Environmental Science Nano This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Polystyrene nanoplastics accumulate in ZFL cell Cite this: Environ. Sci.: Nano, 2020, lysosomes and in zebrafish larvae after acute exposure, inducing a synergistic immune response Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. 7, 2410 in vitro without affecting larval survival in vivo† Irene Brandts,ab Marlid Garcia-Ordoñez,ab Lluis Tort,b Mariana Teles*ab and Nerea Roher *ab The presence of small-sized plastic particles in marine and freshwater environments is a global problem but their long-term impact on ecosystems and human health is still far from being understood. Nanoplastics (<1000 nm) could pose a real and uncontrolled ecological challenge due to their smaller size and sharp ability to penetrate living organisms at any trophic level. Few studies evaluate the impact of nanoplastics in vivo on the immune system of aquatic organisms, while most of them assessed the impact on indirect markers of immune response such as regulation of gene expression, ROS production or DNA genotoxicity, among others. Moreover, the study of the effects of nanoplastics on aquatic vertebrate species in vivo is still scarce. In this context, we seek to shed light on the underlying effects of polystyrene nanoplastics (PS-NPs) on the immune response in a model fish species (Danio rerio, zebrafish) after an acute exposure, with a combination of in vitro and in vivo experiments. Our results show that PS-NPs (65 nm) are efficiently taken up by zebrafish liver cells, accumulating mainly in lysosomes. Furthermore, the expression of immune genes presents a synergy when cells were simultaneously exposed to PS-NPs, at a low dose and early time point (12 h) and challenged with a viral stimulus (polyIJI:C)). Moreover, zebrafish larvae also internalize PS-NPs, accumulating them in the gut and pancreas. However, at concentrations of Received 25th May 2020, up to 50 mg l−1 in an acute exposure (48 h), PS-NPs do not interfere with the survival of the larvae after a Accepted 30th June 2020 lethal bacterial challenge (Aeromonas hydrophila). This study addresses the relevant environmental DOI: 10.1039/d0en00553c question of whether a living organism exposed to PS-NPs can cope with a real immune threat. We show that, although PS-NPs can induce an immune response, the survival of zebrafish larvae challenged with a rsc.li/es-nano bacterial infection after an acute exposure to PS-NP is not decimated with respect to unexposed larvae. Environmental significance Small sized plastic particles have been reported around the planet in all kinds of environments and it is generally accepted that microplastics are able to cause adverse effects on organisms. However, the understanding of the effects and possible implications of nanoplastics on vertebrate species is still scarce. The impact of polystyrene nanoplastics (PS-NPs) on indirect markers of immune response (such as regulation of gene expression, ROS production or DNA genotoxicity) has been to some extent previously assessed. Nevertheless, a key question remains unexplored: can an organism exposed to nanoplastics successfully cope with an additional immune challenge? This is the first study to provide data on how the immune system of a vertebrate species might respond to a combined challenge of nanoplastics and a pathogen in the surrounding environment. We show that an acute exposure of zebrafish larvae to PS-NPs at high concentrations has no effect on the survival rate of the larvae after a bacterial infection. Thus, this study provides novel insight beyond the current understanding of the potential interplay between pathogen and nanoplastic contamination. 1. Introduction a Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de After more than 50 years of plastic production, with 360 Barcelona, 08193 Barcelona, Spain. E-mail: nerea.roher@uab.cat, million tonnes manufactured solely in 2018,1 and the mariana.teles@uab.cat subsequent accumulation in the natural environment, the b Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain impact of plastics in ecosystems worldwide is just starting to † Electronic supplementary information (ESI) available. See DOI: 10.1039/ be understood. Although nanoplastics, defined as particles d0en00553c ranging from 1 nm to 1000 nm resulting from degradation of 2410 | Environ. Sci.: Nano, 2020, 7, 2410–2422 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Paper plastic objects,2,3 have already been detected in the At the present time, assessment of environmental environment,4 their precise quantification in natural aquatic concentrations of nanoplastics is largely speculative, systems and the establishment of environmentally relevant primarily due to the lack of efficient analytical methods.15 In concentration are still under debate. There is a general one relevant study by Gallego-Urrea et al.16 measured 107–109 This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. consensus on how microplastics are able to cause adverse particles per ml (100–250 nm size) in solution, using effects on marine and freshwater species (see Chae et al.5 for nanoparticle tracking analysis (NTA), in different sample sites a review). However, there are still many unanswered questions of Scandinavian waters.16 The exposure concentrations used in nanoplastic research, regarding for example their final in our study fall within this particle range and were chosen destiny once absorbed/ingested, their long-term effects or taking into account the concentrations used in relevant their interaction with microbial communities. One of these studies with PS-NPs published in the recent literature6,8,14,17 relevant knowledge gaps is whether nanoplastic pollution can and our own initial in vitro results, aimed at assessing the Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. impair the capacity of organisms to fight against pathogens. interaction of the zebrafish liver (ZFL) cell line with PS-NPs. This publication aims to start the unravelling of this last Even though the tested concentrations could be in the higher question, combining an in vitro and in vivo approach. range of estimated environmental nanoplastic Zebrafish (Danio rerio) is considered a good model for eco- concentrations, they allow an approximation to the potential toxicological studies as well as a tool to understand eco- response of zebrafish to a double challenge with PS-NPs and physiological adaptations in vertebrates. The versatility of a pathogen. this model, together with the availability of molecular and Our results present evidence that PS-NPs are toxic to ZFL cellular tools, has popularized the use of zebrafish. Previous cells at doses higher than 75 mg l−1 and can accumulate studies exploring the effects of polystyrene nanoplastics (PS- rapidly inside liver cells, specifically in lysosomes. We show NPs) on zebrafish have described a broad physiological that PS-NPs at low doses (5 mg l−1) and early stimulation response, not particularly specific to a given tissue or cell times (12 h) synergistically activated the expression of anti- type, but more typical of a general stress response (e.g. viral genes, while low doses and a longer exposure time did oxidative stress, complement system or glucose metabolism). not have any effect. Moreover, we did not observe this Alterations in behaviour, measured by a decrease6 or synergistic interaction at a high dose (50 mg l−1) and early increase7 in locomotor activity, changes in glucose stimulation time. Similarly, in the in vivo experiment, PS-NPs metabolism and cortisol levels,7 intestinal and skin damage accumulated in the zebrafish larvae intestine and pancreas, triggering a pro-inflammatory response8 and production of but this accumulation did not impair the larvae's defence oxidative stress and lipid accumulation,9 have been mechanisms against a bacterial lethal challenge with documented in zebrafish after waterborne exposure to PS- Aeromonas hydrophila. Thus, zebrafish larvae pre-exposed to NPs. Other studies have evaluated the effects of direct PS-NPs had the same levels of survival as unexposed larvae. injection of PS-NPs on zebrafish embryos, describing changes in the transcriptome10 or production of reactive oxygen 2. Materials and methods species (ROS) and apoptosis.11 Moreover, PS-NPs have been previously found to interfere Characterization of polystyrene nanoplastics (PS-NPs) by DLS with the immune system of zebrafish8,10,12 as well as other and electronic microscopy aquatic species.13,14 In D. rerio, PS-NPs have been shown to Fluorescently labelled PS-NPs (FSDG001 Dragon Green, Bangs upregulate the expression of inflammatory cytokines,8 Ltd.) were purchased from Bang Laboratories (Fisher, IN, activate the complement system and oxidative stress USA). PS-NPs were provided as a 1% (w/w) suspension in mechanisms10 and provoke DNA damage, induced by ROS water with 2 mM NaN3. The particle size distribution and and apoptosis.11 In this context, we seek to address the zeta potential were measured by dynamic light scattering question of whether their interaction with the immune (DLS) using a Zetasizer Nano ZS (Malvern Instruments, UK), system can affect the survival of populations in their natural under different incubation conditions: pure water (Sigma), environments, where organisms are frequently exposed to PBS (Sigma), cell culture medium (DMEM, Gibco) and E3 pathogens. To our knowledge, barely any studies on the embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, combination of PS-NPs and an immune challenge have been 0.33 mM MgSO4 and 0.1% methylene blue). Emission performed. In a recent study, Sendra et al.14 evaluated the scanning electron microscopy (FESEM, Zeiss Merlin) was response of Mytilus galloprovincialis haemocytes in vitro when used to determine the external morphology and physical infected with Vibrio splendidus, following an exposure to PS- dimensions of the PS-NPs in pure water and cell culture NPs. They reported that the invertebrate's haemocytes medium. Exposure solutions were prepared by resuspending showed resilience when infected with the bacteria, being able the stock solution of PS-NPs in PBS and further diluting in to recover their phagocytic capacity. In the present work, we each medium, at 100 μg ml−1. For electron microscopy, 20 μl focus the attention in this direction, to determine whether of each solution were pipetted onto silicon chips and air- the combination of an acute exposure to PS-NPs and a dried O/N. A palladium–gold coating was applied prior to subsequent lethal bacterial challenge can shed light on the observation. Images were analysed using the Fiji open source underlying toxic effects of PS-NPs on aquatic organisms. image processing package,18 measuring the dimensions of a This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 2410–2422 | 2411 View Article Online Paper Environmental Science: Nano minimum of 150 particles for each condition. Size Uptake of PS-NPs by ZFL assessed using flow cytometry and distribution histograms were generated using Prism 7.01 confocal microscopy and in RTGut by microscopy (GraphPad software). To test cellular uptake, fluorescently labelled PS-NPs were added to ZFL cultures at 70% confluence after 2 h of This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Decay of PS-NP fluorescence in vitro and in cell culture incubation in minimal medium, at the doses and times Fluorescently-labelled nanoplastics could leach fluorophores, indicated below. Representative cytometry plots with the since they are not covalently linked to the PS-NPs.19 Catarino gating strategy and the threshold of fluorescence are shown et al.20 suggested careful testing of the fluorescent particle in ESI† Fig. S1. uptake in order to avoid wrong conclusions. To establish the For dose–response assays, cultures were incubated for 20 true internal accumulation of fluorescent PS-NPs, the h with PS-NPs at 0.05, 0.5, 5, 10, 25, 50, 100, 250, 500 and 1000 mg l−1, at 28 °C. For time-course assays, cultures were Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. following experiment was set up: 1 ml of PS-NP suspension (1000, 100 and 50 mg l−1) was centrifuged at 50 000 × g for 60 incubated with PS-NPs at 25, 50 and 75 mg l−1 for 6, 12, 24, min, then the supernatant (SN) was transferred to a new 48 and 72 h, at 28 °C. For positive control of uptake, Atto-488 Eppendorf tube and the pellet (P) resuspended in 1 ml of conjugated TNFα was used, as its uptake has been previously PBS. The fluorescence intensity in the different fractions (P, characterized by the research group in this cell line.21 Both SN and stock solution) was evaluated in solution using a Cary dose–response and time-course experiments were performed Eclipse fluorescence spectrophotometer (Agilent). At the in triplicate. Post treatment, cells were washed in PBS and lowest tested concentration, approximately 25% of the incubated at 28 °C with 1 mg ml−1 trypsin (Gibco) for 15 min. fluorescence remained in SN and we can therefore assume This strong trypsinization step aimed to remove PS-NPs that it leached from the PS-NPs. To further confirm that the attached to the cell surface.21 Then, two volumes of complete fluorescence inside the cells was not due to leached medium were added, and cells were retrieved by fluorophore, ZFL cells were treated with the PS-NP original centrifugation at 300 × g for 5 min. Pellets were resuspended suspension, SN and P and the uptake was monitored by in PBS for flow cytometry (FACSCalibur BD), and 10 000 cytometry as explained below. events were counted. Data were analysed using Flowing Software 2.5.1 (University of Turku, Finland) and plotted with Prism 7.01 (GraphPad). One-way ANOVA followed by Zebrafish liver (ZFL) and RTGut cell culture Dunnett's multiple comparison test was performed, ZFL cells (CRL-2643, ATCC) were cultured at 28 °C, 5% CO2 comparing treatment and control means. To confirm that the in Dulbecco's modified Eagle's medium (DMEM) 4.5 g l−1 fluorescent PS-NPs were inside the cells, confocal microscopy glucose, supplemented with 0.01 mg ml−1 insulin, 50 ng ml−1 was performed (Leica SP5). ZFL cells were seeded on ibidi 35 EGF, 5% (v/v) antibiotic/antimycotic solution, 10% (v/v) heat mm glass bottom dishes (Ibidi GmbH, Germany). The next inactivated fetal bovine serum (FBS) and 0.5% (v/v) heat- day, cells at approximately 60% confluence were placed in inactivated trout serum (TS) as described in Torrealba et al.21 minimal medium. PS-NPs were added 2–3 h later at a RTGut cells were obtained from Dr. Carolina Tafalla's concentration of 5 or 50 mg l−1 and cells were incubated for laboratory and cultured as described by Kawano et al.22 24 h at 28 °C. Cells were then washed and the medium Briefly, cells were kept at 20 °C in Leibovitz's L-15 Medium replaced with new minimal medium. RTGut cells were GlutaMAX™ (Gibco) #31415029), supplemented with heat- treated in the same way for confocal microscopy but inactivated FBS 10% (v/v) and 1% antibiotic/antimycotic. incubated with PS-NPs at 10 and 25 mg l−1, at 20 °C. The cells were stained with Hoechst (nuclei), either WGA Alexa PS-NP cytotoxicity studies in ZFL Fluor555 or Cell Mask Deep Red (membrane) and LysoTracker Red (lysosomes). Images were analysed using Cytotoxic and cytostatic effects of PS-NPs on ZFL were Imaris software v9.3 (Bitplane) and Image J. assessed using the MTT assay. After 2.5 h in minimal medium (0–0.5% FBS; 2% antibiotic/antimycotic), cultures were incubated with PS-NPs at 0, 0.05, 0.5, 5, 10, 25, 50, 100, Lipid peroxidation 250, 500 and 1000 mg l−1 for 20 h at 28 °C. A 0.2 mM sodium ZFL cells at 60% confluence were cultured in minimal azide control was also included. Cells were then washed in medium (0.5% FBS; 2% A/A) for 2–3 h and then exposed to PBS and the MTT substrate (Sigma-Aldrich) was added to PS-NPs at 5 mg l−1 and 50 mg l−1 for 24 h. After the exposure, 10% of the total volume and further incubated at 28 °C for cells were stimulated with poly(I:C) (25 μg ml−1), a synthetic 1–2 h. The solution was removed, the cells were solubilized analog of dsRNA virus, for 16 h. Controls were: poly(I:C) 25 in DMSO and the lysate was read on a Victor 3 (PerkinElmer) μg ml−1 (Sigma-Aldrich) and control cells with neither viral at 550 nm. The experiment was repeated three times. Data stimulus nor PS-NP exposure. Moreover, each PS-NP exposure were normalized using Prism 7.01 (GraphPad) such that the time and concentration had a respective control group, with control readings were set at 100%. One-way ANOVA was no viral stimulus. After completing the exposure, ZFL cells performed with Dunnett's multiple comparison test, were washed with PBS and total lipid peroxidation was comparing treatment and control means. assessed using the lipid peroxidation (MDA) assay kit (Sigma) 2412 | Environ. Sci.: Nano, 2020, 7, 2410–2422 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Paper following the manufacturer's instructions. The experiment Principles for Research Involving Animals (EU 2010/63) and was repeated twice. previously authorized by the Ethics Committee of the Universitat Autònoma de Barcelona (UAB, CEEH number RNA extraction and gene expression analysis of ZFL cells 1582). This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. treated with PS-NPs ZFL cells were plated and grown to 60% confluence, cultured Uptake of PS-NPs by zebrafish larvae assessed by fluorescence in minimal medium for 2–3 h and then exposed to PS-NPs ± microscopy poly I:C as described above, for the lipid peroxidation assay. Groups of 14 larvae (n = 14 per condition) were distributed For gene expression analysis, an additional exposure time to on 96-well plates (ThermoFisher) with one larva per well PS-NPs of 12 h was added, together with the 24 h exposure containing 200 μl E3 medium or fluorescent PS-NPs at 5, 50 and 100 mg l−1. Mortality was recorded for 96 h and zebrafish Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. previously described. Total RNA was extracted using TriReagent (Sigma-Aldrich) following the manufacturer's larvae were observed using a fluorescence stereomicroscope instructions. RNA was quantified using a nanodrop ND-1000 (Nikon SMZ800) coupled with a camera (Nikon DS-Fi2). (Thermo Fisher Scientific) and integrity was checked on an Agilent 2100 Bioanalyser using the RNA 6000 Nano Lab-Chip A. hydrophila culture and zebrafish larvae infection kit (Agilent Technologies). The experiment was repeated, and Experimental infections were performed as previously four complete sets of high-quality RNA from two independent described.25 Briefly, bacteria were grown on LB agar plates experiments were selected for cDNA synthesis using 1 μg of overnight at 28 °C, collected from the plates in an Eppendorf total RNA and the iScript cDNA synthesis kit (Bio-Rad). RT- tube, washed with PBS and finally resuspended with E3 to qPCR was performed in a CFX384 touch real-time PCR obtain a stock solution containing approximately 1010 colony- detection system (Bio-Rad) using the iTaq universal SYBR forming units (CFUs) per ml (OD620nm = 1.3). Dilutions at the green supermix kit (Bio-Rad) following the manufacturer's desired concentration were all prepared from the stock instructions. In brief, each PCR mixture consisted of 5 μl solution. A. hydrophila infection was carried out by bath SYBR green supermix, 0.4 μM specific primers (ESI† Table immersion according to Ji et al.25 Groups of 72 larvae (n = 72 S1), 2 μl diluted cDNA and 2.6 μl water (Sigma-Aldrich) in a per condition) were distributed on 96-well plates final volume of 10 μl. Primers for the assessed genes had (ThermoFisher) with one larva per well containing 200 μl E3 been previously designed and tested by the research team in medium or PS-NPs. For infection, the bacteria were diluted the ZFL cell line.23 A reference gene (elongation factor 1alpha from the stock solution using E3 medium in serial 100-fold (ef1-α)) and three gene markers of the innate immune dilutions from 10−1 to 10−9. The bacterial dilutions of 10−7, response to viral infection (IFN-induced protein Mx (mx), 10−8 and 10−9 (100 μl of each) from the stock solution were viperin (vig1) and grass-carp-reovirus-induced gene 2 (gig2)) inoculated on LB plates and incubated overnight at 28 °C to were used. All the samples were run in triplicate, and data calculate the real CFUs during the infection. The survival were analysed using the Livak method.24 For statistical curves were analysed, and statistical differences were analysis used, a one-way ANOVA test, followed by Dunnett's assessed using the log-rank test (GraphPad Prism v7.0). multiple comparisons for each treatment versus control, was conducted; p < 0.05 was considered statistically significant in all analyses (GraphPad Prism v7.0). 3. Results and discussion Characterization of PS-NPs in biological relevant buffers Zebrafish husbandry and breeding The size and shape of fluorescently labelled PS-NP particles Wild type zebrafish (D. rerio) were kept in a re-circulating were evaluated under different conditions by scanning aquarium with water temperature maintained between 26 electronic microscopy and DLS (Fig. 1a–c). We performed and 28 °C. The lighting conditions were 14 : 10 h (light : dark) incubations at 24 and 72 h in different biological media: and adult fish were fed twice a day at a rate of 2% phosphate buffer saline (PBS), cell culture medium (DMEM) bodyweight. Ammonia, nitrite, pH and nitrate levels were and larvae water (E3) (Fig. 1a). DLS results showed a particle measured once a week. Ammonia and nitrite levels were kept size of 65.1 ± 0.6 nm in water, 61.3 ± 1.4 nm in PBS, 67.2 ± below the detection level and pH maintained between 6.8 1.2 nm in DMEM and 59.6 ± 0.4 nm in E3 after 24 h and 72 and 7.5. The nitrate levels were maintained to be <100 mg h. No major changes were observed using DLS under any l−1. For in-tank breeding, one female and three males were conditions with respect to pure sterile water. No aggregation transferred to a breeding tank in the late afternoon. The of the particles over time was found (PDI less than 0.09) and divider was removed on the next morning after the onset of the zeta potential was stable and did not change significantly light. Embryos were collected after 1–2 h and cultured in over time, ranging between −20 and −32 mV and thus embryo medium (E3 medium) in a Petri dish (Deltalab). indicating a stable dispersion of the PS-NPs in suspension. Fertilized eggs were separated from unfertilized eggs using a Further confirmation of their characteristics was performed plastic pipette (Deltalab). All experiments involving zebrafish by direct sizing of FESEM images, showing values of 65.2 ± (D. rerio) were performed following International Guiding 6.5 nm (water) and 64.9 ± 7 nm (DMEM) (Fig. 1b and c). This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 2410–2422 | 2413 View Article Online Paper Environmental Science: Nano This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. Fig. 1 Characterization of fluorescently labelled polystyrene nanoparticles (PS-NPs) in different biological media. (a) PS-NP properties (size, PDI and ζ potential) assessed by dynamic light scattering, in pure water, PBS, cell culture medium (DMEM) and E3 embryo medium (E3). Field emission scanning electron microscopy representative images and size distribution histograms (n = 150) in (b) pure water and (c) DMEM. (d) Cytotoxicity of PS-NPs in zebrafish liver cells (ZFL). Cell viability after 24 h of incubation with PS-NPs at increasing concentrations (0.05 to 1000 mg l−1). Untreated cells and NaN3 treated cells were used as controls (C). An additional toxicity control with NaN3 was included, with the potential maximum concentration of NaN3 present at the highest concentration of PS-NPs. Data show the mean ± standard deviation (SD) of three independent experiments. Significant differences from the control are indicated as: *p < 0.05. Moreover, the expected spherical morphology was observed Fig. 1, the mean size, aggregation and morphology did not by FESEM under both conditions (Fig. 1b and c). As shown in change significantly after 24 and 72 h of incubation in 2414 | Environ. Sci.: Nano, 2020, 7, 2410–2422 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Paper DMEM nor E3. Overall, common biological buffers and data was available on any zebrafish cell line. In a recent study media did not change the physico-chemical properties of PS- by Cortés et al.,27 no toxicity was reported at concentrations NPs. The same commercially available PS-NPs were used by of up to 200 mg l−1 PS-NPs, after 24 h of treatment. It is Cui et al.,26 who reported a similar zeta potential and mean worth mentioning that the LD50 calculation methodology is This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. size in a slightly different zebrafish incubation medium. Pitt originally designed to calculate lethal doses of drugs or et al.6 reported a similar zeta potential but different mean chemicals. Due to this, and since no specific adaptation to size (34.8 ± 10 nm). This difference is probably due to the nanoplastic toxicity has been made, we sustain that these different compositions of the zebrafish incubation media. LD50 calculations should be interpreted with caution. To further study PS-NPs' interaction dynamics with living Considering the results of the viability and uptake assays as cells, we used PS-NPs labelled with a green fluorophore (Dragon well as the previous literature, we decided to use 5 and 50 Green). This fluorophore is not covalently linked to the PS-NPs mg l−1 as a representative non-toxic low and high PS-NP dose. Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. and could be drained from the NPs. In order to discard In all the cytometry experiments, a positive control of uncontrolled loss of fluorescence, we evaluated the fluorophore fluorescent nanoparticles with a well-characterised both by fluorimetry (ESI† Fig. S2a) and cytometry (ESI† Fig. S2b internalization behaviour was included.21 and c). The fluorescence signal at three different concentrations Results for the PS-NPs endocytosis showed that 100% of (1000, 100 and 50 mg l−1) was stable under the assayed ZFL cells took up the fluorescent PS-NPs after 6 h of conditions and the amount of leached fluorophore reached a incubation (Fig. 2a and c). Johnston et al.30 showed that 20 maximum of 25% after dilution and drastic manipulation (ESI† nm fluorescent PS-NPs were rapidly internalized in Fig. S2a). Although there is an apparent increase of fluorescence mammalian hepatocytes, as after a 30 min incubation the loss as the PS-NP concentration decreases, this can be explained fluorescence was located inside the cells. The ZFL by the inner filter effect, a well-known phenomenon that causes internalization dynamics follows a dose–response pattern, a decrease of fluorescence intensity at high doses and a better with cells incubated at higher doses and longer times yield at low doses. Therefore, the fluorescence measured at 50 presenting higher fluorescence (Fig. 2b–d). Internalization mg l−1 is more precise to obtain real percentages of fluorescence was confirmed by confocal microscopy at 5 and 50 mg l−1 PS- loss. When the distinct fractions (PS-NPs; P, pellet; SN, NPs (Fig. 2e; see ESI† Fig. S4 for the control). PS-NPs were supernatant) were used to evaluate the internalization of PS-NPs internalized and found in the cytosol as quite large clusters by ZFL cells (ESI† Fig. S2b and c), we observed that a remnant of (Fig. 2e and f, white arrows). At 50 mg l−1, there was a 24.7 ± 2.4% of cells were fluorescent when incubated with SN, massive accumulation in the cytosol (Fig. 2e) even though the while when incubated with the P fraction positive fluorescent cell viability was barely affected (Fig. 1d). Co-staining with cells were 78.2 ± 1.2%. According to Catarino et al., fluorescent Cell Mask and 3D reconstruction using Imaris software nanoplastics could have a significant loss of fluorescence and confirmed that PS-NPs were totally embedded inside the this draining has to be characterised using appropriate cytosol (Fig. 2f). Confocal image analysis indicated that ≈9 controls.20 In the present study, most of the observed PS-NP agglomerates of different sizes were observed per cell fluorescence is stably associated with PS-NPs and the (9.23 ± 3.4 agglomerates per cell at 5 mg l−1). Moreover, co- fluorescence signal computed as a positive fluorescence signal staining with Cell Mask (magenta), Hoechst (blue) and by cytometry and confocal microscopy is truly bound to PS-NPs. LysoTracker (red), a lysosome specific fluorophore, suggested that PS-NPs (green) accumulated in acidic lysosomes (Fig. 2g, white arrows). Lysosomes are dense spherical In vitro toxicity, uptake and accumulation of PS-NPs in ZFL organelles, but they can display considerable variation in size cells and shape as a result of differences in the materials that have Liver is the main detoxification organ in vertebrates and been taken up31. The 3D image reconstruction showed that accumulation of PS-NPs in zebrafish liver has previously been PS-NPs and lysosomes co-localize in quite large lysosomal reported.6,9 Understanding the interaction of PS-NPs with structures (Fig. 2h). To our knowledge, few studies have liver cells would help to better understand how aquatic studied the internalization dynamics of nanoplastics in cells vertebrates cope with the presence of nanoplastics in their of aquatic vertebrates. Different authors have shown that natural environment. Therefore, in order to evaluate the microplastics and larger nanoplastics (larger than 200 nm) endocytosis and potential toxic effects of PS-NPs, we choose are not easily internalised but particles of 100 nm or less are ZFL cells as an in vitro model. ZFL cells exposed to PS-NPs readily taken up by the endocytic cell machinery.32,33 showed a significant decrease in viability (68.8 ± 11.6%) after Johnston et al. described that 20 nm PS-NPs did not 20 h at concentrations of 100 mg l−1 and higher (Fig. 1d), accumulate in lysosomes but suggested that in human and although at 75 mg l−1 we already observed a non-significant rat cell lines NPs ended up in the mitochondria.30 Recently, decrease trend in cell viability. The calculated LD50 of PS-NPs it has been demonstrated that granulocytes from the mussel in ZFL cells was 188,8 mg l−1 (ESI† Fig. S3), extremely high M. galloprovincialis internalized 50 nm PS-NPs and compared to other toxics or genotoxics such as KBrO3 or accumulated them in lysosomes.14 In our work, we showed MMS.27 Other studies using different fish cell lines showed for the first time the internalization dynamics of PS-NPs in that toxicity is highly dependent on the cell line28,29 but no aquatic vertebrate hepatocytes and we demonstrated that This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 2410–2422 | 2415 View Article Online Paper Environmental Science: Nano they reached the lysosomal compartment (Fig. 2f). The final intracellular fate of nanoplastics could be different depending on the species, size, charge and plastic polymer composition, pointing out the need for more studies focused This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. on nanoplastic internalization dynamics in vertebrates. Transcriptional changes in zebrafish liver cells associated with PS-NP and poly(I:C) treatment To gain insight into the effects of PS-NPs alone and in combination with an immune stimulus, we used poly(I:C), a Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. synthetic dsRNA compound, to mimic a viral infection. The assessed target genes (mx, vig1 and gig2) are canonical genes related to the antiviral response that codify for interferon induced proteins34 and their upregulation in ZFL cells in response to poly(I:C), mounting a typical and strong antiviral response, has been previously characterised.23,35,36 As shown in Fig. 3, ZFL cells respond to poly(I:C) by upregulating antiviral genes such as mx, vig1 and gig2, at similar levels to those reported previously.21,23,35 When ZFL cells were pre- exposed for 12 h to PS-NPs and then treated with poly(I:C), a clear synergistic effect was observed (Fig. 3). Interestingly, when the cells were pre-exposed to PS-NPs for 24 h instead of 12 h, this synergistic effect was not observed, showing a typical poly(I:C) stimulation pattern. To further assess whether cells remained in a normal state after 24 h, we assessed lipid peroxidation levels after the PS-NP exposure and subsequent poly(I:C) treatment. No alterations in cell peroxidation status due to PS-NP exposure were observed (ESI† Fig. S5). Lipid peroxidation is a direct consequence of ROS production and we saw that after a 24 h exposure cells can deal with stress and maintain homeostasis. Altogether, data suggest that PS-NPs are internalized, accumulating in lysosomes, and initially provoke a strong and general stress state, thus potentiating the anti-viral response system. It could be speculated that PS-NP aggregation at higher doses could play a role in the uptake dynamics and, in turn, lead to Fig. 2 Uptake of fluorescently labelled PS-NPs by ZFL cells. Dose–response: differences in gene expression. However, we discarded this (a) percentage of fluorescent positive cells; (b) mean fluorescence intensity hypothesis, since the PS-NP stability data shown in Fig. 1 (MFI). Cells were incubated for 20 h with PS-NPs at an increasing indicate very low aggregation in DMEM after 72 h of concentration range (from 0.05 to 1000 mg l−1). Control (Ctrl) is ZFL cells incubation (PDI value of 0.07 versus 0.03 in water). A second without PS-NPs; a positive control of fluorescent nanoparticles (TNFα, in red) hypothesis is that interferences in the endocytosis and was included. Time-course: (c) percentage of fluorescent positive cells; (d) mean fluorescence intensity (MFI). Cells incubated for 6–72 h with PS-NPs at associated mechanisms could be affected by the PS-NP dose 25 (light green), 50 (mid green) and 75 (dark green) mg l−1. A positive control and exposure time. TLR3, the main receptor for poly(I:C) of fluorescent nanoparticles (TNFα, in red) was included. Data represent the responsible for the initial activation of the immune response, mean ± SD of three independent experiments. One-way ANOVA was is located in the endosomal membrane,34 the same performed with Dunnett's multiple comparison test, comparing treatment subcellular compartment where the PS-NPs traffic and and control means. Significant differences are indicated as: *p < 0.05; **, p < 0.01. (e) Confocal microscopy images of ZFL cells showing the accumulate. We hypothesise that at a low PS-NP internalization of PS-NPs, after 24 h of incubation at 5 and 50 mg l−1. Green concentration the antiviral response signals through TLR3 fluorescence corresponds to PS-NPs (white arrows), blue to Hoechst stained and synergises with a cell stress response. At a higher dose or nuclei (N), and magenta to WGA555 or Cell Mask stained plasma membrane longer exposure (leading to an internalization of a higher (PM). (g) Green fluorescence corresponds to PS-NPs; blue and magenta to number of particles), the endosomal architecture might be Hoechst stained nuclei (N) and Cell Mask stained plasma membrane. Red fluorescence corresponds lysosomes (LYS) stained with LysoTracker. White overloaded and the TLR3 pathway and cell stress response arrows point to colocalization of PS-NPs (green) and lysosomes (red). (f) 3D disturbed. Nevertheless, the antiviral response can signal image analysis of PS-NP uptake (z-stack) and whole-membrane alternatively through RIG-1, LGP-2 or MDA5,37,38 thus reconstruction. (h) 3D image analysis of PS-NP uptake (z-stack) and lysosome maintaining the poly(I:C) response at high dose or long co-localization of PS-NPs. 2416 | Environ. Sci.: Nano, 2020, 7, 2410–2422 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Paper size of the endosomal–lysosomal system are massively changed at low versus high dose (ESI† Fig. S6a and b). We observed that at high dose: the number of clusters inside the cell increased (n = 464 at high dose versus n = 950 at low This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. dose) and their size almost tripled (mean 0.1549 ± 0.0058 – high dose versus mean 0.05902 ± 0.001895 – low dose). Coincidentally, Canesi et al.39 found a perturbation on the endolysosomal system of M. galloprovincialis haemocytes after exposure to PS-NPs. They observed a decrease in lysosomal membrane stability (LMS) after exposures to 5 and 50 mg l−1 PS-NPs.39 The endosomal–lysosomal system is known to Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. participate in several aspects of cell physiology, besides its classical role in protein degradation. As Neefjes and collaborators40 say: “endosomal system constitutes a key negotiator between the environment of a cell and its internal affairs”. For example, lysosome overload has an impact on the mRNA levels of several lysosomal proteases41 and is extremely important for a correct regulation of antigen receptor and pattern recognition receptor signalling in the innate immune system.42 On the other hand, a cell stress response in which low doses cause a greater impact than high doses has been previously documented in nanoparticle toxicology. For example, Teles et al.43 observed changes in TOS (total oxidative status) and in mRNA levels of antioxidant-related genes after exposure to gold nanoparticles in Sparus aurata. Changes only occurred at the tested low and intermediate concentrations, revealing a non-monotonic dose–response curve, a pattern of response frequently found for endocrine disrupting chemicals. Moreover, Brandts et al.44 found an increased mRNA expression of genes involved in biotransformation (cypp11, pgp) and immune function (cathepsin) and a decrease in ChE in M. galloprovincialis only at lower PS-NP doses (0.05 and 0.5 mg l−1). In the same Fig. 3 Analysis of gene expression in ZFL cells after PS-NP treatment species, Canesi et al.39 found a greater increase in ROS followed by poly(I:C) treatment. Cells were incubated for 12 or 24 h with PS-NPs followed by 25 μg ml−1 poly(I:C) stimulation for 16 h. production in haemocytes exposed to PS-NPs at low Unexposed control cells (grey), 5 mg l−1 PS-NPs (light green) and 50 concentration (1 mg l−1). This suggests that, under some mg l−1 PS-NPs (dark green). Viperin (vig1), grass-carp-reovirus-induced circumstances, low doses of nanoplastics could cause a gene 2 (gig2) and IFN-induced protein Mx (mx) gene expression is greater impact than higher doses. Maybe a combination of shown. Samples are from two independent experiments and data are scenarios (disturbance of the endolysosomal system and/or expressed as mean ± SD (n = 4). Differences between each treatment mean and control are indicated as *p < 0.05. The legend presented cell stress response) could explain the gene expression underneath the graphics indicates the presence (+) or absence (−) of synergy observed at low dose. However, further work would the different treatments: PBS ± poly(I:C), 5 mg l−1 PS-NPs ± poly(I:C); be necessary to understand the precise role of intracellular 50 mg l−1 PS-NPs ± poly(I:C). PS-NP accumulation and how this affects the immune response. exposure. To further explore this hypothesis, we analysed the average size of the PS-NP clusters inside the ZFL cells after In vivo uptake and accumulation of PS-NPs in zebrafish exposure to 5 and 50 mg l−1 PS-NPs. We found a significant larvae and effects on survival after bacterial infection difference in cluster size between the two concentrations Zebrafish larvae 5 dpf were imaged after 48 h of in vivo (0.2691 ± 0.01395 – low dose versus 0.3873 ± 0.01387 – high exposure to fluorescent PS-NPs (5 and 50 mg l−1) (see Fig. 4a), dose), which could sustain the possibility of a lysosomal to confirm uptake. PS-NPs were taken up by the larvae and overload (ESI† Fig. S6c). In line with this, we also tested accumulated in the intestine and pancreas (Fig. 4b). The RTGut cells, a different fish cell line derived from intestinal distribution of the fluorescence intensity normalized by the epithelia, which could be more conceptually relevant for organ area was 65 ± 8.7% in the gut and 34 ± 8.7% in the waterborne absorption. In this cell line, the morphology and pancreas (Fig. 4c). The zebrafish pancreas reaches its final This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 2410–2422 | 2417 View Article Online Paper Environmental Science: Nano This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. Fig. 4 Zebrafish larvae PS-NP uptake and survival after PS-NPs and infection with A. hydrophila. (a) Exposure and infection chronogram. Zebrafish larvae (3 dpf) were immersed in 5 or 50 mg ml−1 of PS-NPs for 48 h. At 5 dpf, larvae were infected with A. hydrophila by bath immersion, at a dose of 1.85 × 109 cfu per ml. Survival was recorded every 24 h for 6 days. E3, 5 and 50 mg l−1 PS-NP immersed larvae were used as controls. G, gut and P, pancreas. (b) Representative images of biodistribution of fluorescent PS-NPs in zebrafish larvae. Zebrafish larvae (3 dpf) were immersed in 5 or 50 mg ml−1 of PS-NPs for 48 h. E3 immersed larvae were used as controls. (c) Distribution of the fluorescence intensity (%) in the gut and pancreas. Images were analysed with ImageJ and the measured fluorescence was normalized by the organ area. (d) Survival of the PS-NP treated zebrafish larvae (n = 72) challenged with A. hydrophila. Larvae immersed in E3 followed by A. hydrophila challenge was used as a mortality control. Cumulative survival curves were constructed using GraphPad. Significant differences were analysed using the log-rank (Mantel–Cox) test. 2418 | Environ. Sci.: Nano, 2020, 7, 2410–2422 This journal is © The Royal Society of Chemistry 2020 View Article Online Environmental Science: Nano Paper position and shape by 6 dpf, the intestine is open to the concentrations of 5 and 50 mg l−1 for 48 h did not provoke surrounding environment between 3 and 4 dpf,45,46 and the any mortality different from control larvae (Fig. 4c). Low dose larva has a fully functional digestive system at day 5,47 (5 mg l−1) and high dose (50 mg l−1) exposures were tested for therefore imaging at 5 dpf is an optimal window of time. The a subsequent infection with A. hydrophila. As shown in This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. pancreas is located asymmetrically on the right side of the Fig. 4d, the PS-NP exposure did not affect the survival after body and has an elongated shape and characteristic head– infection, as both treated and non-treated larvae had the neck–tail morphology, where the anterior part contains islet same survival curves. Different authors have described how tissue surrounded by exocrine tissue, while the neck and tail exposure to nanoplastics may affect the immune system by consist of exocrine tissue.45 We hypothesise that the larvae interfering in immune related gene expression, detoxifying immersed in the solution of fluorescent PS-NPs could have enzyme activity (catalase etc.), or in ROS production, but this ingested the PS-NPs and absorbed them through the is the first study investigating the effects of nanoplastic Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. intestinal epithelia, distributing them systemically and exposure on the capacity to fight a lethal infection in accumulating also in the pancreas. Nevertheless, we do not zebrafish. At both tested concentrations, zebrafish larvae have definite proof of this uptake route. We do not discard could cope with an acute PS-NP exposure and the that PS-NPs may accumulate in other organs such as the physiological machinery was able to maintain the liver, spleen or brain at later life stages. We actually expected homeostasis. Only one previous work studied the interaction to find fluorescence in the liver, but even at continuous PS- between nanoplastic exposure and pathogenic infection. In NP exposure and saturating doses (60 h, 100 mg l−1) we could the said study, an in vitro infection study using isolated not detect PS-NPs' fluorescence in the zebrafish liver. This mussel haemocytes,14 the authors showed that the pattern of accumulation has been previously reported hemocytes' phagocytic capacity decreased when exposed to although with some differences, likely due to the specific PS-NPs but not when the hemocytes were exposed to a exposure time, dose, size and larvae age. For example, Brun combination of PS-NPs and V. splendidus. Nevertheless, a et al. observed accumulation of 25 nm PS-NPs (20 mg l−1)7,8 chronic or repeated acute exposure could perhaps lead to a in the intestine, exocrine pancreas and gall bladder after 48 h failure of the pathogen response mechanisms. To further of exposure at 120 hpf (5 dpf). It is worth mentioning that understand the effects of PS-NPs on fish, chronic or repeated there is a considerable variation in between studies, with PS- acute exposure should be investigated, in order to determine NPs being located in the intestine and neuromast,7 in the if PS-NPs could affect the immune system in the long-term. heart region,10 in the digestive track and eyes,48 in the gut and gills region49 and in the gastrointestinal tract, gallbladder, liver, pancreas, heart and brain.6 Differences Conclusions with published data could also be attributed to the use of Our study shows that PS-NPs are efficiently taken up by ZFL other ZF strains (e.g. AB/TL wild type as in van Pomeren cells in vitro, tending to accumulate in lysosomes, possibly in et al.,48 Veneman et al.,10 Brun et al.7 or Sökmen et al.11), an attempt of the cell machinery to degrade PS-NPs. The instead of wild type larvae as in our case. We decided to use expression of immune genes was synergistically affected by a wild type zebrafish (no strain) to approach the individual viral stimulus (polyIJI:C)) at low doses and early time points in variability present in a natural population, in order to better ZFL cells. Even though we see this synergistic effect of PS- emulate an environmental situation. NPs and poly(I:C) at 12 h and 5 mg l−1, this effect disappears In aquatic invertebrates, the scenario is similar, and the at a longer exposure time (24 h) as well as a higher dose (50 accumulation pattern of nanoplastics depends on age, dose, mg l−1). Therefore, it suggests that ZFL cells can regain material or size. For example, oyster (Crassostrea gigas) larvae homeostasis and normal function after the PS-NP challenge, have been found to ingest both nanoplastics and presenting the expected response to a viral stimulus. microplastics at different ages, being the younger larvae the Furthermore, zebrafish larvae also incorporated PS-NPs, ones with the highest capacity to ingest 160 nm PS-NPs.50 potentially through ingestion, and accumulated them in the Sea urchin (Paracentrotus lividus) embryos accumulated PS– gut and pancreas. Nevertheless, on exposure to PS-NPs at COOH-NPs inside the embryo's digestive tract without any concentrations of up to 50 mg l−1, PS-NPs did not show toxic effects, while PS–NH2-NPs induced strong toxic effects.51 toxicity nor interference with a normal immune response Finally, mussels (M. galloprovincialis) can accumulate 50 nm against A. hydrophila. Altogether, although PS-NPs can induce PS-NPs after 24 h of waterborne exposure, in the digestive an immune response, the survival of zebrafish larvae system, muscle and gills.14 A recent review by Kögel et al.52 challenged with a bacterial infection after an acute exposure pointed out how different parameters such as charge, size or to PS-NPs is not decimated with respect to unexposed larvae. concentration could affect the uptake and toxicity of nanoplastics in aquatic organisms. In our study, the highest PS-NP concentration tested (100 Authorship mg l−1) in a continuous 96 h exposure provoked 100% mortality, while continuous exposure to 50 mg l−1 provoked IB and NR designed the study. IB and NR designed and around 35% mortality (ESI† Table S2). However, performed the experiments. IB, MT, MGO and NR did the This journal is © The Royal Society of Chemistry 2020 Environ. Sci.: Nano, 2020, 7, 2410–2422 | 2419 View Article Online Paper Environmental Science: Nano data analysis. NR wrote the manuscript and all authors microplastics in zebrafish (Danio rerio) and toxic effects in contributed to the preparation of the final manuscript. liver, Environ. Sci. Technol., 2016, 50, 4054–4060. 10 W. J. Veneman, H. P. Spaink, N. R. Brun, T. Bosker and Conflicts of interest M. G. Vijver, Pathway analysis of systemic transcriptome This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. responses to injected polystyrene particles in zebrafish The authors declare no conflict of interest. larvae, Aquat. Toxicol., 2017, 190, 112–120. 11 T. Ö. Sökmen, E. Sulukan, M. Türkoğlu, A. Baran, M. Acknowledgements Özkaraca and S. B. Ceyhun, Polystyrene nanoplastics (20 This work was supported by grants from the Spanish Ministry nm) are able to bioaccumulate and cause oxidative DNA of Science, European Commission and AGAUR funds to NR damages in the brain tissue of zebrafish embryo (Danio (RTI2018-096957-B-C21, MINECO/FEDER and 2014SGR-345 rerio), Neurotoxicology, 2020, 77, 51–59. Open Access Article. Published on 22 July 2020. Downloaded on 12/9/2020 6:09:43 PM. AGAUR). IB was funded by a pre-doctoral scholarship from 12 Y. Jin, J. Xia, Z. Pan, J. Yang, W. Wang and Z. Fu, Polystyrene UAB (2018FI_B_00711). We thank the LLEB (UAB), Servei de microplastics induce microbiota dysbiosis and inflammation Citometria (SCAC-UAB) and Servei de Miscroscopia (UAB) for in the gut of adult zebrafish, Environ. Pollut., 2018, 235, technical support and Dr. C. Tafalla for the generous gift of 322–329. RTGut cells. 13 E. Bergami, A. Krupinski Emerenciano, M. González- Aravena, C. A. Cárdenas, P. Hernández, J. R. M. C. 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