Environmental Science Nano View Article Online PAPER View Journal Hazard assessment of ingested polystyrene This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: DOI: 10.1039/d1en01199e nanoplastics in Drosophila larvae† Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. Mohamed Alaraby,ab Doaa Abass,ab Josefa Domenech,a Alba Hernándeza and Ricard Marcos *a Micro- and nanoplastics (MNPLs) are intentionally produced for commercial uses (primary MNPLs) or are formed from environmentally aged plastics (secondary MNPLs). Independent of their origin, all of them will finally end up in the environment constituting some of the known emergent pollutants. Despite the inert nature of plastics, questions about their potential biological effects on human health need to find sound answers. In addition, the association between the potentially induced effects and the MNPL size is also required to be known. In this context, we have used our in vivo model of Drosophila larvae and three nanopolystyrene plastics (PSNPLs) sized 50, 200, and 500 nm (PS-50, PS-200, and PS-500) to add new data to better understand the potential health risks of MNPLs. Our model has permitted us to visualize (via transmission electron microscopy, TEM) the journey of the PSNPLs administered via ingestion, their interaction with gut lumen components (including symbiotic microbiota), their uptake by gut enterocytes, their translocation through the intestinal barrier to the hemolymph, and their uptake by hemocytes. This behavior was observed for the three analyzed sizes and, for the largest sizes, changes in size/shape were observed in ingested PSNPLs. Although no relevant toxicity, as measured by the egg-to-adult viability, was Received 27th December 2021, observed, exposure induced a wide molecular response altering the expression of genes involved in the Accepted 1st March 2022 general stress response, in the antioxidant response and even in the genotoxicity response, as well as in genes related to the intestinal damage response. Furthermore, a general induction of ROS production and DOI: 10.1039/d1en01199e DNA damage was also detected. Interestingly, these types of responses were size-dependent with the rsc.li/es-nano small PSNPL size inducing a higher response. Environmental significance Micro/nanoplastics (MNPLs) are wide-spread environmental pollutants requiring new data to know more about the probable health risks associated with their exposure. Since the main exposure route is ingestion, we have tried to answer different unsolved questions using three different sizes of polystyrene nanoplastics (PSNPLs) in an in vivo model of Drosophila. As a novelty, we have extensively characterized their interaction with the intestinal barrier of the Drosophila larvae by using TEM, detailing, step by step, the journey of PSNPLs ending in the enterocyte uptake and posterior translocation. The biologically evaluated effects were oxidative stress, gene expression changes, and genotoxicity. It is important to emphasize that no similar studies on this topic have been found in the literature. Background gyres.1 Unfortunately, and despite the actions that are being carried out, these pollution levels are expected Plastic wastes are polluting every point of our planet. This is to continue to grow in the following years.2 Although special extremely alarming in oceans where millions of tons of emphasis is focused on the environmental effects of gross plastic wastes accumulate, mainly in the subtropical plastic wastes, the effects of micro- and nano-products resulting from plastic degradation are gaining interest, mainly as potential factors affecting human health.3 Micro a Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of and nanoplastics (MNPLs) resulting from the degradation of Biosciences, Universitat Autònoma de Barcelona, Campus of Bellaterra, 08193 plastic items, known as secondary MNPLs, are considered a Cerdanyola del Vallès, Barcelona, Spain. E-mail: ricard.marcos@uab.es; new group of emergent contaminants requiring intensive Fax: +34 93 581 23 87; Tel: +34 93 581 20 52 b Zoology Department, Faculty of Sciences, Sohag University, 82524, Sohag, Egypt efforts to evaluate their potential effects on health. It must be † Electronic supplementary information (ESI) available. See DOI: 10.1039/ stated that there are MNPLs tailored at the micro/nano size d1en01199e to be used for different purposes, which are denoted as This journal is © The Royal Society of Chemistry 2022 Environ. Sci.: Nano View Article Online Paper Environmental Science: Nano primary MNPLs. One example of this type of MNPL is nano/ with exposure to PSMNPLs. Special attention has been microbeads extendedly used in the production of cosmetics. dedicated, via transmission electron microscopy (TEM) Although there are no reliable data on the production of such images, to monitoring their journey into the intestine lumen, MNPLs, the results of a survey indicated that in Europe their their potential interaction with different lumen components, annual production is about 6.8 × 105 metric tons, although their potential uptake by enterocytes, and their translocation this amount could vary for a factor of 10.4 Nevertheless, and through the intestinal barrier to the open circulatory system independent of their origin as primary or secondary MNPLs, of the larvae, reaching hemocytes (as equivalent to they will all finally end up as plastic debris into the lymphocytes in humans). In addition, the effects on the environment. Although the environmental concentrations of deregulation of different genes, as well as the induction of This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. MNPLs are disputable, mainly for NPLs, the concentration of oxidative stress and genotoxicity, as detected by the induction MPLs could reach 3.35 × 106 particles per m3 in sediments of DNA breaks, were also systematically studied. Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. and 5.57 particles per m3 in water, as determined in the German Elbe river.5 Although the number of studies aiming to produce sound Materials and methods data to be used for the risk assessment of MNPL exposure is Physicochemical properties of polystyrene nanoplastics exponentially growing, there are still many gaps mainly Nanopolystyrene particles, as a popular nanoplastic model, related to the identification of their mechanisms of action. In were purchased from Spherotech (Lake Forest, IL, USA). Most this way, it is difficult to obtain shreds of evidence that of the other chemicals and kits used in the different environmental MNPL pollutants can cause human exposure experiments were from Sigma Chemical Co. (St. Louis, MO, and health effects.6,7 Alternatively, in vivo approaches can USA). To study the impact of size on the effects of plastic constitute appropriate ways to find sound data linking MNPL particles, three different sizes of polystyrene nanoplastics exposure and biological effects on the evaluated organisms. (PSNPLs) were selected: i) particles ranging from 50–100 nm Nevertheless, although the reported results using mouse (PS-50; SPH-PP-008-10), ii) particles ranging from 100–200 models suggest that accumulation of MNPLs in mammals nm (PS-200; SPH-PP-015-10), and iii) particles ranging from would likely have negative effects, their long-term 400–600 nm (PS-500; SPH-PP-05-10), according to the consequences are unclear.8 Due to the complexity and the manufacturer's data. As the size range is in the nano range cost of using mammalian models, together with many posed (1–1000 nm), they are all considered polystyrene nanoplastics ethical issues, the use of more simple in vivo models is (PSNPLs). They were supplied as a 0.02% sodium azide gaining interest. Among these alternative in vivo models dispersion and stored at room temperature. To achieve the Drosophila stands out.9 desired concentrations, PSNPLs were dispersed in Milli-Q- Drosophila is a pioneering in vivo model widely used to water. Particle suspensions were always vortexed before use. determine the harmful effects of a wide set of agents, The characterization of the PSNPLs included several including different categories of micro and nano-sized techniques and tools; a JEOL 1400 instrument (JEOL Ltd., materials.9,10 A new field termed Drosophotoxicology was Tokyo, Japan) for transmission electron microscopy (TEM) proposed to highlight the many advantages of this new-old and a Malvern Zetasizer Nano-ZS zen3600 (Malvern, UK) model to evaluate the health effects of environmental instrument were used to detect the size, morphology, pollutants.11 Since about 75% of the genes involved in hydrodynamic size, and charge. In addition, Fourier- human pathologies are homologous to Drosophila, this transform infrared spectroscopy (FTIR) using a Hyperion organism can represent a useful model to identify 2000 micro-spectrometer (Bruker, Billerica MA, USA) was mechanisms associated with different human diseases.12,13 used to confirm that all particles related to polystyrene share Despite the above mentioned, it is surprising that few studies the same functional groups. have applied the Drosophila model to obtain new insights into the potential hazard resulting from MNPL exposure. Only four recent studies have been found in the literature. Toxic effects of polystyrene nanoplastics Two of them evaluated polystyrene (PS) MNPLs, and the other The wild-type strain Canton-S was used in all the two used polyethylene (PET) MNPLs. Exposure to PSMNPLs experiments. To evaluate the toxic effects of PS-50, PS-200, resulted in gut damage, locomotor dysfunction, epigenetic and PS-500 and to determine the appropriate doses to be silencing, and aggravated cadmium toxicity,14 as well as used in the further experiments, Drosophila eggs were somatic mutation and recombination in the wing imaginal exposed to a wide range of concentrations: 0, 32, 160, and disks.15 On the other hand, exposure to PETMNPLs was 800 μg mL−1, which are equivalent to 0, 0.08, 0.4, and 2 mg associated with changes in fertility, the offspring of treated PSNPLs per g food. Exposure lasts for all the developmental flies were smaller than the offspring of controls,16 as well as stages, until adult emergence. A total of 250 eggs per dose decreased oviposition in exposed females and reduction of were seeded in five vials (50 eggs per vial) containing 4 grams triglyceride and glucose content in male flies.17 of instant medium per vial. To hydrate the lyophilized instant In this context, the present study was planned to obtain culture medium, 10 mL of the different concentrations of information on a wide range of potential effects associated PSNPLs were added to each vial. The emergent adults were Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2022 View Article Online Environmental Science: Nano Paper counted, and the viability percentage was calculated for each using 30 larvae (∼50 mg) in the third-instar of development. administered dose, in comparison with the unexposed flies. The obtained RNA pellet was dissolved in Milli-Q water and quantitatively measured with a NanoDrop device. The RNA Interaction of polystyrene nanoplastics with the intestinal samples were used to synthesize cDNA using a transcriptor barrier components first strand cDNA synthesis Roche kit and were subjected to To investigate the interaction of PS-50, PS-200, and PS-500 RT-PCR analysis using a Light Cycler 480 (Roche, Basel, with the different components of the larvae intestine, Switzerland). The housekeeping gene β-actin was used as a including enterocyte uptake, our previous protocol was reference gene. For all the genes, the same reaction followed.18 Thus, about ten treated larvae (2 mg g−1 food), conditions were applied: pre-incubation for 5 min at 95 °C, 1 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. collected after the 5th day of exposure, were carefully washed cycle, and the amplification was repeated 45 times (10 s at 95 °C, 15 s at 61 °C, and 72 °C for 25 s). Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. and dissected in phosphate buffer solution to extract the intestinal tracts. After extraction, the intestines were directly immersed for 2 h in a fixation solution, post-fixed in 1% Detection of oxidative stress induction (w/v) osmium tetroxide containing 0.8% (w/v) potassium The levels of oxidative stress induced by the three selected hexacyanoferrate, before washing four times with deionized sizes of PSNPLs were detected in the hemocytes of Drosophila water and sequential dehydration in acetone. Next, Eponate larvae. To proceed, 5th day larvae treated with PS-50, PS-200, 12™ resin (Ted Pella Inc., Redding, CA) was used to embed and PS-500 (0.0, 0.08, 0.4, and 2 mg g−1 food) were dissected the samples and left to polymerize at 60 °C for 48 h. in buffer solution (PBS 1%) to extract the hemolymph. The Ultrathin sections (100 nm in thickness) were obtained by hemolymph was incubated with 5 μM 6-carboxy-2,7′- cutting the resin blocks with a diamond knife (45°, Diatome, dichlorodihydro-fluorescein diacetate (DCFH-DA) for 30 min Biel, Switzerland). Such ultrafine intestinal cuts were placed at room temperature to mark cells containing reactive oxygen on non-coated 200 mesh copper grids and contrasted with species (ROS). For negative and positive controls, the uranyl acetate for 30 min and Reynolds lead citrate for 5 hemolymph was extracted from non-treated larvae and larvae min. Finally, the sections were inspected using a JEOL 1400 treated with 0.5 mM H2O2 for 30 min. Ten fluorescence (100 kV) TEM equipped with a CCD Gatan ES1000w images (fields) were taken using a fluorescence microscope, Erlangshen camera (Gatan Inc, Pleasanton, CA). For these with an excitation wavelength of 485 nm and an emission studies, evaluating the journey of PSNPLs inside the gut, only wavelength of 530 nm (green filter). For the quantitative the higher dose was used just to facilitate their detection. evaluation of fluorescent cells, both control and treated larvae from fluorescence images were quantified using the Detection of polystyrene nanoplastics in the hemolymph ImageJ program. To detect the potential translocation of PSNPLs through the intestinal barrier, their presence in the hemolymph was Detection of genotoxicity using the comet assay investigated. To proceed, samples of 5th day treated larvae The potential induction of DNA damage in hemocytes was were prepared and investigated according to our previous investigated by using the comet assay, which can quantify the protocol.19 Briefly, larvae were picked up with forceps, induction of DNA strand breaks. To proceed, third-instar (72 carefully cleaned, and dissected in buffer solution (PBS 1%) h) larvae treated with two doses (0.4 and 2 mg g−1 food) of to obtain the hemolymph fluid. Hemolymph samples were PS-50, PS-200, and PS-500 were collected, washed, and kept collected and placed upon copper grids, left to dry, and in autoclaved phosphate buffer solution (PBS 1%). investigated with a JEOL 1400 (100 kV) TEM to detect the Unexposed larvae were used as a negative control, while presence of PSNPLs. larvae treated with 4 mM ethyl methanesulfonate (EMS) were used as a positive control. The used protocol has been Detection of gene expression changes previously described.9 Ten μL of hemolymph, containing Mild stress responses to PSNPL exposure can be detected via about 10 000 cells, was added to 90 μL of 0.75% low-melting changes in the expression levels of specific genes In this agarose at 37 °C and mixed carefully. The mixture of cells study we proposed to investigate changes in the expression and agarose was dropped upon the hydrophilic surface of a levels of general stress genes (heat-shock-protein-83, Hsp83, cold Gelbond® film (GBF). After lysis, electrophoresis, and and heat-shock-protein-70, Hsp70), oxidative stress genes (Cu/ neutralization steps, GBFs were stained with SYBERGold Zn superoxide dismutase, Cu/Zn Sod; catalase, Cat, and fluorochrome for 20 min and finally washed with distilled superoxide dismutase-2, Sod2), DNA repair genes water. DNA break induction was measured according to the (8-oxoguanine DNA glycosylase, Ogg1), and two genes percentage of DNA in the tail using the Komet 5.5 image- involved in the response to physical stress induced in the analysis system (Kinetic Imaging Ltd., Liverpool, UK) fitted intestinal barrier (dual oxidase, Duox, and prophenoloxidase- with an Olympus BX50 fluorescence microscope equipped 2, PPO2). To this end, real-time RT-PCR was used to with a 480–550 nm wide-band excitation filter and a 590 nm investigate gene expression levels as previously reported.9 barrier filter. Three replicates of GBFs of 300 randomly RNA was extracted following the manufacturer's protocol selected cells per dose were used. This journal is © The Royal Society of Chemistry 2022 Environ. Sci.: Nano View Article Online Paper Environmental Science: Nano Statistical analysis functional groups. The obtained results are shown in Fig. 1. The normality and the homogeneity of the data were checked The TEM images showed that all the studied PSNPLs agree before performing the statistical analysis, using the with the range of sizes indicated by the supplier and show a Kolmogorov–Smirnov & Shapiro–Wilk test and Levene's test, spherical shape. The average diameter for PS-50, according to respectively. For data with normal distribution and equal TEM, was 61.40 ± 14.72, for PS-200 the obtained average was variance, Student's t-test and the one-way ANOVA were 145.56 ± 9.0, and finally, for PS5-00 the average diameter was applied. The nonparametric analysis (Mann–Whitney U-test) 415.22 ± 25.25. The spectra obtained from FTIR confirm that was used for data with unequal variance or abnormal all chosen PSNPLs have the same functional groups of polystyrene. The peaks that appeared below 3000 cm−1 refer This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. distribution. Significant differences were considered at P ≤ 0.05. Data were calculated as mean ± standard error (SE). to CH2, while those above 3000 cm−1 refer to the benzene ring, as confirmed by the strong peaks at 1500 cm−1. These Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. Results peaks are indicative of the functional groups that are identical to those of PS. Zetasizer data showed a trend similar Physicochemical characteristics of PS-50, PS-200, and PS-500 to that obtained with TEM. The averages for the To determine the effect of size, one PSNPL <100 nm (PS-50) hydrodynamic sizes were 73.74 ± 1.88, 171.6 ± 3.94, and 488.9 and two PSNPLs >100 nm (PS-200 and PS-500) were ± 8.624 for PS-50, PS-200, and PS-500, respectively. All PSNPLs characterized using different available tools like transmission showed negative charges with zeta potential values of −49.7 ± electron microscopy (TEM), Malvern Zetasizer measurement, 0.66, −49.6 ± 0.41, and −53.0 ± 1.99 for PS-50, PS-200, and PS- and FTIR spectroscopy to determine the size, morphology, 500, respectively. These zeta potential values indicate the degree of aggregation, hydrodynamic size, charge, and good dispersion of the different PSNPLs. Fig. 1 Physicochemical characteristics of different sized PSNPLs (PS-50, PS-200, and PS-500). The diameter averages of PS-50 (61.40 ± 14.72), PS-200 (145.56 ± 9.19), and PS-500 (415.22 ± 25.25) (D–F) were determined from different representative TEM images (A–C). To confirm the chemical composition of PSNPLs, Fourier transform infrared spectroscopy (FTIR) was applied to measure their functional groups (G). A Malvern Zetasizer instrument was used to determine the hydrodynamic size and zeta potential of the different PSNPLs (H). Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2022 View Article Online Environmental Science: Nano Paper Polystyrene nanoplastic toxicity internalization of PS-200 is presented in Fig. 3A–F. Thus, PS- To detect the toxicity of the different sized PSNPLs, a defined 200 particles are distributed inside the midgut lumen, number of eggs of Drosophila were seeded upon an instant surrounding symbiotic midgut bacteria, near the peritrophic medium, previously wetted with different concentrations of membrane (3A). Interestingly, some PS-200 can penetrate the PSNPLs. The emergent adults were harvested and counted to bacteria membrane to internalize inside the midgut bacteria calculate the percentage of adult survival after PSNPL (3B). PS-200 particles are also taken up by enterocytic cells exposure. The obtained results (ESI† Fig. S1) show that none facilitated by the presence of luminal vacuoles that appeared of the different sized PSNPLs tested induced toxicity. No to engulf these particles to translocate them into enterocytes crossing the peritrophic membrane (3C and 3D). The This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. morphological changes or delays in the emergence of adults were observed associated with PSNPL exposure. particles just passing through the peritrophic membrane can be seen between the enterocyte microvilli and cytoplasm (3D Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. and 3E). Finally, PS-200 can translocate towards the open Monitoring of PSNPLs along the intestinal tract circulatory system, as detected in the hemolymph (3F). The The behavior of PSNPLs during their transit through the monitoring of PS-500 particles from ingestion to their intestine can provide important information on their translocation into the open circulatory system is presented in potential biological effects. To this end, we have described Fig. 4(A–D). After oral administration of PS-500 particles, step by step all this long way starting from their entrance into their existence inside the midgut lumen can be detected the intestinal lumen, their interaction with different lumen easily due to their large size (4A). PS-500 can be detected near components, their interaction with the intestinal barrier, the peritrophic membrane to be ready to enter enterocytes their uptake and distribution inside enterocytes, and their (4B) or surrounding midgut bacteria (4C). The internalization potential translocation through the barrier reaching the into the enterocytes forming the intestinal barrier can be hemolymph. All this information is shown in Fig. 2–4. The associated with the previous attachment to the peritrophic monitoring of the smallest particles (PS-50) is presented in membrane or the engulfment by luminal vacuoles (4D). After Fig. 2(A–F). Thus, the particles are distributed in the midgut their uptake by the intestinal barrier, PS-500 particles are lumen (2A) and are observed to interact with the peritrophic distributed into the enterocyte cytoplasm (4E), being able to membrane, which is the first line of the intestinal barrier translocate towards the hemolymph (4F). As a summary, our defense. Interestingly PSNPLs are observed to be surrounded results show that PSNPLs, independent of their size, can by extracellular membranes acting as carriers for their penetrate and cross the intestinal barrier reaching the open internalization (2B). PS-50 particles succeed to internalize circulatory system as detected in the hemolymph. into the enterocytes and are observed inside the cytoplasm especially surrounding the mitochondria (2C and 2D, respectively). PS-50 can translocate through the intestinal PSNPL midgut erosion barrier as observed in the hemolymph of exposed larvae as Since the midgut lumen environment can affect the integrity individual particles (2E) or as aggregates (2F). The large size of the administered PSNPLs, potential changes in their initial of polystyrene particles, whether PS-200 or PS-500, facilitated size were evaluated. The large size of the selected PSNPLs, their detection during their internalization process. The especially PS-200 and PS-500, helps us to measure a sufficient Fig. 2 Monitoring of ingested PS-50 from their ingestion until their translocation to the hemolymph compartment. PS-50 particles are distributed inside the midgut lumen (A), attached to the peritrophic membrane (B), inside the enterocyte cytoplasm (C), surrounding the mitochondria (D) and, finally, reaching the hemolymph (E and F). This journal is © The Royal Society of Chemistry 2022 Environ. Sci.: Nano View Article Online Paper Environmental Science: Nano This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. Fig. 3 Monitoring of PS-200 from their ingestion until their translocation to the hemolymph compartment. PS-200 particles are detected in the midgut lumen and enterocytes (A), surrounding/inside midgut bacteria (B), surrounded by/inside midgut vacuoles (C), near the peritrophic membrane or between microvilli (D), inside the enterocyte cytoplasm (E) and, finally, reaching the hemolymph (F). Fig. 4 Monitoring of PS-500 from their ingestion until their translocation to the hemolymph compartment. As occurs with the other sizes, PS-500 particles are detected inside the midgut lumen (A), surrounding the peritrophic membrane (B), surrounding midgut bacteria (C), inside the cytoplasm of enterocytes (D), surrounded by midgut vacuoles (E) and, finally, reaching the hemolymph (F). number (70 particles per size) to determine their size both PSNPL exposure modulates gene expression levels inside the midgut lumen and in previous oral administration (Fig. 5, A–F). The measured mean sizes inside the midgut The molecular response against any xenobiotic agents could lumen (141.90 ± 39.73 nm and 319.79 ± 97.45 nm) were give us an indication of their biological impact, acting as an compared with their initial sizes (145.56 ± 9.19 nm and early alarm of disturbance in the internal hemostasis 415.22 ± 25.25 nm) for PS-200 and PS-500 (5A and 5B, balance. Accordingly, we checked the molecular response respectively). Measurements of the distribution of sizes for associated with PSNPL exposure in Drosophila larvae. A set of PS-200 and PS-500 inside the midgut lumen (5C and 5D) were genes, previously evaluated in front of other nanomaterials,20 done according to the representative TEM images (5E and were selected to determine the potential effects of PS-50, PS- 5F). In summary, our results have shown that PLNPLs with 200, and PS-500 (Fig. 6). The obtained results are indicated in larger sizes are eroded inside the midgut lumen. This would the ESI.† The conserved stress genes, involved in the general indicate that when PSNPLs increase in size, they are more stress response (Hsp70 and Hsp83), are the first signal of prone to erode during their intestinal transit, possibly due to molecular response, and their expression was significantly chemical and mechanical stress. down-regulated with all the PSNPLs evaluated, regardless of Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2022 View Article Online Environmental Science: Nano Paper This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. Fig. 5 PSNPL biodegradation inside the midgut lumen. Measurements of PS-200 and PS-500 diameters were carried out both in the administered dispersion and inside the midgut lumen, to check their ability to erode. The average diameter of PS-200 in solution (145.56 ± 9.19 nm) was a bit larger than that observed in the midgut lumen (141.90 ± 39.73 nm) (A). Regarding PS-500, the average in solution (415.22 ± 25.25 nm) was larger than that inside the midgut lumen (319.79 ± 97.45 nm) (B). The distribution of diameters for both P-200 and PS-500 is shown in C and D, respectively. These measurements were obtained from 70 PSNPLs of each size, whether in solution or in the midgut lumen. Representative TEM images are shown (E and F, respectively). Statistical significance **P < 0.01, ***P < 0.001. Due to the lack of homogeneous variance and their abnormal distribution (especially for PS-200 data), the Mann–Whitney U-test was used for the statistical analysis. their size; nevertheless, PS-50 produced more marked effects Drosophila larvae. Although the exposure to PS-200 and PS- than PS-200 and PS-500. Regarding the antioxidant genes 500 induced a low effect upon the expression of Duox, PS-50 (Cat, Sod2, and Cu/Zn Sod), the results showed that the mediated a significant down-regulation for both genes Duox exposure to PSNPLs, independent of their size, stimulated and PPO2 at all tested doses. Finally, changes in the significantly the down-regulation of all the studied genes at expression levels of the base excision repair (BER) gene Ogg1 all selected doses. The interaction of the internalized PSNPLs were determined showing that, regardless of their size, with the intestinal barrier, after oral administration, caused a PSNPL exposure induced the down-regulation of Ogg1 at all down-regulation in the expression of Duox and PPO2 in evaluated doses. Fig. 6 Gene expression changes associated with polystyrene exposure. Different gene categories were investigated including stress genes: Hsp70 and Hsp83; antioxidant genes: Cat, Sod2, and Cu/Zn Sod; DNA repair gene: Ogg1 (F); and intestinal physical stress genes: Duox and PPO. Treated (120 h) and non-treated larvae were collected and stored at −80 °C. Changes in gene expression were detected using real time RT-PCR. The expression levels in PSNPL exposed larvae were compared with those in unexposed ones. Three different replicates per dose were used. (a) P < 0.05, (b) P < 0.01, and (c) P < 0.001. White color for PS-50, grey for PS-200, and black for PS-500. This journal is © The Royal Society of Chemistry 2022 Environ. Sci.: Nano View Article Online Paper Environmental Science: Nano Oxidative stress induction after PSNPL exposure Discussion A key factor highlighting the negative impacts of exposure to The term MNPLs hides two hot and unresolved topics, the PSNPLs is their ability to stimulate an oxidative stress status, frontiers between micro and nano ranges and the chemical via increasing the levels of reactive oxygen species (ROS). The nature of the plastics. Unfortunately, no agreement exists results (Fig. 7A) indicate that PSNPLs, independent of their regarding these two points. If we accept the large experience size, can elevate the ROS levels in hemocytes in a dose- gained in the field of engineered nanomaterials, where such dependent manner. This is especially observed for the materials are considered to range between 1 and 100 nm, by smaller PSNPLs (PS-50 and PS-200) (Fig. 7C). extension nanoplastics should cover the same range. Less This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. agreement exists in the range covered by microplastics since many authors have considered that this size covers materials Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. Genotoxicity of PSNPLs as evaluated with the comet assay ranging from 1 μm to 5 mm, which assumes that the higher In the hazard assessment of any type of exposure, the sizes escape from the micro-level. This complex scenario has induction of genotoxicity is of pivotal relevance. This is been well presented by Hartmann et al.21 who proposed to mainly related to the well-known association between DNA categorize MNPLs according to the conventional units of size: lesion induction and relevant consequences for human nano (1–1000 nm) and micro (1–1000 μm), although they health. Among the different ways to detect the induction of realized that this proposal clashed with the accepted DNA damage, the comet assay stands out, which detects the definition of nanoplastics (1–100 nm). To overcome this induction of DNA breaks. The obtained results show that the situation, they proposed to divide the nano range (1–1000 exposure to the different sized PSNPLs (PS-50, PS-200, and nm) between nanoplastics (1–100 nm) and submicron- PS-500) induced significant increases in the levels of DNA plastics (100–1000 nm). Since the size is an important factor damage, at all tested doses (Fig. 7B). Although all different modulating the effects of nanomaterials,22 we have selected PSNPLs mediated the genotoxic impact, the exposure to the in our study three different MNPL sizes: PS-50, PS-200, and smaller size (PS-50) induced higher levels of DNA damage PS-500. According to the above-mentioned classification, PS- (Fig. 7B). 50 belongs to the nanoplastic range, while PS-200 and PS-500 Fig. 7 The oxidative stress (A) and genotoxicity (B) induced in Drosophila larvae hemocytes after exposure (120 h) to different sizes (PS-50, PS- 200, or PS-500) of PSNPLs in comparison with unexposed ones. Hydrogen peroxide and ethyl methanesulfonate (EMS) are used as positive controls. Representative fluorescence images for ROS detection. *P < 0.05, **P < 0.01 and ***P < 0.001. White color for PS-50, grey for PS-200, and black for PS-500. Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2022 View Article Online Environmental Science: Nano Paper will match with the category of submicron-plastics. ingested TiO2 nanoparticles in Drosophila larvae.29 In this Nevertheless, to standardize the nomenclatures all of them context, it is important to note that we found some PSNPLs are grouped as PS nanoplastics (PSNPLs). Regarding the completely or partially surrounded with intestinal vacuoles chemical composition of the selected plastics, we have and having rough shapes. Nevertheless, the knowledge on avoided the complexity of secondary microplastics by the biological effects of such morphological changes is far to selecting pristine PSNPLs. The sizes, surface be deciphered. The potential degradation ability of MNPLs by functionalization, and labeling of these commercial MNPLs intestinal microbiota is a hot topic waiting for sound data. have made them considered as standard MNPLs. Although some results indicated that members of the phyla Our results showed that PSNPLs, regardless of their size, Actinobacteria and Firmicutes from Lumbricus terrestris were This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. were unable to decrease adult emergence or reduce the able to reduce the size of polyethylene MPLs,32 there is a lack viability of Drosophila, at all tested doses. This lack of toxic of rigorous studies to see if this does occur in humans or in Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. effects agrees with other two reported studies using other species.33 Drosophila and different PSMNPL sizes.14,15 This lack of The advantage of our in vivo model of the Drosophila toxicity is not entirely surprising because PSNPLs do not intestine is that we can visualize the uptake of PSNPLs by the produce toxic effects when administered to different human intestinal cells, as well as their distribution inside the cell cultures, including enterocytic Caco-2 cells.23 enterocytes constituting the intestine wall. This has not been Since the main exposure route for MNPLs is ingestion,24 reported, until now, in other well-used simple in vivo models there is a need for systematic studies to understand what like Caenorhabditis elegans and zebrafish.34 According to our occurs during their journey through the intestinal tract.25 results, PSMNPLs distribute inside the cytoplasm of The Drosophila intestine is considered an emerging and enterocytes and are observed surrounding the mitochondria. versatile model system to study intestinal epithelial This looks like what was observed in Caco-2 cells, where this homeostasis and alterations induced by environmental interaction induces morphological changes in the agents.26 In this context, we have gained experience in the mitochondria23 and mitochondrial dysfunction resulting monitoring of nanomaterials through the Drosophila from membrane disruption.35 intestinal transit which has been applied to the case of Once internalized in the intestinal barrier, independent of MNPLs.20,27 The intestines of exposed larvae are obtained, the size, PSMNPLs succeed to translocate through the and different transversal cuts are used to decipher the fate of intestinal barrier completing their journey reaching the open the ingested MNPLs, by using TEM methodologies. This circulatory system, where they are detected in the approach has permitted us to visualize the engulfment of hemolymph. The presence of MNPLs in the hemolymph has MNPLs by luminal membranes, possibly originating from the been repeatedly observed in bivalves like Mitillus,36 peritrophic membrane, as a potential mechanism of uptake supporting their real ability to cross intestinal barriers. Once by the intestinal cells. It must be remembered that the in the hemolymph, PSNPLs can reach the different tissues peritrophic membrane in Drosophila is considered the first and organs. In this context, it is not rare to find that PSNPLs defense line, having a similar functional role to the mucus are incorporated into hemocytes, due to their phagocytic layer in the human intestine. More interesting is the activity. Thus, our positive findings in Drosophila have also observation of the interaction/uptake of MNPLs by the been observed in the hemocytes of the silkworm Bombyx mori intestinal microbiota which could influence the functionality larvae37 and Mitillus.36 Interestingly, several studies have of that microbiome. Thus, their potential effects on gut reported the uptake of PSMNPLs by hemocytes of different microbiota dysbiosis are an attractive proposal in need of organisms, but working ex vivo. In this case, they obtain the verification.28 It should be indicated that Drosophila has been hemocytes of freshwater zebra mussels which are treated with proposed as a simple model to study the microbiome,29 and different concentrations of PSMNPLs to demonstrate their perturbations of its microbiome can result in states of phagocytic activity against MNPLs.38 Interestingly this approach instability or disease in Drosophila.30 has also been applied to Drosophila.39 In all the cases the The ingested PSNPLs can be subjected to interactions with phagocytosis of PSMNPLs by hemocytes was demonstrated. All midgut bacteria and with different intestinal enzymes, as well these data confirm the ability of PSMNPLs to translocate as with the acidic intestinal environment, potentially leading through the intestinal barrier and their capacity to be to decreases in their initial size; thereby, plastic particles can incorporated, in a first instance, by hemocytes and possibly by be eroded. These effects were confirmed in our study via cells from other organs/tissues. These results would agree with measuring the diameters of PS particles in the midgut lumen those obtained with PSNPLs in an in vitro model of the intestinal and by comparing them with their diameters before oral barrier, where a coculture of human intestinal cells Caco-2 administration. In this context, Yang et al.31 found that the (enterocytes) and HT-29 (goblet cells) was grown in an insert midgut' microorganisms plastic-eating mealworms can with a porous base. This insert was placed in a well with Raji- biodegrade polystyrene and form pits and cavities on its B cells (lymphocytes) simulating the blood compartment. surface under enzyme activities secreted by the worm. In Using fluorescent PSNPLs and confocal microscopy, the addition, in recent work, we have reported the tendency of translocation via the intestinal barrier model and their intestinal digestive enzymes to induce biodegradation of uptake by Raji-B cells were demonstrated.40 This journal is © The Royal Society of Chemistry 2022 Environ. Sci.: Nano View Article Online Paper Environmental Science: Nano At the molecular level, exposure to PSNPLs exhibits Nevertheless, and despite the relevance of the induction of observable effects upon the expression of genes related to the DNA damage, the potential genotoxic risk of MNPL exposure maintenance of the intestinal barrier. The expression of Duox presents many gaps and inconsistencies, mainly in in vivo and PPO2 genes was significantly deregulated after exposure models. Since it is well demonstrated that DNA damage is to PSNPLs, regardless of their size, confirming their linked to drastic health consequences for humans,50 internalization via the intestinal barrier. Previously, we have obtaining sound genotoxicity data associated with MNPL reported the deregulation of this set of genes when exposures is of paramount relevance. In our study, we have Drosophila larvae were exposed to different evaluated the ability of PSNPLs to induce DNA breaks, which nanomaterials.20,27,41 Interestingly the induction of intestinal was evaluated in hemocytes using the comet assay. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. damage after exposure to PSMNPLs was also observed using Significant increases in the levels of DNA damage after trypan blue staining, which stained differentially the PSNPL exposure were observed. Although the effects were Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. damaged and undamaged intestines of exposed and independent of the applied doses, an effect of size was unexposed larvae.14 detected, with an indirect relationship between the damage The distribution of PSNPLs in the intestinal and levels and the PSNPL size. These genotoxic effects of PSNPLs hemolymph compartments could produce toxic impacts at can be associated with the recently reported findings also in the level of cells, organs, or even on the whole organism. The Drosophila. In that case, PSMPLs were used, and the toxic effects of ingested PSMNPLs have been mediated by genotoxic effects were detected using the wing spot assay that ROS production in Daphnia pulex, especially by the smaller measures the induction of somatic mutation and ones (100 nm), with a direct effect of the administered recombination.15 Interestingly, in a recent study on human doses.42 This type of mechanism has also been found to be lung epithelial A549 cells it has been reported that, despite involved in exposed Daphnia,43 C. elegans,44 and also in the non-genotoxic potential of pristine plastics, the UV Wistar albino rats.45 In agreement, we also found that despite biodegraded particles produce DNA damage.35 This could size differences, ROS induction was directly associated with explain the genotoxic impact of PSNPLs in Drosophila larvae the exposure dose. The effects of PS-50 and PS-200 are more where they biodegraded and eroded the plastic material prominent than those caused by PS-500. Any change in the inside the gut lumen of the larvae. Noteworthily, the smaller redox balance recalls the antioxidant machinery to store PSNPS (PS-50 or PS-200 nm) produce more DNA damage, internal hemostasis.46 In our case, the exposure to PSNPLs, especially at the highest dose, than the PSNPLs with a larger independent of the size, significantly inhibited antioxidant size (PS-500). It should be noted that these findings correlate gene expression (Cat, Sod2, and Cu/Zn Sod) at all tested with their ability to cause oxidative stress. It is important to doses, confirming the sensitivity of the antioxidant defense mention that the relationship of oxidative stress with of Drosophila to PSNPLs. Similar results were obtained when genotoxicity and mutagenicity is well known.51,52 Moreover, Daphnia pulex was exposed to PSMNPLs, where the the exposure to PSNPLs caused down-regulation of the Ogg1 expression of Cat, Sod2, and Cu/Zn Sod was significantly gene expression, mainly at the highest dose. It must be decreased. This indicates the ability of PSMNPLs to induce remembered that the DNA glycosylase (Ogg1) enzyme overproduction of ROS coinciding with the inhibition of the constitutes the main defense line against the genotoxicity antioxidant defense pathway.42 Interestingly, the expression caused by oxidative DNA damage.53 Taking all together, our of Cu/Zn Sod was size-dependent, displaying the highest results would confirm the genotoxic potential of PSNPLs in expression with the exposure to PS-50, and the lowest internal cells, once administered via ingestion. This would expression with the exposure to PS-500. indicate that PSNPLs, mainly those with small size, can Regarding the expression of the general stress (Hsp) genes, translocate via the intestinal barrier reaching the hemolymph they act as the first line of defense at the molecular level after with enough concentration to induce significant levels of environmental pollutant exposure. In our case, the exposure DNA damage. to the different sized PSNPLs inhibited the expression of This study demonstrates the goodness of the Drosophila Hsp70 and Hsp83, at all tested doses. This downregulation larvae intestinal model to monitor the journey of ingested agrees with that observed in goldfish where deregulation of MNPLs into the larval body. The sequence of pictures, Hsp70 expression was observed after the exposure to two showing their presence in the gut lumen and their different PSMPLs, where such deregulations were size- interactions with different lumen components, gives us dependent, like in our study.47 This would indicate that unequivocal visual information on such types of interactions. PSMNPLs with small sizes mediate more prominent stress Furthermore, the model allows the detection of the than those with higher sizes. In addition, the decrease in the translocation of MNPLs via the intestinal barrier by their expression of Hsp genes was associated with damaged presence in the hemolymph, as well as in the hemocytes. intestinal epithelial cells in rats48 or with the presence of Their potential interaction with other cells from different potentially hazardous content inside the gut lumen, also in tissues/organs remains to be solved. Although no relevant rats.49 toxicity, as measured by the egg-to-adult viability, was It is assumed that oxidative stress resulting from MNPL observed independent of the PSNPL size, we obtained exposure could initiate a genotoxic cascade of events. molecular pieces of evidence of general stress induction, as Environ. Sci.: Nano This journal is © The Royal Society of Chemistry 2022 View Article Online Environmental Science: Nano Paper well as of intestinal damage. Furthermore, significant 7 Y. M. Cho and K. H. Choi, The current status of studies of induction of oxidative stress and DNA damage was observed human exposure assessment of microplastics and their in hemocytes. These effects were associated mainly with the health effects: a rapid systematic review, Environ. Anal. PSNPL size, with higher effects induced by smaller sizes. Health Toxicol., 2021, 36, e2021004. 8 C. Q. Y. Yong, S. Valiyaveetill and B. L. Tang, Toxicity of Author contributions microplastics and nanoplastics in mammalian systems, Int. J. Environ. Res. Public Health, 2020, 17(5), 1509. RM and AH planned the experiments. MA, DA, and JD carried 9 M. Alaraby, B. Annangi, R. Marcos and A. Hernández, out the experimental part. MA and DA analyzed the data, Drosophila melanogaster as a suitable in vivo model to This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. carried out the statistical analysis, and prepared the tables/ determine potential side effects of nanomaterials: A review, figures. MA, DA, RM, and AH wrote the final manuscript. J. Toxicol. Environ. Health, Part B, 2016, 19, 65–104. Open Access Article. Published on 12 April 2022. Downloaded on 4/15/2022 1:33:48 PM. 10 C. T. Ng, L. E. Yu, C. N. Ong, B. H. Bay and G. H. Baeg, The Conflicts of interest use of Drosophila melanogaster as a model organism to study immune-nanotoxicity, Nanotoxicology, 2019, 13(4), 429–446. 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