In Vitro Genotoxicity of Polystyrene Nanoparticles on the Human Fibroblast Hs27 Cell Line

Several studies have provided information on environmental nanoplastic particles/debris, but the in vitro cyto-genotoxicity is still insufficiently characterized. The aim of this study is to analyze the effects of polystyrene nanoparticles (PNPs) in the Hs27 cell line. The viability of Hs27 cells was determined following exposure at different time windows and PNP concentrations. The genotoxic effects of the PNPs were evaluated by the cytokinesis-block micronucleus (CBMN) assay after exposure to PNPs. We performed ROS analysis on HS27 cells to detect reactive oxygen species at different times and treatments in the presence of PNPs alone and PNPs added to the Crocus sativus L. extract. The different parameters of the CBMN test showed DNA damage, resulting in the increased formation of micronuclei and nuclear buds. We noted a greater increase in ROS production in the short treatment times, in contrast, PNPs added to Crocus sativus extract showed the ability to reduce ROS production. Finally, the SEM-EDX analysis showed a three-dimensional structure of the PNPs with an elemental composition given by C and O. This work defines PNP toxicity resulting in DNA damage and underlines the emerging problem of polystyrene nanoparticles, which extends transversely from the environment to humans; further studies are needed to clarify the internalization process.


Introduction
Global plastic production to date is highly related to the environmental pollution by plastic materials [1]. Microplastics (MPs), as fragments <5 mm, but also as fragments with lower dimensions (below 1 mm), are released into the environment [2][3][4].
Plastics are synthetic or semi-synthetic polymeric materials obtained from natural components such as cellulose, oil, and coal that are used in the most disparate products for their manageability and their rapid production. They are excellent insulators, resistant to corrosion and degradation, which are not optimal for the fate of the environment. Normally produced at a high temperature and by cooling, the individual monomers bind together and form long carbon chains. The most important and It will take time for the scientific community to build up the body of hazard and environmental exposure data for a full risk assessment of microplastics and NPs of the types applied in cosmetics and personal care product formulations.
In this context, we investigated the cyto-genotoxic potential of the PNPs after exposing the Hs27 human foreskin fibroblast cell line to different concentrations in the culture medium; following the treatment, we evaluated the viability and metabolic activity of the cells by the MTS assay test of cell proliferation associated with a preliminary screening improved by growth curve. Moreover, we detected reactive oxygen species (ROS) production with PNPs alone and PNPs added with an antioxidant extract of Crocus sativus L. stigmas. To estimate the PNP genotoxic potential, we used the cytokinesis-block micronucleus (CBMN) assay. Finally, we carried out PNP morphological analysis through scanning electron microscopy (SEM) equipped with an x-ray microanalysis system to obtain a chemical and semiquantitative characterization of the single elements of the PNPs.

Cell Culture
The in vitro toxicological study was conducted in a cell line, the fibroblast Hs27 (human foreskin, cultures from Public Health England, supplied by Sigma-Aldrich Srl, Milan, Italy).
Cell culture media, trypsin, and all reagents used, unless otherwise indicated, were purchased from Euroclone SpA. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 UI/mL Penicillin/Streptomycin, 2 mM L-glutamine, in a HERAEUS incubator (Hera cell 150, Thermo Electron Corporation, Langenselbold, Germany) set with the following parameters: 5% CO 2 atmosphere, 37 • C temperature. The culture maintenance was carried out under sterile conditions under a biological laminar flow hood. The cells were detached with 0.05% trypsin-0.02% EDTA.

Polystyrene Nanoparticles
The polystyrene nanoparticles (PNPs) were purchased to Sigma Aldrich (catalogue No. 43,302). The particle size was 100 nm, the diameter was 0.100 mm, and the density 1.05 gr/cm 3 . The particles are in aqueous suspension (10% WT).

Saffron: Crocus sativus L. Stigmas Extract
Plant material (Crocus sativus L.) was kindly furnished by local farmers in the area of the "Zafferano dell'Aquila PDO" consortium, Navelli, AQ (Italy). Plant extraction (stigmas) was performed according to [36], in our case, however, the extraction was carried out in aqueous solution [37].

Cell-Growth Curve
The cells were seeded at a density of 10,000 cells/cm 2 in a six multi-well (35 mm in diameter) and when 90% of confluence was achieved, they were counted in a Bürker camera with the dye exclusion Trypan Blue, diluted 1:10. The determination was carried out for 4, 24, and 48 h at different concentrations of 5, 25, and 75 µg/mL of PNPs. In particular, the cells were treated with the different concentrations of PNPs, then readings were taken for each concentration at different times of exposure (4, 24, and 48 h).
Each experimental condition represents a technical triplicate, data refer to the mean and standard error of three independent experiments.

ROS (Reactive Oxygen Species) Detection
Cellular ROS concentration was determined according to the "Total ROS Assay Kit 520 nm". Briefly, 10,000 cells/cm 2 were seeded in 96-well plates, after 24 h, cells were incubated at 37 • C for 60 min with ROS stain 1X (Thermo Fisher Scientific Inc., Waltham, MA, USA ref. 88-5930) resuspended in dimethyl sulfoxide (DMSO). After incubation, the medium was removed, DMEM was added in the control cells and DMEM containing 5, 25, and 75 µg/mL of PNPs in treated cells. The H 2 O 2 (150 µM) was added as the positive control. Another plate was performed with the same conditions (5, 25, and 75 µg/mL of PNPs), in which an antioxidant extract of Crocus sativus stigmas was added at 25 µg/mL final concentration [37].
Both plates were read at different times in a microplate reader (Perkin-Elmer Victor 3) (λexc 490, λemi535) at T0, T15, T30, T45, T60 min, and T24 h. The fluorescence data T0-T24 h were evaluated for statistical analysis. Each experimental condition represents a technical triplicate, data refer to the mean and standard error.

Cytokinesis-Block Micronucleus (CBMN) Assay
CBMN was carried out with slight modifications according to the protocol of Fenech [39] and OECD guidelines [40]. The Hs27 cell line was seeded in each flask with 2.5 × 10 5 cells/flask, and after 24 h of culture, the cells were exposed to different concentrations (5 µg/mL, 25 µg/mL, and 75 µg/mL) of PNPs for 48 h. Colchicine was used as a positive control at 5 µg/mL. Cytochalasin B (3 µg/mL) no longer than 24 h after stimulation by PNPs was added to the cell cultures.
Cells were harvested after an additional 24 h and centrifuged for 8 min at 1100 rpm; next, the supernatant was removed, and cells treated for 1 min with 0.075 M KCl hypotonic solution.
Following, the cells were processed and analyzed according to the criteria of Fenech guidelines [39]. Three biological replicates for each sample were used for CBMN analysis with three technical replicates (slides) each.
For each experimental condition, we calculated the cytokinesis block proliferation index (CBPI) to determine the frequency of mononuclear cells, bi-and multinucleated, using the following formula: ((N • mononucleated cells) + (2 × N • binucleated cells) + (3 × N • multinucleated cells))/(total number of cells). Furthermore, for each experimental condition we evaluated the total cells and nuclear buds (NBUDs) as a biomarker of genotoxicity.

Analysis of Polystyrene Particles (PNPs) by Scanning Electron Microscopy SEM
The study of the morphology and the elemental analysis of PNPs were carried out by scanning electron microscopy (Gemini Field Emission SEM 500, ZEISS, Milan, Italy) equipped with an x-ray microanalysis system (EDS Oxford Inca 250 x-act) at the Center of Microscopies, University of L'Aquila.
For PNP characterization, the sample (1 µL) was deposited on a dedicated sample carrier (stub) and then dehydrated in air. Finally, a thin film (5 nm) of chromium was deposited onto the sample using Sputter Quorum 150T ES to make it conductive for measurement purposes.
The SEM observations were carried out at different magnifications, and morphological analysis of the particles was performed simultaneously to obtain the EDS microanalysis of the selected particles.

Statistical Analysis
For the data statistical analysis, we used the Student's t test (unpaired) with post-hoc correction, comparing the value of the treated cells with the respective untreated control, through independent tests. For statistically significant values, * = p < 0.05; ** = p < 0.005; *** = p < 0.0005.
The data were analyzed using the GraphPad Prism software, version 6.0 (© 1995-2015 GraphPad Software, Inc. San Diego, CA 92108). Three independent experiments were performed for all assays applied.

Cell Growth Curve and MTS
Preliminary results are reported in the growth curve for the Hs27 cell line with PNP treatment at different concentrations ( Figure 1). In Figure 1, it can be seen that with respect to the control in every experimental condition up to 4 h, there was no significant proliferation decrease, regardless of the concentration. Differences appeared after 24 h, but control and the highest concentration still showed almost identical results. The exposure of the cells to 75 µg/mL of PNPs at 48 h showed a sudden decrease. The trend of the growth curve was due to the possible tendency of the nanoparticles to aggregate (PNPs in this work as in general the nanoparticles do [30]), this explains why at the lower concentrations there were no statistically significant results; on the contrary, at the concentration of 75 µg/mL and at a longer time of incubation (48 h), we can assume that PNP particles aggregates tend to enhance and interact with cell proliferation. For the data statistical analysis, we used the Student's t test (unpaired) with post-hoc correction, comparing the value of the treated cells with the respective untreated control, through independent tests. For statistically significant values, * = p < 0.05; ** = p < 0.005; *** = p < 0.0005.
The data were analyzed using the GraphPad Prism software, version 6.0 (© 1995-2015 GraphPad Software, Inc. San Diego, CA 92108). Three independent experiments were performed for all assays applied.

Cell Growth Curve and MTS
Preliminary results are reported in the growth curve for the Hs27 cell line with PNP treatment at different concentrations ( Figure 1). In Figure 1, it can be seen that with respect to the control in every experimental condition up to 4 h, there was no significant proliferation decrease, regardless of the concentration. Differences appeared after 24 h, but control and the highest concentration still showed almost identical results. The exposure of the cells to 75 μg/mL of PNPs at 48 h showed a sudden decrease. The trend of the growth curve was due to the possible tendency of the nanoparticles to aggregate (PNPs in this work as in general the nanoparticles do [30]), this explains why at the lower concentrations there were no statistically significant results; on the contrary, at the concentration of 75 μg/mL and at a longer time of incubation (48 h), we can assume that PNP particles aggregates tend to enhance and interact with cell proliferation. The cytotoxicity of the PNPs was measured by the MTS cell viability test, which evaluates the metabolic activation of cultured cells, Hs27, after treatment at different concentrations.
The test determines whether cells increase their metabolic activity, measuring the reduction of MTS by a formazan soluble in the culture medium, as MTS reduction only occurs in viable and metabolically active cells. Compared to the control cells, the data at 4 h showed a significant viability increase at 75 μg/mL (about 33%). After 24 h, again, the treatment at 75 μg/mL of PNPs was statistically significant; finally at 48 h, the results showed an increase only at 5 μg/mL PNPs, which was about 20%. Triton-X-100 0.1% was used as a positive control and induced a significant decrease The cytotoxicity of the PNPs was measured by the MTS cell viability test, which evaluates the metabolic activation of Hs27 cells after treatment at different concentrations.
The test determines whether cells increase their metabolic activity, measuring the reduction of MTS by a formazan soluble in the culture medium, as MTS reduction only occurs in viable and metabolically active cells. Compared to the control cells, the data at 4 h showed a significant viability increase at 75 µg/mL (about 33%). After 24 h, again, the treatment at 75 µg/mL of PNPs was statistically significant; finally at 48 h, the results showed an increase only at 5 µg/mL PNPs, which was about 20%.
Triton-X-100 0.1% was used as a positive control and induced a significant decrease in viability after 4, 24, and 48 h treatment in PNPs ( Figure 2). We speculate that the observed viability trend is not dose dependent and that there is no significant variation in cell viability. in viability after 4, 24, and 48 h treatment in PNPs ( Figure 2). We speculate that the observed viability trend is not dose dependent and that there is no significant variation in cell viability.

Tests of Micronuclei with Block of Cytokinesis with Cytochalasin B "CBMN Assay"
From the data obtained from the micronucleus test, we calculated the CBPI index "Cytokinesis Block Proliferation Index" to evaluate the cellular proliferation progression and therefore the cytostatic and cytotoxic effects, followed by the different concentrations of PNPs (5, 25, and 75 μg/mL).
Compared to the control condition, the CBPI obtained from the cells incubated with the PNPs was, in every condition data, not statistically significant (Figure 3a). Regarding the induction of micronuclei (BNMN), we observed a significant increase dose-dependent at 25 μg/mL and 75 μg/mL where we observed an increase of about 38% and 52%, respectively (Figure 3b). Furthermore, we analyzed the presence of NBUDs (Figure 3c), which originate from the nucleus as extroflections of nucleoplasmic material or as micronuclei connected to the nucleus by a bridge [40]. Our result shows a significant decrease at 5 μg/mL (about 30%) with respect to the control, and on the contrary, an increase at 75 μg/mL of about 50%. In Figure 4, we can see DNA damage as micronuclei and NBUDs in Hs27 cells after PNP treatment.

Tests of Micronuclei with Block of Cytokinesis with Cytochalasin B "CBMN Assay"
From the data obtained from the micronucleus test, we calculated the CBPI index "Cytokinesis Block Proliferation Index" to evaluate the cellular proliferation progression and therefore the cytostatic and cytotoxic effects, followed by the different concentrations of PNPs (5, 25, and 75 µg/mL).
Compared to the control condition, the CBPI obtained from the cells incubated with the PNPs was, in every condition data, not statistically significant (Figure 3a). Regarding the induction of micronuclei (BNMN), we observed a significant increase dose-dependent at 25 µg/mL and 75 µg/mL where we observed an increase of about 38% and 52%, respectively ( Figure 3b). Furthermore, we analyzed the presence of NBUDs (Figure 3c), which originate from the nucleus as extroflections of nucleoplasmic material or as micronuclei connected to the nucleus by a bridge [40]. Our result shows a significant decrease at 5 µg/mL (about 30%) with respect to the control, and on the contrary, an increase at 75 µg/mL of about 50%. In Figure 4, we can see DNA damage as micronuclei and NBUDs in Hs27 cells after PNP treatment.

ROS Detection
Time course experiments were performed to comparatively evaluate the possible ROS production in Hs27 cells at different concentrations of PNPs (5, 25, and 75 μg/mL) and PNPs added with an antioxidant extract of Crocus sativus stigmas (25 μg/mL). Figure 5a shows that treatment with 5 μg/mL NPs induces highly significant stimulation of ROS production in the cell line, starting from T15 min/T0 of treatment. At T30 min/T0, we statistically increased in ROS production at 5 μg/mL (p < 0.0005) and 25 μg/mL (p < 0.05) concentrations. The increase ROS level at T1 h/T0 was still statistically significant with respect to the control at 5 μg/mL (p < 0.005) and 25 μg/mL (p < 0.05) concentrations, but we also noticed that the same concentrations with respect to T30 min/T0 had a strong decrease in ROS production. At T24 h/T0, there are no significant variations in ROS production with respect to the control cells. In Figure 5b, we reported the level of ROS production with PNPs added together with saffron extract: we observed that in the presence of the extract, the ROS production was lower. Significant data obtained by comparing the PNP treatment and PNPs added with saffron are reported in Table 1, and it is noticeable that there was a significant decrease in reactive oxygen species at 5 μg/mL and T15 min/T0, T30 min/T0, and T1 h/T0. In particular at T30 min/T0, we had a ROS decrease by about 30%. Regarding the 25 μg/mL concentration, the results were statistically significant at T30 min/T0 and T1 h/T0 and a ROS reduction of about 18% and 22%, respectively.

ROS Detection
Time course experiments were performed to comparatively evaluate the possible ROS production in Hs27 cells at different concentrations of PNPs (5, 25, and 75 µg/mL) and PNPs added with an antioxidant extract of Crocus sativus stigmas (25 µg/mL). Figure 5a shows that treatment with 5 µg/mL NPs induces highly significant stimulation of ROS production in the cell line, starting from T15 min/T0 of treatment. At T30 min/T0, we statistically increased in ROS production at 5 µg/mL (p < 0.0005) and 25 µg/mL (p < 0.05) concentrations. The increase ROS level at T1 h/T0 was still statistically significant with respect to the control at 5 µg/mL (p < 0.005) and 25 µg/mL (p < 0.05) concentrations, but we also noticed that the same concentrations with respect to T30 min/T0 had a strong decrease in ROS production. At T24 h/T0, there are no significant variations in ROS production with respect to the control cells. In Figure 5b, we reported the level of ROS production with PNPs added together with saffron extract: we observed that in the presence of the extract, the ROS production was lower. Significant data obtained by comparing the PNP treatment and PNPs added with saffron are reported in Table 1, and it is noticeable that there was a significant decrease in reactive oxygen species at 5 µg/mL and T15 min/T0, T30 min/T0, and T1 h/T0. In particular at T30 min/T0, we had a ROS decrease by about 30%. Regarding the 25 µg/mL concentration, the results were statistically significant at T30 min/T0 and T1 h/T0 and a ROS reduction of about 18% and 22%, respectively.

Analysis of PNPs by SEM Scanning Electron Microscopy
PNPs information were obtained by evaluating their morphology and elemental composition. With electron microscopy, we undertook a morphological analysis through different images to test the particle size ( Figure 6). Regarding the composition, an investigation was made with EDS microanalysis to assess whether there were impurities such as heavy metals that could affect the experiment. During the analysis of the sample, properly treated, we noticed that polystyrene nanospheres tended to form a reticular structure thanks to their homogeneous shape. Furthermore, it could be seen that the average particle size was around 100 nm, according to the manufacturer's specifications ( Figure 6).

Analysis of PNPs by SEM Scanning Electron Microscopy
PNPs information were obtained by evaluating their morphology and elemental composition. With electron microscopy, we undertook a morphological analysis through different images to test the particle size ( Figure 6). Regarding the composition, an investigation was made with EDS microanalysis to assess whether there were impurities such as heavy metals that could affect the experiment. During the analysis of the sample, properly treated, we noticed that polystyrene nanospheres tended to form a reticular structure thanks to their homogeneous shape. Furthermore, it could be seen that the average particle size was around 100 nm, according to the manufacturer's specifications ( Figure 6).

Analysis of PNPs by SEM Scanning Electron Microscopy
PNPs information were obtained by evaluating their morphology and elemental composition. With electron microscopy, we undertook a morphological analysis through different images to test the particle size ( Figure 6). Regarding the composition, an investigation was made with EDS microanalysis to assess whether there were impurities such as heavy metals that could affect the experiment. During the analysis of the sample, properly treated, we noticed that polystyrene nanospheres tended to form a reticular structure thanks to their homogeneous shape. Furthermore, it could be seen that the average particle size was around 100 nm, according to the manufacturer's specifications ( Figure 6). Taking advantage of EDX spectroscopy (energy dispersive x-ray analysis), we evaluated the elemental composition of the sample (Figure 7). The technique provides information on the elemental composition, hence the spectrum only shows the presence of the carbon elements, reinforcing the idea that the only component was polystyrene and oxygen due to the presence of the water residue. Taking advantage of EDX spectroscopy (energy dispersive x-ray analysis), we evaluated the elemental composition of the sample (Figure 7). The technique provides information on the elemental composition, hence the spectrum only shows the presence of the carbon elements, reinforcing the idea that the only component was polystyrene and oxygen due to the presence of the water residue.

Discussion
Global plastic production increases annually [41], with an estimated 4.8 to 12.7 million metric tons of plastic entering the oceans each year [42], posing a threat to seabirds [43], fish [44], turtles [45], and marine mammals [46]. Dispersed plastic is a new emergency for environmental health, and the greatest danger is derived from the products of their degradation. NPs are dispersed in the soil, air, and water; in particular PNPs are the most subjected to degradation. Some new evidence of the toxic potential of PNPs has emerged from the present study, particularly with regard to the genotoxicity.
Tests of the viability of MTS cells by evaluating the metabolic activity of Hs 27 cells exclude an inhibitory action of PNPs on metabolic activity; this activity increased after PNP treatment most likely as a response to cellular stress. This hypothesis is supported by the results of the CBMN tests regarding the treatments with the lowest concentrations of NPs. Genotoxic damage was observed at concentrations above 5 μg/mL, which produced results comparable to MTS tests. Thus, high concentrations of PNPs seem to be necessary to produce appreciable cell damage in relation to exposure times. These in vitro data are quite indicative of the genotoxicity of PNPs and provide indirect evidence of the ability of PNPs to penetrate cells, as widely reported for other particles of similar size to NPs [30] and PNPs [47].
From our results, it is clear that by analyzing the metabolic activity in relation to the production of ROS, treatment with PNPs is able to determine oxidative stress inside the cells. In agreement with the literature [30], the result obtained by us show a high production of ROS within the first 30 min and a decrease afterward, due to the detoxification systems that the cell puts in place. Moreover, we observed that the ROS production decreased when PNPs were added together with the saffron extract. Hence, the free radical scavenging ability of saffron [48] is also expressed in human fibroblasts in which oxidative stress is produced by PNPs. This ability is related to the phenolic/flavonoid contents of saffron Crocus sativus L. stigmas known to play a role in preventing oxidative damage caused by free radicals and inhibiting hydrolytic and oxidative enzymes [49]. Thus, the Hs27 human fibroblasts exposed to PNPs suffer damage both in terms of genotoxicity and oxidative stress and the antioxidant power of saffron extract may be able to contrast the ROS formation.

Discussion
Global plastic production increases annually [41], with an estimated 4.8 to 12.7 million metric tons of plastic entering the oceans each year [42], posing a threat to seabirds [43], fish [44], turtles [45], and marine mammals [46]. Dispersed plastic is a new emergency for environmental health, and the greatest danger is derived from the products of their degradation. NPs are dispersed in the soil, air, and water; in particular PNPs are the most subjected to degradation. Some new evidence of the toxic potential of PNPs has emerged from the present study, particularly with regard to the genotoxicity.
Tests of the viability of MTS cells by evaluating the metabolic activity of Hs 27 cells exclude an inhibitory action of PNPs on metabolic activity; this activity increased after PNP treatment most likely as a response to cellular stress. This hypothesis is supported by the results of the CBMN tests regarding the treatments with the lowest concentrations of NPs. Genotoxic damage was observed at concentrations above 5 µg/mL, which produced results comparable to MTS tests. Thus, high concentrations of PNPs seem to be necessary to produce appreciable cell damage in relation to exposure times. These in vitro data are quite indicative of the genotoxicity of PNPs and provide indirect evidence of the ability of PNPs to penetrate cells, as widely reported for other particles of similar size to NPs [30] and PNPs [47].
From our results, it is clear that by analyzing the metabolic activity in relation to the production of ROS, treatment with PNPs is able to determine oxidative stress inside the cells. In agreement with the literature [30], the result obtained by us show a high production of ROS within the first 30 min and a decrease afterward, due to the detoxification systems that the cell puts in place. Moreover, we observed that the ROS production decreased when PNPs were added together with the saffron extract. Hence, the free radical scavenging ability of saffron [48] is also expressed in human fibroblasts in which oxidative stress is produced by PNPs. This ability is related to the phenolic/flavonoid contents of saffron Crocus sativus L. stigmas known to play a role in preventing oxidative damage caused by free radicals and inhibiting hydrolytic and oxidative enzymes [49]. Thus, the Hs27 human fibroblasts exposed to PNPs suffer damage both in terms of genotoxicity and oxidative stress and the antioxidant power of saffron extract may be able to contrast the ROS formation.
According to the SEM-EDX analysis, PNPs are composed exclusively of C and O, and therefore the physical-chemical properties, and consequently the toxic effects are attributable to the size, shape, surface properties, reactivity, and solubility, all characteristics that influence the ability to induce damage within the cells [50]. The more that particles reach a nanosize, the more their surface area exposed with reactive chemical groups extends.
Our current approach to study the toxicological potential of PNPs raises some important points such as to determine how the particles are internalized by the cells; particles with dimensions of about 100-200 nm are internalized through endocytosis mechanisms, in contrast, larger ones are absorbed through phagocytosis [30,51]. The available information on the toxicity of PNPs in vivo is poor [30,52,53]. In this regard, considering environmental pollution, adverse factors could be invoked for NPs and PNPs such as the risk that they can adsorb, concentrate, and release environmental pollutants into the organisms, thus acting as transporters [54][55][56].