell dedifferentiation associated treated with increasing doses of PSNPLs (10, 25, 75, and 100 μg/mL). with long-term MNPLs exposure. These transformation markers are After exposures lasting for 24 h, the cells were counted and the per­ descriptive of specific features acquired by transformed cells during the centages of viable cells with respect to the non-exposed control were oncogenic process and align with relevant hallmarks of carcinogenesis, represented. such as the cells’ invasive potential (Hanahan and Weinberg, 2011). To assess these carcinogenic-related endpoints, we have chosen a 2.4. PSNPLs internalization in PTP cells pre-transformed cell model of mouse embryonic cells (MEFs), sensitive to oxidative damage induction. In our long-term experiment, such model The quantification of the PSNPLs internalization after acute exposure cells were exposed to PSNPLs for 6 months. As indicated, the selected was carried out by flow cytometry. PTP cells were exposed to 25 μg/mL cells already exhibited a characteristic tumor-associated phenotype as y-PSNPLs for 24, 48, or 72 h. Then, the cells were washed with PBS 1X, evidenced by their acquired spindle-like morphology, invasiveness po­ trypsinized, centrifuged, and resuspended in PBS 1X at a final concen­ tential, and altered differentiation status (Bach et al., 2016). Therefore, tration of 1 × 106 cells/mL. 200 μL of the cell suspension were trans­ the sensitized genetic background of our cell model may facilitate the ferred to a 96-well plate. Propidium iodide (1:1000) was added to identification of mild effects that typically can be unnoticed in a normal discriminate alive cells within the total population. CytoFlex (Beckman cellular context. After characterizing the short-term dynamics of the Coulter Inc., CA, US) was used for the analysis. Approximately 10,000 nanoplastic uptake in our system, we explored both molecular and events per sample out of the living population were analyzed. The functional biomarkers of in vitro carcinogenesis at the end of the PSNPLs internalization was extrapolated from the mean fluorescence 6-month exposure period. intensity. 2 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 2.5. Intracellular PSNPLs detection conditions were 95 ◦ C for 1 min, followed by 55 cycles of 95 ◦ C for 1 min, and 65 ◦ C for 30 s. Cycle threshold (Ct) values were calculated with TEM was used to determine potential changes in the cellular struc­ the Lightcycler software package and then normalized with U6 values. ture induced by PSNPLs uptake. After a 24, 48, or 72 h exposure to 25 μg/mL PSNPLs, cells were washed with PBS 1X, trypsinized, pelleted, 2.10. Anchorage-independent growth ability measurement and fixed in 2.5 % (v/v) glutaraldehyde (EM grade, Merck, Darmstadt, Germany) and 2 % (w/v) paraformaldehyde (EMS, Hatfield, USA), in The anchorage-independent growth potential of chronically exposed 0.1 M cacodylate buffer (PB, Sigma-Aldrich, Germany) at pH of 7.4. PTP cells to PSNPLs was determined by analyzing the ability to form Then, sampling processing was carried out following conventional colonies in a soft-agar medium. Briefly, single-cell suspensions were procedures, as previously reported (Annangi et al., 2015). A Jeol 1400 obtained by filtering cells through a 40 µm mesh. Then, a suspension of TEM (Jeol LTD, Tokyo, Japan) equipped with a CCD GATAN ES1000W 65,000 cells in 1.75 mL of DMEM containing 10 % of FBS and 2.5 µg/mL Erlangshen camera was used to take representative cell images. of Plasmocin was prepared and mixed in a 1:1:1 ratio with 2x DMEM containing 20 % of FBS, 2 % non-essential amino acids, 2 % L-Glu 200 2.6. In vitro chronic PSNPLs exposure mM, and 2 % penicillin-streptomycin and with 1.2 % of bacto-agar (DIFCO, MD, USA). Dispensing 1.5 mL of the mixture, triplicates of PTP cells were chronically exposed for 6 months to 25 µg/mL 20,000 cells each were prepared in each well of a 6-well plate over a 0.6 PSNPLs. Replicates of exposed cells (PSNPLs) and non-exposed passage- % base agar (supplemented with 2x DMEM). Once the agar was dry, the matched controls (CT) were maintained in two separate T-25 flasks and plates were kept in the incubator for 21 days. Then, the colonies were grown under the culture conditions previously described. To ensure a stained by incubating with 1 mg/mL of (2-p-iodophenyl)− 3–3(p- constant exposure condition in the long-term exposure experiments, the nitrophenyl)− 5-phenyl tetrazolium chloride (INT; Sigma-Aldrich, cell culture medium was replaced every 2–3 days with new media Germany). For quantification, the plates were scanned with an HP containing the selected concentration of PSNPLs. Scanjet G4050, and the number of colonies was determined using the colony cell counter enumerator software OpenCFU (3.9.0). 2.7. Total RNA extraction 2.11. Invasion and migration ability detection Total RNA from control and PSNPLs-exposed PTP cells was extracted using TRI® Reagent (Sigma-Aldrich, Germany) and following the Invasion and migration assays were performed to evaluate the manufacturer’s instructions. DNA contamination was removed using invasive potential of PTP cells long-term exposed to PSNPLs. To that RNase-free DNase I (Turbo DNA-free Kit; Invitrogen, CA, USA). aim, cells at 80 % confluency were deprived of FBS for 24 h. The following day, each 8 µm pore size polycarbonate membrane 24 mm 2.8. mRNA retrotranscription and expression evaluation by Real-Time- transwell insert (Costar-Corning, NY, USA) was coated with a 180 μL 1:2 qPCR dilution of Matrigel® (Costar-Corning, NY, USA) in FBS free DMEM/F12 with 0.1 % BSA. The Matrigel® mixture dried for 1 h in the cell incu­ From total RNA, the first-strand cDNA synthesis was performed using bator at 37 ºC. After that, 2.5 mL DMEM/F12 complemented with 15 % 1 μg of total RNA and the high-capacity RNA-to-cDNA Kit (Applied FBS was added to the bottom chamber of the transwell as the chemo­ Biosystems, CA, USA). Real-time PCR analysis on a LightCycler 480 was attractant medium. A single-cell suspension containing 600,000 FBS- used to determine the relative expression of panels of stress-, EMT- or deprived MEF cells in 1.5 mL of FBS free DMEM/F12 with 0.1 % BSA stemness-related genes, with Actb as the housekeeping control. Each 20 was added on top of the transwell Matrigel®-coated membrane. Then, μL of reaction volume contained 5 μL of cDNA, 10 μL of 2 × LightCycler the plates were incubated at 37 ºC for 48 h. To determine the proportion 480 SYBR Green I Master (Roche, Manheim, Germany), 3 μL of H2O, and of invading cells, the cell monolayer and Matrigel® mix on top of the 1 μL of each primer pair at a final concentration of 500 nM. The cycling transwell were removed with a cotton swab, and the cells adhered to the parameters began with 95 ◦ C for 5 min, then 45 cycles of 95 ◦ C for 10 s, bottom part of the membrane were fixed with 1 mL methanol (VWR 61 ◦ C for 15 s, and 72 ◦ C for 25 s. The target genes and their corre­ International, Ireland) and stained with 0.2 % crystal violet (Sigma- sponding primer sequences are listed in the supplementary Table S1. Aldrich, Germany). After sequential washes, the membranes were The LightCycler software package was used to calculate cycle time allowed to dry and then photographed with a Zeiss Observer A1 mi­ values which were then normalized by the Actb data. croscope. The proportion of migrating or invading cells was measured with ImageJ software. 2.9. microRNA retrotranscription and expression evaluation by Real- Time-qPCR 2.12. Tumorsphere formation ability detection Changes in the microRNAs’ expression were evaluated by Real-Time Exposed and control PTP cells were seeded at a density of 2500 cells/ RT-PCR. To proceed, a mix including 80 ng of total RNA, 1 μL of 10X mL on 96-well ultra-low-attachment plates (Corning, Costar-Corning, poly(A) polymerase buffer, 10 mM of ATP, 1 μM of RT-primer (Sigma- NY, USA) in serum-free DMEM/F12 supplemented with B27, 20 ng/ Aldrich, Germany), 0.1 mM of each deoxynucleotide (dATP, dCTP, mL basic fibroblast growth factor (bFGF) (both from Life Technologies, dGTP, and dTTP) (VWR International, Ireland), 100 units of MulV NY, USA), epithelial growth factor, and 4 µg/mL heparin (both from reverse transcriptase (New England Biolabs, USA), and 1 unit of Poly(A) Sigma-Aldrich, Germany). Cells were incubated for 6 days in a humid­ polymerase (New England, Biolabs USA) was prepared at a final volume ified atmosphere of 5 % CO2 and 95 % air at 37 ºC. Then, the observed of 10 μL. The mix was incubated for 1 h at 37 ºC before to an enzyme tumorspheres were counted and photographed using a Zeiss Observer activation step at 95 ºC for 5 min. The sequence of the RT-primer was 5′ - A1 microscope. Tumorspheres’ mean size was determined with ImageJ CAGGTCCAGTTTTTTTTTTTTTTTVN, where V is A, C, and G and N are software. A, C, G, and T. The cDNA of a panel of 33 microRNAs was amplified by RT-qPCR on a LightCycler480. Each 10 μL of reaction contained 3 μL of 3. Results cDNA, 5 μL of 2X LightCycler 480 SYBR Green I Master (Roche, Ger­ many), 250 nM of each primer (Sigma-Aldrich, Germany), and 1 μL of 3.1. PSNPLs characterization H2O. The target miRNAs and the sequences of the primers used for the amplification are listed in the Supplementary Table S2. Cycling According to the measurements taken from TEM images (Fig. 1A), 3 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 quantified by flow cytometry in living PTP cells exposed to 25 μg/mL y- PSNPLs at 24, 48, and 72 h (Fig. 2B). Our results evidence that nano­ plastic uptake reaches its maximum at 48 h, although internalized levels are highly significant at all time points studied when compared to non- exposed cells. At a cellular level, PSNPLs’ localization was further explored by TEM. As seen in Fig. 3, vacuoles loaded with sphere-like particles (indicated with black or white arrows) are found in exposed PTP cells, but not in the control cells. We identified the observed particles as PSNPLs. Interestingly, we consistently found bigger vacuoles after the 48 h of exposure, smaller ones after 72 h, and the smallest ones were observed 24 h after the exposure. This observation agrees with the variations in the fluorescence intensity levels detected by flow cytometry. 3.3. Stress-related gene expression changes After the sustained 6-month exposure of PTP cells to PSNPLs (25 μg/ mL), we evaluated different endpoints related to the oncogenic process. Fig. 1. : Nanoplastic characterization. (A) Representative TEM images of We analyzed the gene expression pattern of stress-related genes, PSNPLs dispersed in distilled water or DMEM (B) PSNPLs characterization by including Keap, Glut-1, Gstp-1, Nrf2, Pgp, Sod1, and Sod2. From this gene TEM (median size) and Zetasizer Nano ZS (mean ± SD). Each material was diluted at a concentration of 100 μg/mL for characterization. panel, we could observe that Gstp-1 was the only upregulated gene, while the expression of Keap, Nrf2, Pgp, Sod1, and Sod2 was significantly downregulated (Fig. 4). Taken together, these results show that stress PSNPLs are round-shaped particles with equivalent sizes when diluted response is altered by chronic PSNPL exposure which may render the both in distilled water or in DMEM. The approximate 50 nm diameter cells more susceptible to oxidative and stress-related damage. measured in our samples agrees with the size indicated by the manu­ facturer. The nanoplastic particles were further characterized by a Zetasizer device (Fig. 1B). The polydispersity index (PdI) and the Z- 3.4. microRNA expression deregulation potential values indicate that the samples are highly stable mono- dispersions in water, while they show a propensity to aggregation The expression of a battery of 33 microRNAs related to oncogenesis when diluted in DMEM. The characterization of y-PSNPLs (data not was assessed in chronically exposed PTP cells. Out of the whole set, miR- shown) demonstrated that both, the fluorescently labeled and non- 33a, miR-124, miR-126, miR-132, miR-224, miR-505, miR-541, and labeled nanoplastic, display comparable features, as was previously re­ miR-544a were not expressed in our system. Among the expressed ported by us (Cortés et al., 2020). microRNAs, many presented significant differences in their levels when comparing PSNPL-exposed and non-exposed cells (Fig. 5). Most of these microRNAs were upregulated (miR-21, miR-23a, miR-25, miR-30c, miR- 3.2. PSNPLs short-term cytotoxicity, internalization, and cellular 30d, miR-96, miR-135b, miR-148b, miR-155, miR-199b, miR-200a, localization miR-210, miR-218, and miR-502) while only miR-34a and miR-203a appeared significantly downregulated. Among those overexpressed PTP cells were exposed for 24 h to increasing doses of PSNPLs (0, 10, microRNAs, miR-21, miR-23a, miR-30d, miR-96, miR-210, and miR-502 25, 75, and 100 μg/mL), showing low levels of cytotoxicity (Fig. 2A). showed higher expression levels (more than 5 folds of expression Although cell viability is over 80 % for all tested doses, we selected compared to control cells). 25 μg/mL for the rest of the experimental procedures involving both short- and long-term exposures. This concentration is in the low-dose 3.5. Oncogenic phenotype exacerbation range and, therefore, appropriate when aiming to recreate realistic exposure conditions. Several biomarkers representative of features developed by the cells Despite the lack of cytotoxic effects, results showed a very significant over the oncogenic process were studied in PSNPLs long-term exposed uptake of PSNPLs by the cells. The mean fluorescence intensity was PTP cells, to determine if the transformed phenotype of the cells was Fig. 2. : Nanoplastic cytotoxicity and internalization in PTP cells. (A) Cell viability of PTP cells exposed to increasing doses of PSNPLs for 24 h. Data are presented as the number of exposed cells relative to the non-exposed control ± SEM (B) y-PSNPLs internalization in PTP cells exposed for 24, 48, or 72 h to 25 μg/mL indicated as mean fluorescence intensity values ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test (**P ≤ 0.01; ***P ≤ 0.001). 4 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 Fig. 3. : Representative TEM images of PTP cells after 24, 48, or 72 h of exposure to 25 μg/mL PSNPLs. Vacuoles loaded with spherical particles are indicated by black and white arrows. The red square indicates the area where the image has been zoomed in. Fig. 4. : Gene expression analysis by RT-qPCR for a panel of stress response-related genes. Data are shown as the fold change of expression in exposed PTP cells compared to non-exposed controls per gene. Values are presented as mean ± SEM for each gene analyzed by the student’s t-test (*P ≤ 0.05). 5 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 endpoints associated with the development of an aggressive tumoral phenotype were explored. Nanoplastic-exposed cells displayed an increased potential to grow independently of anchorage forming col­ onies in the soft-agar assay (Figs. 6B and 6 C), and presented higher migration (Figs. 6D and 6E), and invasion (Fig. 6 F and 6 G) potentials than control PTP cells. These results indicate that, under chronic expo­ sure settings, PSNPLs induce the development of a significantly aggressive oncogenic phenotype. 3.6. Stemness biomarkers alteration To further characterize the oncogenic phenotype induced by PSNPLs Fig. 5. : Expression analysis for a battery of microRNA by RT-qPCR. Data are long-term exposure, several stemness biomarkers were evaluated. As plotted as fold changes of PSNPLs-exposed cells’ expression compared to con­ seen in Fig. 7A, the expression analysis of a panel of pluripotent markers trols. Values represent mean ± SEM. Results were analyzed with the student’s t- showed that exposed PTP cells presented increased levels of Klf4, Nanog, test for each miRNA (* P < 0.05). Notch2, and Oct3/4 as well as decreased levels of Sox2. Therefore, at a molecular level, stem-related markers are altered by PSNPLs exposure. aggravated by PSNPLs exposure. On the one hand, we evaluated five This deregulation is consistent with the result obtained when the genes related to EMT and tumor progression to a metastatic phenotype. tumorsphere formation ability of PTP cells was determined. The ability Interestingly, Slug and Zeb1 expression was downregulated in exposed of the cells to grow to form tumorspheres is associated with the presence PTP cells, while Snail and Twist were overexpressed (Fig. 6 A). There­ of stem or progenitor cells in tumoral populations. Although no differ­ fore, there are molecular alterations induced by PSNPLs exposure that ences in the number of tumorspheres were found, we could observe that may eventually lead to EMT deregulation. At a functional level, different PSNPL-exposed cells formed spheres of a significantly bigger diameter than those generated by non-exposed PTP cells. All these results together Fig. 6. : Evaluation of the in vitro transformed phenotype of PTP cells after the long-term nanoplastic exposure. (A) Expression levels for a set of EMT-related genes. Values are presented as fold changes in exposed cells compared to non-exposed controls. (B) Representative images of colonies growing independently of anchorage in the soft-agar assay. (C) The number of colonies quantified in the soft-agar assay. (D) Representative images of the cells able to translocate to the basal side of the transwell membrane in the migration assay. (E) The proportion of migrating cells is represented as the percentage of exposed cells quantified in the basal part of the membrane compared to controls. (F) Representative images of invading cells observed in the basal side of the transwell membrane. (G) Quantification of the percentage of invading PTP cells after the exposure compared to non-exposed cells. Quantified data are represented as mean ± SEM. Results were analyzed with the student’s t-test for each endpoint (* P < 0.05; *** P < 0.001). 6 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 Fig. 7. : Study of stem-related features. (A) Gene expression analysis of a set of pluripotent markers. Values are shown as fold changes in exposed PTP cells compared to non-exposed cells. (B) Representative images of the tumorspheres formed by PSNPL-exposed cells and control cells. (C) Tumorspheres diameter showing the increment in the size of exposed PTP cells -derived tumorspheres. Quantified data are presented as mean ± SEM. Results were analyzed with the student’s t-test for each endpoint (* P < 0.05; ** P < 0.01). hint at a potential role of PSNPLs in inducing the dedifferentiation of the long-term bioassays to understanding the mechanistic basis for culture under our exposure conditions. chemical-induced cancers (Bhat et al., 2020). Using such new ap­ proaches, plausible modes of action (MOA) for environmental agents, 4. Discussion mainly for emergent pollutants such as MNPLs, can be determined using in vitro experiments. Considering that tumor induction is a multistep The potential health risks associated with human exposure to MNPLs process requiring prolonged exposures, long-term experimental ap­ is a hot topic being reviewed in different recent issues (Rubio et al., proaches, such those used in this study, are needed to go deeper into the 2020b; Facciolà et al., 2021; Danopoulos et al., 2021; Das et al., 2021). determination of the potential carcinogenic risk of MNPLs exposure. The lack of powerful enough tools to determine the exposure levels to Interestingly, our results clearly show that many of the used bio­ MNPLs in humans prevents the use of human biomonitoring approaches, markers of our battery of cancer hallmarks are exacerbated after long- as the most direct way to determine the human risks linked to such term exposure to MNPLs. Such cancer hallmarks constitute a reference exposures. In this way, most of the information we have in our hands framework, summarizing the abilities of cancer cells to escape control results from in vitro studies using human/mammalian cell lines, (Hanahan and Weinberg, 2011). At this point, we wish to highlight the although some in vivo studies using mammalian models have also been usefulness of our PTP cell model, highly sensitive to the action of agents carried out (Yong et al., 2020). with tumorigenic potential, and the use of concurrent controls to iden­ Most of the reported in vitro studies lack relevant information, mainly tify if observed changes could be associated with the long-term growing about the exposure type and on the evaluated endpoints, which are conditions. MEF cells, knockdown for different genes, have shown their useful requirements for their extrapolation to human health risks usefulness to discover mechanisms involved in oncogenesis. This occurs assessment (Brachner et al., 2020). The most used in vitro exposure ap­ with FRA1, a transcription factor frequently overexpressed in epithelial proaches involve high concentrations and short exposure times, which is tumors, playing an essential role in the in vitro cell transformation far away from the real exposure scenario, where individuals are induced by arsenic (Barguilla et al., 2020); or with the Atg5 (autopha­ chronically exposed to low concentrations. Thus, in general, there is a gy-related gene 5) knockdown, playing a role on the levels of lack of studies using long-term exposure conditions, which better reflect inflammation-related proteins and apoptosis induced by polystyrene the in vivo situation (Yong et al., 2020; Llewellyn et al., 2021). This nanoplastics (Wang et al., 2021). In the present study, PTP cells have general criticism also serves for the case of MNPLs, where very few in been shown to dramatically accumulate large amounts of polystyrene vitro studies using long-term exposures have been published until now nanoplastics, mainly after exposures lasting for 48 h. This easy uptake (Visalli et al., 2021; Domenech et al., 2021; Gopinath et al., 2021). In reaffirms the usefulness of our selected cell model to evaluate the effects this context, our study with exposures lasting for 6 months must be of MNPLs. highlighted for its novelty and effort to simulate a more realistic expo­ As previously mentioned, the more relevant finding of our study is sure scenario. It should be mentioned that, although this type of that most of the used hallmarks of cancer are exacerbated, indepen­ experimental approach involves a time/economic consuming design, dently if they are associated with an early tumoral phenotype (changes this type of long-term exposure scenario is very relevant to identifying in stress-related genes or microRNA deregulation), advanced tumoral potential hazards affecting human health. It is easy to understand that, phenotype (growing independently of anchorage ability and migration faced with the experimental complexity indicated above, many re­ capacity), or an aggressive tumoral phenotype (invasion ability, changes searchers avoid it. However, if we agree with its usefulness and rele­ in pluripotency markers, and ability to grow to form tumorspheres). All vance, efforts using long-term approaches must be done. this information constitutes a relevant warning on the potential carci­ Regarding the potential health effects associated with MNPLs expo­ nogenic risk associated with long-term exposures to MNPLs, at least sure, the carcinogenic risk stands up. Although the standard way to regarding the PSNPLs evaluated in this study. Despite the lack of studies determine the carcinogenic risk of any agent implies the use of lab aiming to directly determine the carcinogenicity of MNPLs some studies mammals (OECD, 2018), the risk assessment of potential carcinogens have determined adverse outcome pathways (AOP) associated with their has evolved from simply counting tumors present in animals to exposures. Thus, using Daphnia, a proposed AOP for polystyrene 7 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 nanoplastics included ROS production and oxidative stress as the mo­ observed an increased expression of hepatic HNF4A and CYP2E1. lecular initiating events, followed by changes in specific signaling and Accordingly, an AOP relevant to PSMPLs was proposed, where their metabolism pathways. These processes lead to effects such as growth potential risks of liver steatosis, fibrosis, and cancer were implicated inhibition and altered reproductive outputs (Liu et al., 2021a). (Cheng et al., 2022). These results would confirm our findings pointing In our study, changes in the expression levels of different EMT out the potential carcinogenic risk of long-term PSNPs exposures. markers (Slug, Snail, Twist, Zeb) have been found associated with long- In addition to the potential direct carcinogenic effects of MNPLs, term exposure to PSNPLs. The mesenchymal phenotype is closely asso­ many studies point out the potential synergies between MNPLs and ciated with malignant transformation and linked to the migration carcinogenic pollutants. The adsorption of carcinogenic polycyclic aro­ phenotype. Although no previous studies are using MNPLs, such changes matic hydrocarbons (PHA) onto microplastics has been proposed as a have been reported to occur associated with exposure to different potential mechanism to increase the probable cancer risk of ingested plasticizers such as bisphenol A and phthalates (Lee et al., 2017). PAHs-enriched MNPLs (Sharma et al., 2020). Similarly, the co-exposure Interestingly, changes in cell migration ability were also observed in of tetracycline and polystyrene nanoplastics also exacerbates different human umbilical vein endothelial cells exposed to polystyrene micro­ biomarkers in juvenile grass carp. The authors described enhanced plastics, together with the inhibition of angiogenic tube formation, oxidative damage under co-exposure conditions, as well as increased suppression of angiogenic signaling pathways, and inhibitory activity expression levels of MMP2, MMP9, and IL-8 both in liver and intestine against wound healing (Lee et al., 2021). tissues. Since mRNA expression of MMP2 is positively correlated with microRNAs usually act post-transcriptionally to suppress the TGFβ1, IL8, and MMP9 in liver hepatocellular carcinoma and colon expression of many targeted genes, and different studies have demon­ adenocarcinoma, that study points out an increased cancer risk associ­ strated that they are crucial players in numerous biological processes ated with such co-exposure (Liu et al., 2022). such as inflammation, proliferation, differentiation, development, and In conclusion, our results add relevant information supporting a apoptosis, among others (Chekhun, 2019). In the used battery of potential carcinogenic risk associated with MNPLs exposure, when long- microRNAs, related to cell transformation processes, we have found a term exposures take place. Due to the relevance of these findings, further large impact on miRNA expression profiling after our long-term expo­ experimental approaches are required to decipher the link between sure to PSNPLs. Interestingly, the expression of mir-21, mir 23a, mir-96, MNPLs exposure and cancer risk. and mir-210 was also altered in BEAS-2B cells exposed to cigarette smoke condensate (Ballesteros et al., 2021a) and zinc-oxide and tita­ Environmental implication nium dioxide nanoparticles (Ballesteros et al., 2021b), agents able to produce cell transformation in a long-term exposure scenario. These two Environmental carcinogenesis is an old topic but updated according studies used the same battery of microRNAs associated with cell trans­ the presence of new emergent environmental contaminants, such as formation processes that have been used in the present study. Interest­ micro/nanoplastics. For such pollutants, is urgent to determine their ingly, in vivo dysregulation of different microRNAs has been observed in potential carcinogenic risk. Due to the lack of information on this topic, Caenorhabditis elegans exposed to polystyrene nanoparticles, and has and to avoid the use of experimental mammals, we have designed a been associated with various biological processes and signaling path­ battery of in vitro approaches containing different cancer hallmarks. ways (Qu et al., 2019). Moreover, these dysregulated microRNAs were Interestingly, our approach using nanopolystyrene as a model and a long observed to be involved in the resistance to the toxicity of polystyrene term (6 months) exposure demonstrate exacerbated tumoral phenotypes nanoparticles. Using this in vivo model, the overexpression of mir-35 was after exposure. Our data constitutes a relevant warning on the potential also observed (Li et al., 2020), and it is worth recalling that this carcinogenic risk associated with long-term exposures to MNPLs. microRNA was also upregulated in our study. In the intestine of C. elegans, NDK-1 was identified as the direct target of mir-35, and the CRediT authorship contribution statement kinase suppressors of Ras (KSR-1 and KSR-2) were further identified as downstream targets of intestinal NDK-1. This information would support RM and AH planned the experiments. IB, SB, JD, and LR carried out the view indicating a tumoral risk associated with PSNPLs. More inter­ the experimental part. IB analyzed the data, carried out the statistical estingly, and to confirm the goodness of our results, three others of the analysis, and prepared tables/figures. IB, RM, and AH wrote the final microRNA dysregulated in our study (mi-794, mi-38, and mi-76) have manuscript. also been found dysregulated in C. elegans exposed to nanopolystyrene (Qiu et al., 2020; Yang et al., 2020; Liu et al., 2021b). All this reinforces Declaration of Competing Interest the usefulness of the used microRNAs battery to pick up those micro-RNAs responding to the long-term exposure to MNPLs. The authors declare that they have no known competing financial Our results also show that PSNPLs exposure targeted stem cells. This interests or personal relationships that could have appeared to influence is reflected by the altered expression of different pluripotent markers the work reported in this paper. such as Klf4, Nanog, Notch2, Oct3/4, and Sox2 genes which play a dominant role in regulating pluripotency (Hadjimichael et al., 2015). No Data Availability other studies have used such biomarkers to unravel the mechanisms involved in MNPLs exposure. Furthermore, such long-term exposure was Data will be made available on request. able to increase the size of the induced tumorspheres, which are asso­ ciated with the presence of stem-like cells in the cell population. Acknowledgments Tumorsphere analysis is widely used to analyze the self-renewal capa­ bility of cancer stem cells and to detect both pro-tumoral and We wish to thank Mr. C. Valiente for his technical assistance. The anti-tumoral agents/effects (Lee et al., 2016). An alternative way to authors I. Barguilla S. Ballesteros, and J. Domenech were supported by evaluate the effects on stem cells is the use of mini-organoid cultures PhD fellowships from the Universitat Autònoma de Barcelona [PIF- obtained from various types of stem cells. Such ex vivo models are UAB]. L. Rubio was supported by the Fondo Nacional de Innovación y promising tools for a more comprehensive understanding of the under­ Desarrollo Científico y Tecnológico (FONDOCYT) República Domi­ lying mechanisms involved in in vitro toxicities (Bredeck et al., 2022). nicana (Project 2018-2019-2B2-093). This work was partially supported Interestingly, a very recent study has evaluated the effects of PSMPLs on by the Spanish Ministry of Science and Innovation [PID2020-116789, liver organoids generated from human pluripotent stem cells, as an RB-C43]. This project has received funding from the European Union’s alternative model to the human liver. Using that model, the authors Horizon 2020 research and innovation programme under grant 8 I. Barguilla et al. Journal of Hazardous Materials 438 (2022) 129470 agreement No 965196 (PlasticHeal). Domenech, J., Hernández, A., Rubio, L., Marcos, R., Cortés, C., 2020. Interactions of polystyrene nanoplastics with in vitro models of the human intestinal barrier. Arch. Toxicol. 94 (9), 2997–3012. https://doi.org/10.1007/s00204-020-02805-3. Domenech, J., de Britto, M., Velázquez, A., Pastor, S., Hernández, A., Marcos, R., Author contribution statement Cortés, C., 2021. Long-term effects of polystyrene nanoplastics in human intestinal Caco-2 cells. Biomolecules 11, 1442. https://doi.org/10.3390/biom11101442. Domenech, J., Marcos, R., 2021. Pathways of human exposure to microplastics, and RM and AH planned the experiments. IB, SB, JD, and LR carried out estimation of the total burden. Curr. Opin. Food Sci. 39, 144–151. https://doi.org/ the experimental part. IB analyzed the data, carried out the statistical 10.1016/j.cofs.2021.01.004. Facciolà, A., Visalli, G., Pruiti Ciarello, M., Di Pietro, A., 2021. 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