UVB-aged microplastics and cellular damage : An in vitro study

UVB-aged microplastics and cellular damage : An in vitro study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article UVB-aged microplastics and cellular damage : An in vitro study SEBASTIANO LA MAESTRA, Mirko Benvenuti, Stefano Alberti, Linda Ferrea, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3951751/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jun, 2024 Read the published version in Archives of Environmental Contamination and Toxicology → Version 1 posted 4 You are reading this latest preprint version Abstract Plastics are synthetic organic compounds whose widespread use generates enormous waste. Different processes, such as mechanical abrasion, microbiological activity, and UVB irradiation, can fragment the plastic material and generate microplastics (MPs). MPs are ubiquitous, and various organisms, including humans, can ingest or inhale them, with potential adverse health effects. The differences between UV-aged and virgin particles were studied to evaluate the genotoxic damage and oxidative stress induced by polystyrene MPs with 1 and 5 µm sizes on the monocyte-like cell line (THP-1). Fourier transform infrared spectroscopy and Ζ-potential measurements were used to characterise MP particles after UVB exposure. Cells exposed to MPs show a widespread change in the cellular environment with the generation of Reactive Oxidative Species (ROS), as indicated by the increased malondialdehyde (MDA) level. The occurrence of genotoxic damage is correlated to the smaller size and ageing state of the MPs. The biochemical and genomic alterations observed in this in vitro study suggest that MPs, ubiquitous pollutants, following natural degradation and oxidation processes can cause various adverse effects on the health of the exposed population, making it necessary to carry out further studies to better define the real risk. Microplastics UVB aging DNA damage oxidative stress aneuploidy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plastics are organic polymeric compounds synthesised from natural resources such as gas, oil, and its derivatives. These polymers have many applications in different sectors, including packaging, construction, automotive, and electronics. Due to their advantageous characteristics and low production costs, manufacturing increases yearly (Hu et al., 2023 ). Extensive use generates enormous quantities of plastic waste. It has been calculated that from 1950 to 2015, about 6.3 billion tons of plastic waste were generated, and 26 billion tons will be produced by 2050 (Geyer et al., 2017 ; Guglielmi, 2017 ). Commonly used plastics are polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polystyrene (PS), and polyurethane (PU). All these polymers are characterised by environmental persistence due to their molecular stability, which may be referenced to covalent bonds involving C-C and C-H groups. These groups may undergo degradation reactions triggered by heat and light (UVB) (Wilkinson et al., 2017 ), causing instability and breaking into small fragments. Moreover, environmental MPs are degraded by different metabolic microorganisms (Miri et al., 2022 ). Depending on their size, plastic particles can be classified as MPs with dimensions between 0.001 and 5 mm and nanoplastics (NPs) smaller than 0.1 µm (Kubowicz and Booth, 2017 ). MP pollutants can be generated by mechanical abrasion (Song et al., 2017 ) weathering on environmentally dispersed macroplastics or by releasing small fragments from clothing, car tyres, paint coatings, pre-production dust, pellet spills, or also found in various preparations, such as cosmetic products (Vethaak and Legler, 2021 ). According to their source, MPs can be classified into primary or secondary. Primary MPs originate as small particles released directly into the environment. They are derived from textiles, medical products, and personal care products. Secondary MPs result from the degradation process of large amounts of plastic waste (Eriksen et al., 2013 ; Alomar et al., 2017 ). Due to their small size, MPs can be considered ubiquitous and carried through the marine and terrestrial environment and into the atmosphere (Lim, 2021 ). Moreover, their small size makes them easily ingested by aquatic organisms perpetuated throughout the trophic chain (Lim, 2021 ; Desforges et al., 2015 ; Nelms et al., 2018 ). Although MPs have also been found in different environmental matrices, such as in drinking water, WHO (2019) stated that there are not enough studies to define the toxicity of these particles on human health. On the other hand, humans may ingest or inhale MPs, potentially affecting health (Vethaak and Legler, 2021 ). Once ingested, MPs cross the gastrointestinal tract epithelium gaps to disseminate into the circulatory system to be transported to the lymph nodes, liver, and spleen. Several subjects showed the presence of MPs < 2 µm in blood and different organs, such as the human placenta's fetal, maternal, and chorioamnionitis membrane sides (Ragusa et al., 2021 ), although it is unclear whether MPs with larger sizes follow the same pathways (Revel et al., 2018 ). Inhaled MPs with a size < 2.5 µm can cross the respiratory barrier (Liao et al., 2011 ) by alveolar macrophages to disseminate at the systemic level (Vethaak and Leslie, 2016 ). MPs have been observed to lead to intestinal barrier dysfunction, microbiota imbalance, alteration in triglyceride synthesis and lipogenesis and reduction in intestinal mucus secretion (Rahman et al., 2021 ). Ingested MPs can be eliminated in the stool or urine (Massardo, et al., 2024 ). A recent study suggests the renal system can eliminate MPs above 10 nm in the urine after glomerulus filtration. MPs with larger dimensions could cross the renal tubules by exocytosis from the efferent artery and endocytosis from the proximal convoluted tubule (Pironti et al., 2022 ). On the other hand, ingested MPs can cross the gastrointestinal system, damage the epithelia, or be evacuated through the feces (Yan et al., 2022 ). Moreover, MPs can carry a series of toxic and other polluted compounds that could increase the risk of adverse effects (Rist et al., 2018 ). In vitro studies showed that MPs induce oxidative stress, damaging mitochondrial membranes, generating unbalanced membrane potential (Wang et al., 2021 ). ROS are underlying the genotoxicity processes and are involved in the multi-step process of carcinogenesis (De Sá Junior et al., 2017 ) and chronic disease. Human studies reported that MPs and NPs may induce DNA damage, such as micronuclei (MN) formation, chromosomal aberration, DNA strand breaks, and genotoxicity (Ballesteros et al., 2020 ). The present study aims to assess genotoxic damage and oxidative stress induced by polystyrene MPs 1 and 5 µm in size on THP-1 cells to identify the differences between UV-aged and non-aged particles. Different assays were performed to assess cytotoxicity, oxidative stress, genotoxicity, and aneuploidy. Materials and Methods Microplastics oxidation PS virgin MPs of 1 µm and 5 µm were acquired by Cospheric (Santa Barbara, California 93160 United States), and their oxidation was performed by UVB lamp exposure at 318 nm (57 V, 20 W G13, Philips) started in the presence of 40% hydrogen peroxide (Batel, et al., 2016 ). In particular, microplastic suspensions (10 mg/ml) were placed in a glass disk containing an equal volume of H 2 O 2 , UVB-irradiated for 96 h and periodically agitated. At the end of treatment, the MPs suspension was precipitated by centrifuging (12,000 rpm for 10 min), washed in Milli-Q water, and dried by SpeedVac. Precipitated microplastics were weighted and resuspended in phosphate buffer saline (PBS) at known concentrations. Before each use, the microplastic samples were resuspended using ultrasonic devices. ζ-potential ζ-potential (surface charge) measurements on microplastic samples were performed using Dynamic Light Scattering (DLS) analyses, with a Zetasizer Instrument, Nano ZS90 Series (Malvern Panalytical, Malvern, UK), working with He-Ne laser (emission λ = 633 nm). Samples were subjected to 40 kHz sonication before each analysis. Measurements were performed at a fixed temperature (T = 25°C) using a Peltier thermostatic system, with an equilibrating time set to 120 s. MPs were dispersed in deionised water to reach a final concentration of 0.1 mg mL − 1 . Measurements were performed in triplicate, and each measurement consisted of 20 runs. Fourier Transform Infrared Spectroscopy Fourier Transform Infrared Spectroscopy (FTIR) measurements were performed on Perkin Elmer Spectrum 65 FT-IR, which was equipped with an attenuated total reflectance (ATR) accessory. The spectra were collected in the range of 4,000–500 cm − 1 ; each spectrum results from 10 accumulations of 15 seconds each. The spectra obtained from FTIR analysis were used to determine the carbonyl index (CI) as reported by different studies (Zhang et al. 2021 ; Hu et al., 2023 ) and calculated as the ratio between the absorbance of the carbonyl peak and the absorbance of the methylene peak, considered as reference. In particular, for PS samples, the carbonyl peak is usually calculated at 1,730 cm − 1 and the methylene at 1,452 cm − 1 .. Furthermore, the same analysis allowed us to calculate the oxidation index (I ox ) (Vicente et al., 2009 ) as: $${I}_{ox}=\frac{\int {Abs\left(t\right)}_{C=O}}{\int {Abs\left(t\right)}_{ref}}-\frac{\int {Abs\left(0\right)}_{C=O}}{\int {Abs\left(0\right)}_{ref}}$$ Where: Abs(t) C=O absorbance of C = O (1,615–1,840 cm − 1 ) at reaction time t Abs(t) ref absorbance of reference peaks (1,471–1,522 cm − 1 ) at reaction time t Abs(0) C=O absorbance of C = O (1,615–1,840 cm − 1 ) for unmodified polystyrene Abs(0) ref absorbance of reference peaks (1,471–1,522 cm − 1 ) for unmodified polystyrene. This parameter can be used as a reference for the ageing of the material under study and quantifies this process as a function of the oxidised groups. Cell culture THP-1 cell line was obtained from the European Collection of Cell Cultures (ECACC). Monocytes derived from peripheral blood patients with acute monocytic leukaemia were used to mimic the toxicological responses of systemic monocytes/macrophages. Briefly, the THP-1 cell was maintained in a complete culture medium (RPMI, 10% FBS, 10% Glutamine and 1% Penicillin/Streptomycin) and split every two days or used for different assays. Cell viability MTT assay measures the insoluble formazan derived from tetrazolium salt and generated by the activity of mitochondria of viable cells. The assay was performed in cells cultured for 24 h in 96-well microplates (1.5 x 10 4 ). Eight wells were inoculated with oxidated or virgin microplastics at sizes of 5 and 1 µm at different concentrations (25, 50, 100, 250, and 500 µg mL − 1 ) with two different exposure times (24 and 48h). After exposure, 0.5 mg mL − 1 of MTT was added to each well, and the microplates were reincubated at 37°C for 3 h. Dimetil sulfoxide was used to solubilise the purple-coloured formazan crystals, proportional to the number of metabolic active cells. Measurement was performed at 570 nm using a microplate spectro-photometer reader (Tecan Italia, Milan, Italy). The values obtained were compared to the negative control, assigned 100% vitality. Comet assay The alkaline comet assay was performed using a protocol suggested by Singh et al. ( 1988 ) with some modifications (La Maestra et al. , 2020 ) . Briefly, the THP-1 cells were seeded in 96 well plates at the density of 1.5 x 10 4 cells/well and exposed to microplastic, both oxidised and non-oxidised at two different sizes (1 and 5 µm), and two concentrations (25 and 50 µg mL − 1 ) per 24 h. After incubation, the viability of cells was checked by trypan blue exclusion test. Viability of less than 80% was considered an exclusion factor of the sample. After that, about 20,000 cells were embedded in 75 µL of 0.5% low melting-point agarose, coated onto slides, covered with a coverslip, and allowed to solidify at 4°C, followed by a second layer of low melting-point agarose. The slides were immersed in cold lysis solutions (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid, 10 mM Tris, pH 10, 1% Triton X-100, and 10% dimethyl sulfoxide) overnight, rinsed in alkaline solution (0.3 M NaOH, 1 mM ethylenediaminetetraacetic acid, pH 13) and placed horizontally in an electrophoresis chamber in which it was performed, in fresh alkaline solution, electrophoresis (30 min at 25 V (0.66 V/cm), adjusted to 300 mA). After electrophoresis, the slides were gently removed and washed in a neutralisation buffer (0.4 M Tris-HCl, pH 7.5) for 5 min and stained with propidium iodide (2 µg mL − 1 ). One hundred random nuclei were acquired with a fluorescence microscope and a digital camera at a magnification of 200×. The analysis was conducted using CASP (Comet assay software project, http://www.casp.sourceforge.net ), and the results were expressed in terms of the percentage of DNA in the tail (TDNA %). Cytokinesis-block micronucleus assay Cytokinesis-block micronucleus assay (CBMN) was performed in duplicate to highlight breakage or loss of chromosomes following exposure to microplastic, as described by Fenech ( 2000 ). Briefly, 10 x 10 4 THP-1 cells were seeded onto 15 ml tubes and exposed to equal microplastics and concentrations used for the comet assay. After 24 h exposure, cells added with 4 µg/mL cytochalasin B. THP-1 cells were maintained in culture for an additional 28 h and then washed and resuspended in 0.075 M KCl hypotonic solution for 2 min, centrifugate 10 min at 1,200 rpm and prefixed in 3:5 methanol/acetic acid, and washed twice with a 6:1 methanol/acetic acid fixative solution. Microscopic slides were obtained by smearing each sample. Subsequently, the slides underwent acid hydrolysis for 1 h ( HCl 5 N), rinsed in distilled water and deoxyribonucleic acid specifically stained with Schiff’s reagent (Sigma Chemical Co., St. Louis, MO) for 30 min, washed in distilled water, and left for 5 min in running tap water in order to intensify the pink colour. Finally, the samples were washed, blotted dry, and mounted. One thousand cells from each sample were examined under an optical microscope at 1000× magnification to score the presence of micronucleated cells and binucleate cells. Fisher’s exact test was performed to determine a statistically significant difference between different treatments and the negative control. Malondialdehyde (MDA) Briefly, 15 x 10 4 THP-1 cells were seeded onto 15 ml tubes and exposed to concentrations MPs reported above. After 24 h exposure, the cells were collected by centrifuging 10 min at 2,200 rpm, and each cell aliquot was tested for lipid peroxidation by concentration of TBARS as described by Ohkawa et al. ( 1979 ). Therefore, 200 µl of 8.1% sodium dodecyl sulfate, 1,500 µl of 20% acetic acid solution (pH 3.5), and 1,500 µl of 0.8% aqueous solution thiobarbituric acid were added to the cells. Distilled water was added to reach a volume of 4 ml, and the samples were heated at 95°C for 60 min. After, 4 ml of n-butanol and pyridine (15:1 v/v) were added, shaken vigorously, and centrifugated at 4,000 rpm for 10 min. The organic layer was taken, and a fluorometric measurement was made (Ex 533 nm; Em 553 nm). Moreover, an external standard, MDA, was used, and the results were expressed as nanomoles MDA equivalent per cell number. Statistical analyses The analyses were performed by JMP software. The results regarding multiple individual experiments were expressed as means ± SD, and data were analysed by one-way analysis of variance (ANOVA) with post hoc testing using the Bonferroni test. A P -value of < 0.05 was considered as statistically significant. Fisher’s exact test was performed with a statistically significant difference between each treatment and the control with respect to the frequency of micronucleated cells. Results Fourier Transform Infrared Spectroscopy (FTIR) Figure 1 (A, B) reports FTIR spectra obtained for virgin and oxidated MPs of both dimensions, 1 µm and 5 µm MPs, respectively. Differently, Fig. 1 C reports the comparison of virgin samples with PS standard reference spectrum. The analyses of the MPs highlighted a broad band centred around 3500 cm − 1 , only in the photo-aged PS, typical of the stretching vibration of the O-H groups. This band most likely expresses the increase in the number of oxygenated features. Furthermore, at the wavelength of 1650–1750 cm − 1 and 1100–1350 cm − 1 it is possible to report peaks attributable to C = O vibrations, as reported by (Hu et al., 2023 ). All spectra detect signals around 3020 cm − 1 , due to the stretching vibration of the C-H bond of the aromatic groups, along with additional signals at 2910 cm − 1 , due to the stretching of the C-H bonds of the methylene groups. The presence of aromatic groups is further highlighted with the set of low but typical signals between 1750–2100 cm − 1 , which are also slightly affected by the ageing process, likely due to the oxidation of the aromatic group. Figure 2 shows the evolution of CI, highlighting an increase in the index value as the ageing process progressed for both PS sizes. This result provides further evidence of the oxidation process, particularly of the mechanisms that consider the presence of oxygen-containing functional groups due to the UV ageing action. As further evidence of the oxidation process, the I ox , as suggested by Vicente et al. ( 2009 ), was roughly calculated for aged samples and compared with a standard PS sample (Table 1 ). Even though an absolute range of I ox values is unavailable (I ox values are reported between 0,0 and 2,0), we can see the difference between the aged samples and the standard PS. Table 1 I ox value in UVB ageing MPs Sample I ox PS 1 µm 0,95 PS 5 µm 0,90 Standard PS 0,00 ζ-potential Eventually, Z-potential values found for investigated samples are reported in Fig. 3. Even though values do not agree with the ones reported by the manufacturer (specifications about the experimental set-up were not reported, so the measurement conditions are unknown), Z-pot values are negative for all samples. Figure 3Z -potential measurement in investigated samples. Aged samples present a much negative value of Z-pot, which can be safely attributed to more oxygen-containing functional groups that make the surface of the MPs more negative. This outcome confirms the presence of a different surface condition characterising aged MPs concerning the bare samples. Cell viability MTT assays performed on THP-1 cells at increasing doses of microplastics (25, 50, 100, 250 and 500 ug/ml), both oxidised and virgin, with contact times of 24H and 48h, did not show any effect on viability, even at higher doses and at longer exposure times. For this reason, the results were not reported in the text. DNA Damage Comet assay was performed to evaluate genotoxic damage induced by exposition of THP-1 cells at two different sizes of MPs (1 and 5 µm) and concentrations (25 and 50 µg/ml) when oxidated by UVB exposition or in their native state. Genotoxic damage in THP-1 (Fig. 4 ) was expressed as a percentage of DNA in the tail (%TailDNA) after 24h or 48h of exposure, and the results were compared with untreated cells (Ctrl). The assay showed a significant increase in %TailDNA when the cells were exposed to MPs. In particular, after 24h, cells exposed to 1 µm MPs ox showed 5.9-fold DNA damage in the samples treated with 25 µg/ml and 7.2 in the samples treated with 50 µg/ml (P < 0.001). A moderate increase in damage (P < 0.05) was reported in all cells exposed to 1 µm MPs v , with fold variations of 2.4 at 25 µg/ml and 2.2 at 50 µg/ml. When the cells were exposed to 5 µm MPs, an increase in DNA damage was observed but with a lower magnitude. In general, the DNA damage riched a fold change around 2 (P < 0.05) in all samples tested. Forty-eight hours of exposition showed a similar DNA damage trend, albeit the magnitude was higher in all tested samples, as shown in Fig. 4 . Micronuclei The photograph in Fig. 5 provides examples of internalisation MPs in THP-1 cells and the same example of micronucleus. Figure 5 Photomicrographs obtained under an optical microscope representative of THP-1 cells exposed to 5 µm MPs (right panel) and 1 µm MPs (left panel), respectively. In the image, the intracellular MPs are indicated by dashed arrows, while solid arrows indicate the presence of micronuclei. 100X magnification. Figure 6 Frequency of micronuclei (MN), cytokinesis-block proliferation index (CBPI) in THP-1 cell lines either untreated (CTRL-) or exposed to methanesulfonate (CTRL+) or to MPs of varying size (1 or 5 µm) when oxidate or virgin. Treatment with MPs elicited increased MN formation, although not all differences reached statistical significance. A higher statistically significant change was observed in THP-1 cells exposed to 1µm MPs V with a dose-dependent response. In particular, cells treated with 1µm MPs V at 25 µg/ml reported a fold change of 3.1 (P < 0.05) when at 50 µg/ml of 5-fold (P < 0.01). Micronucleus formation increased in cells in contact with 1µm MPs ox at higher concentration as 4 fold change (P < 0.05). MDA As shown in Fig. 7, thiobarbituric acid reactive substances (TBARS) generation significantly increased in THP-1 when exposed to 1µm MPs V at concentrations of 25 µg (P < 0.05) and 50 µg (P < 0.01). Differently, a significant increase was only at the concentration of 50 µg for MPsOx 5µm (P < 0.05). Figure 7. TBARS levels, expressed as nmol/mg protein, in THP-1 cells after contact for 24 h with two concentrations of MPs (25 and 50 µg/ml) of different size (5 or 1 µm) and status (virgin or oxidate). The columns report the means + SD of triplicate analyses. Statistical analysis: *P < 0.05 and **P < 0.01 vs. controls. Discussion The results of the present study show that UVB triggers oxidative processes that alter MPs surface structure. Ageing changes their morphology, colour, size, and surface charge. (Hu et al., 2023 ). The lab-based model for UVB ageing simulated the solar irradiation of plastic particles dispersed in the environmental water. UVB ageing process can increase the release of toxic components such as phthalates and bisphenol-A (Weis and Alava, 2023 ), or increased absorption of environmental organic pollutants enhances the toxicity of MPs dispersed in different environmental matrices (Bhagat et al., 2022 ). PS is reported to have a negative surface charge, which increases after ageing (Pelley et al. , 2008), as obtained by our measure. This behaviour may be explained by the oxidation of the carbon atoms induced by hydroxyl groups to carbonyl, carboxylic, and carbon dioxide (Zhang et al., 2021 ). The particle surface charge is one of the critical determinants of biological injury that influences cellular uptake efficiency through other factors such as aggregation/agglomeration, protein corona formation, and composition (Jeon et al., 2018 ). Our in vitro study evaluated the effects of both oxidate and virgin MPs at two different sizes in THP-1 cell line to reproduce the systemic effect when the particles are taken by ingestion or inhalation. The results indicate the fact that MPs are able to generate widespread damage in the cellular environment. The action of MPs depends mainly on size, surface features, and dose. In fact, although our viability test has not highlighted a significant mortality increase at the different concentrations tested, an impairment of oxidative stress and genotoxic damage was identified after MPs cell exposure. In particular, ingested or inhaled MPs can cross the epithelial barrier, diffuse into the circulatory or lymphatic systems, and, by macrophages, deposit in different organs such as the intestine, liver, and kidney (Barceló et al., 2023 ). MPs can cross membrane cells, triggering negative consequences in different cellular structures. The observed redox imbalance, expressed as MDA generation, reflects the ability of small-size MPs v to generate ROS. In this case, the generation of ROS in the cellular environment can be due to mitochondrial membrane damage. In fact, it was observed that MDA increases as the MPs sizes decrease (Jeong et al., 2016 ). Furthermore, ROS generation can be attributed to direct lysosomal damage induced by attempts of these organelles to digest the foreign body or to the excessive production of intracellular ROS that can denature lysosome membranes. PS is a stable, inert material that has a slight negative charge. Following oxidative processes, the surface charge acquires a more negative potential. The enrichment of charges determines more electrochemical interactions, facilitating bonds with serum proteins and triggering protein corona formation. Corona structures critically impact biological systems, offering new identities to MPs, such as cellular internalisation and interaction targets (Cao et al., 2022 ). Indeed, genotoxic tests indicate that the deleterious effects of MPs are mainly due to their smaller size and ageing state. Specifically, the comet test results show that oxidised particles are more capable of causing DNA damage. This could be related to the more significant negative surface charges on MPs ox, which promote greater interaction with serum proteins, increasing their bio-availability. Indeed, it is believed that both the primary physicochemical properties of MPs and those acquired in biological systems play a predominant role in cellular pathogenesis. On the other hand, the aneugenic and clastogenic effects observed in THP-1 cells after MPs exposition highlight that the particles of 1 µm increase the genomic instability. The segregation error can be most likely attributable to the disturbance that the microparticles cause in the organisation of the mitotic spindle. These results are supported by Çobanoğlu et al., ( 2021 ), reporting an MN frequency increase in human peripheral blood lymphocytes exposed to PE in vitro , concluding that MPs can cause genetic instabilities after chronic exposure. Nevertheless, the mechanisms by which MPs cause MN formation have yet to be investigated (Shamy et al., 2002 ; Somorovská et al., 1999 ; Ballesteros et al., 2020 ; Laffon et al., 2002 ). MPs represent a ubiquitous pollutant of significant importance for health. The increase in plastic waste inevitably determines its dispersion into the environment, where it undergoes various degradation processes, which lead to continuous fragmentation. UVB irradiation is one of the first causes of the agng of these materials, which inevitably alters their structure. Particles that are increasingly smaller and have an increasingly negative surface charge can trigger biochemical and structural alterations within the cellular compartment, laying the foundations for establishing even important pathological events. In the general population, exposure to MPs can occur directly (contact, inhalation, or ingestion) or indirectly (ascending of MPs in the trophic chain), and these particles can be excreted or accumulate in various tissues, increasing the toxicological risk dictated by exposure to different xenobiotics environmental. This phenomenon may cause different diseases, considering current estimates suggest that an individual ingests between 0.1 and 5 grams of plastic material per week. In conclusion, further studies would be necessary to evaluate the effects of MPs in vivo and also through biomonitoring studies to be performed on the general population in order to identify the long-term effects of this recent class of persistent pollutants. Declarations Authors’ contributions S.L.M. designed and wrote the manuscript. M.B., S.L.M., and L.F. performed in vitro experiments. S.A. performed chemical evaluation and characterization. S.L.M. and F.D., analyzed the data. S.L.M., S.A. and F.D. assisted with the manuscript revision. S.L.M. supervised the study. Declaration of conflicting interests : The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding : No funding was received for conducting this study. References Alomar C, Sureda A, Capó X, Guijarro B, Tejada S, Deudero S (2017) Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. 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Environ Int 146106274. https://doi.org/10.1016/j.envint.2020.106274 Rahman A, Sarkar A, Yadav OP, Achari G, Slobodnik J (2021) Potential human health risks due to environmental exposure to nano- and microplastics and knowledge gaps: A scoping review. Sci Total Environ 757143872. https://doi.org/10.1016/j.scitotenv.2020.143872 Revel M, Châtel A, Mouneyrac C (2018) Micro(nano)plastics: a threat to human health? Curr Opin Environ Sci Health 1:17–23. https://doi.org/10.1016/j.coesh.2017.10.003 Rist S, Carney Almroth B, Hartmann NB, Karlsson TM (2018) A critical perspective on early communications concerning human health aspects of microplastics. Sci Total Environ 626:720–726. https://doi.org/10.1016/j.scitotenv.2018.01.092 Shamy MY, Osman HH, Kandeel KM, Abdel-Moneim NM, El SK (2002) DNA single strand breaks induced by low levels of occupational exposure to styrene: the gap between standards and reality. J Environ Pathol Toxicol Oncol 21:57–61. https://doi.org/10.1615/JEnvironPatholToxicolOncol.v21.i1.40 Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 175,184 – 91 https://doi.org/10.1016/0014-4827(88)90265-0 Somorovská M, Jahnová E, Tulinská J, Zámecníková M, Sarmanová J, Terenová A, Vodicková L, Lísková A, Vallová B, Soucek P, Hemminki K, Norppa H, Hirvonen A, Tates AD, Fuortes L, Dusinská M, Vodicka P (1999) Biomonitoring of occupational exposure to styrene in a plastics lamination plant. Mutat Res 428255–428269. https://doi.org/10.1016/s1383-5742(99)00052-6 Song YK, Hong SH, Jang M, Han GM, Jung SW, Shim WJ (2017) Combined Effects of UV Exposure Duration and Mechanical Abrasion on Microplastic Fragmentation by Polymer Type. Environ Sci Technol 51:4368–4376. https://doi.org/10.1021/acs.est.6b06155 Vethaak AD, Legler J (2021) Microplastics and human health. Science 371:672–674. https://doi.org/10.1126/science.abe5041 Vethaak AD, Leslie HA (2016) Plastic Debris Is a Human Health Issue. Environ Sci Technol 50:6825–6826. https://doi.org/10.1021/acs.est.6b02569 Vicente JS, Gejo JL, Rothenbacher S, Sarojiniamma S, Gogritchiani E, Wörner M, Kasperb G, Braun AM (2009) Oxidation of PS Aerosols by VUV-Photolysis and/or Ozone. Photochem Photobiol Sci 8:944–952. https://doi.org/10.1039/b902749a Wang YL, Lee YH, Hsu YH, Chiu IJ, Huang CC, Huang CC, Chia ZC, Lee CP, Lin YF, Chiu HW (2021) The Kidney-Related Effects of PS Microplastics on Human Kidney Proximal Tubular Epithelial Cells HK-2 and Male C57BL/6 Mice. Environ Health Perspect 129:57003. https://doi.org/10.1289/ehp7612 Weis JS, Alava JJ (2023) (Micro)Plastics Are Toxic Pollutants. Toxics . 11,935. https://doi.org/10.3390/toxics11110935 Wilkinson J, Hooda PS, Barker J, Barton S, Swinden J (2017) Occurrence, fate and transformation of emerging contaminants in water: An overarching review of the field. Environ Pollut 231:954–970. https://doi.org/10.1016/j.envpol.2017.08.032 World Health Organization (2019) Microplastics in drinking-water. https://iris.who.int/handle/10665/326499 Yan Z, Liu Y, Zhang T, Zhang F (2022) Hongqiang Ren, and Yan Zhang Analysis of Microplastics in Human Feces Reveals a Correlation between Fecal Microplastics and Inflammatory Bowel Disease Status. Environ Sci Technol 56:1414–1421. https://doi.org/10.1021/acs.est.1c03924 Zhang Y, Peng Y, Peng C, Wang P, Lu Y, He X, Wang L (2021) Comparison of Detection Methods of Microplastics in Landfill Mineralized Refuse and Selection of Degradation Degree Indexes. Environ Sci Technol 55:13802–13811. https://doi.org/10.1021/acs.est.1c02772 Cite Share Download PDF Status: Published Journal Publication published 19 Jun, 2024 Read the published version in Archives of Environmental Contamination and Toxicology → Version 1 posted Reviewers agreed at journal 06 Apr, 2024 Reviewers invited by journal 06 Apr, 2024 Editor assigned by journal 12 Feb, 2024 First submitted to journal 12 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3951751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272502260,"identity":"1e06e820-7f31-4cad-a700-3c618dddb284","order_by":0,"name":"SEBASTIANO LA MAESTRA","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-4939-2124","institution":"University of Genoa Department of Health Sciences: Universita degli Studi di Genova Dipartimento di Scienze della Salute","correspondingAuthor":true,"prefix":"","firstName":"SEBASTIANO","middleName":"LA","lastName":"MAESTRA","suffix":""},{"id":272502261,"identity":"97755546-3cb9-421e-872c-3af02537af15","order_by":1,"name":"Mirko Benvenuti","email":"","orcid":"","institution":"University of Genoa: Universita degli Studi di Genova","correspondingAuthor":false,"prefix":"","firstName":"Mirko","middleName":"","lastName":"Benvenuti","suffix":""},{"id":272502262,"identity":"a6c28eda-f9ed-454a-8410-af24f117efd6","order_by":2,"name":"Stefano Alberti","email":"","orcid":"","institution":"University of Genoa: Universita degli Studi di Genova","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Alberti","suffix":""},{"id":272502263,"identity":"67e954f8-c736-4707-8638-feeaff26195c","order_by":3,"name":"Linda Ferrea","email":"","orcid":"","institution":"University of Genoa: Universita degli Studi di Genova","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Ferrea","suffix":""},{"id":272502264,"identity":"cda01fae-4a58-4322-b065-6bff3f33568b","order_by":4,"name":"Francesco D'Agostini","email":"","orcid":"","institution":"University of Genoa: Universita degli Studi di Genova","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"D'Agostini","suffix":""}],"badges":[],"createdAt":"2024-02-12 19:19:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3951751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3951751/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00244-024-01073-x","type":"published","date":"2024-06-19T15:46:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51110499,"identity":"16eec511-e3d0-4092-b35b-2a733ceb39d4","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254415,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra in the MPs range 4,000-600 cm\u003csup\u003e-1\u003c/sup\u003e. A) black line 1 μm V and red 1 μm Ox; B) black line 5 μm V and red 5 μm Ox; C) comparison of MPs reference spectra with different MPs samples.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/c752a13bf1fd6ec1a5ffc496.png"},{"id":51110501,"identity":"1ec48026-fe0c-4dbf-8cc6-c5b6d44e468f","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67196,"visible":true,"origin":"","legend":"\u003cp\u003eCarbonyl Index of MPs samples\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/028d64b575cc72d92e03fdcf.png"},{"id":51110498,"identity":"3d552318-c922-47bc-a6ec-54e5080f3002","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55589,"visible":true,"origin":"","legend":"\u003cp\u003eZ-potential measurement in investigated samples.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/1a07fa50d1215324e09804b8.png"},{"id":51110500,"identity":"fa551d3c-192f-4dab-a7c9-acb2fcc738f8","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123719,"visible":true,"origin":"","legend":"\u003cp\u003eDNA damage (% TDNA) obtained by comet assay in THP-1 cells when exposed to 1 µm and 5 µm MPs, oxidate (ox) o virgin (v) at two different concentrations (25 and 50 µg/ml) and two times of contact (24 and 48h). The columns report the means + SD of triplicate analyses. Statistical analysis: * P \u0026lt; 0.05, ** P \u0026lt; 0.01 and ***P \u0026lt; 0.001 vs. controls.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/9a8371faafcfd36ea4794eb6.png"},{"id":51110502,"identity":"35af0fe5-2694-46d5-9bcb-b1379297e380","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":160295,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs obtained under an optical microscope representative of THP-1 cells exposed to 5 µm MPs (right panel) and 1 µm MPs (left panel), respectively. In the image, the intracellular MPs are indicated by dashed arrows, while solid arrows indicate the presence of micronuclei. 100X magnification.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/64eb438d23847d5a6c2bc9a7.png"},{"id":51110820,"identity":"11547607-29ed-4f37-9088-784934d5a0fe","added_by":"auto","created_at":"2024-02-14 09:40:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":127204,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of micronuclei (MN), cytokinesis-block proliferation index (CBPI) in THP-1 cell lines either untreated (CTRL-) or exposed to methanesulfonate (CTRL+) or to MPs of varying size (1 or 5 µm) when oxidate or virgin.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/aff52d9d4c50b1b2b87ebba7.png"},{"id":51110504,"identity":"a8ed541a-8e9f-4e60-97ec-7295a3da9cdd","added_by":"auto","created_at":"2024-02-14 09:32:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104050,"visible":true,"origin":"","legend":"\u003cp\u003eTBARS levels, expressed as nmol/mg protein, in THP-1 cells after contact for 24 h with two concentrations of MPs (25 and 50 µg/ml) of different size (5 or 1 µm) and status (virgin or oxidate). The columns report the means + SD of triplicate analyses. Statistical analysis: *P \u0026lt; 0.05 and **P \u0026lt; 0.01 vs. controls.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/7e5846f3c53a994baaefb5fc.png"},{"id":58823618,"identity":"92750404-e9df-42c0-b322-fb089dba33cc","added_by":"auto","created_at":"2024-06-21 17:04:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1360535,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3951751/v1/0478c50f-a405-433c-927f-cc4f16754f5a.pdf"}],"financialInterests":"","formattedTitle":"UVB-aged microplastics and cellular damage : An in vitro study","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlastics are organic polymeric compounds synthesised from natural resources such as gas, oil, and its derivatives. These polymers have many applications in different sectors, including packaging, construction, automotive, and electronics. Due to their advantageous characteristics and low production costs, manufacturing increases yearly (Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Extensive use generates enormous quantities of plastic waste. It has been calculated that from 1950 to 2015, about 6.3\u0026nbsp;billion tons of plastic waste were generated, and 26\u0026nbsp;billion tons will be produced by 2050 (Geyer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guglielmi, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCommonly used plastics are polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polystyrene (PS), and polyurethane (PU). All these polymers are characterised by environmental persistence due to their molecular stability, which may be referenced to covalent bonds involving C-C and C-H groups. These groups may undergo degradation reactions triggered by heat and light (UVB) (Wilkinson et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), causing instability and breaking into small fragments. Moreover, environmental MPs are degraded by different metabolic microorganisms (Miri et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Depending on their size, plastic particles can be classified as MPs with dimensions between 0.001 and 5 mm and nanoplastics (NPs) smaller than 0.1 \u0026micro;m (Kubowicz and Booth, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMP pollutants can be generated by mechanical abrasion (Song et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) weathering on environmentally dispersed macroplastics or by releasing small fragments from clothing, car tyres, paint coatings, pre-production dust, pellet spills, or also found in various preparations, such as cosmetic products (Vethaak and Legler, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to their source, MPs can be classified into primary or secondary. Primary MPs originate as small particles released directly into the environment. They are derived from textiles, medical products, and personal care products. Secondary MPs result from the degradation process of large amounts of plastic waste (Eriksen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Alomar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to their small size, MPs can be considered ubiquitous and carried through the marine and terrestrial environment and into the atmosphere (Lim, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, their small size makes them easily ingested by aquatic organisms perpetuated throughout the trophic chain (Lim, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Desforges et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nelms et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough MPs have also been found in different environmental matrices, such as in drinking water, WHO (2019) stated that there are not enough studies to define the toxicity of these particles on human health.\u003c/p\u003e \u003cp\u003eOn the other hand, humans may ingest or inhale MPs, potentially affecting health (Vethaak and Legler, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Once ingested, MPs cross the gastrointestinal tract epithelium gaps to disseminate into the circulatory system to be transported to the lymph nodes, liver, and spleen. Several subjects showed the presence of MPs\u0026thinsp;\u0026lt;\u0026thinsp;2 \u0026micro;m in blood and different organs, such as the human placenta's fetal, maternal, and chorioamnionitis membrane sides (Ragusa et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), although it is unclear whether MPs with larger sizes follow the same pathways (Revel et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInhaled MPs with a size\u0026thinsp;\u0026lt;\u0026thinsp;2.5 \u0026micro;m can cross the respiratory barrier (Liao et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) by alveolar macrophages to disseminate at the systemic level (Vethaak and Leslie, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMPs have been observed to lead to intestinal barrier dysfunction, microbiota imbalance, alteration in triglyceride synthesis and lipogenesis and reduction in intestinal mucus secretion (Rahman et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ingested MPs can be eliminated in the stool or urine (Massardo, et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A recent study suggests the renal system can eliminate MPs above 10 nm in the urine after glomerulus filtration. MPs with larger dimensions could cross the renal tubules by exocytosis from the efferent artery and endocytosis from the proximal convoluted tubule (Pironti et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, ingested MPs can cross the gastrointestinal system, damage the epithelia, or be evacuated through the feces (Yan et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, MPs can carry a series of toxic and other polluted compounds that could increase the risk of adverse effects (Rist et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e studies showed that MPs induce oxidative stress, damaging mitochondrial membranes, generating unbalanced membrane potential (Wang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ROS are underlying the genotoxicity processes and are involved in the multi-step process of carcinogenesis (De S\u0026aacute; Junior et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and chronic disease. Human studies reported that MPs and NPs may induce DNA damage, such as micronuclei (MN) formation, chromosomal aberration, DNA strand breaks, and genotoxicity (Ballesteros et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study aims to assess genotoxic damage and oxidative stress induced by polystyrene MPs 1 and 5 \u0026micro;m in size on THP-1 cells to identify the differences between UV-aged and non-aged particles. Different assays were performed to assess cytotoxicity, oxidative stress, genotoxicity, and aneuploidy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroplastics oxidation\u003c/h2\u003e \u003cp\u003ePS virgin MPs of 1 \u0026micro;m and 5 \u0026micro;m were acquired by Cospheric (Santa Barbara, California 93160 United States), and their oxidation was performed by UVB lamp exposure at 318 nm (57 V, 20 W G13, Philips) started in the presence of 40% hydrogen peroxide (Batel, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In particular, microplastic suspensions (10 mg/ml) were placed in a glass disk containing an equal volume of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, UVB-irradiated for 96 h and periodically agitated. At the end of treatment, the MPs suspension was precipitated by centrifuging (12,000 rpm for 10 min), washed in Milli-Q water, and dried by SpeedVac. Precipitated microplastics were weighted and resuspended in phosphate buffer saline (PBS) at known concentrations. Before each use, the microplastic samples were resuspended using ultrasonic devices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eζ-potential\u003c/h2\u003e \u003cp\u003eζ-potential (surface charge) measurements on microplastic samples were performed using Dynamic Light Scattering (DLS) analyses, with a Zetasizer Instrument, Nano ZS90 Series (Malvern Panalytical, Malvern, UK), working with He-Ne laser (emission λ\u0026thinsp;=\u0026thinsp;633 nm). Samples were subjected to 40 kHz sonication before each analysis. Measurements were performed at a fixed temperature (T\u0026thinsp;=\u0026thinsp;25\u0026deg;C) using a Peltier thermostatic system, with an equilibrating time set to 120 s. MPs were dispersed in deionised water to reach a final concentration of 0.1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Measurements were performed in triplicate, and each measurement consisted of 20 runs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy\u003c/h2\u003e \u003cp\u003eFourier Transform Infrared Spectroscopy (FTIR) measurements were performed on Perkin Elmer Spectrum 65 FT-IR, which was equipped with an attenuated total reflectance (ATR) accessory. The spectra were collected in the range of 4,000\u0026ndash;500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; each spectrum results from 10 accumulations of 15 seconds each.\u003c/p\u003e \u003cp\u003eThe spectra obtained from FTIR analysis were used to determine the carbonyl index (CI) as reported by different studies (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and calculated as the ratio between the absorbance of the carbonyl peak and the absorbance of the methylene peak, considered as reference. In particular, for PS samples, the carbonyl peak is usually calculated at 1,730 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the methylene at 1,452 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.. Furthermore, the same analysis allowed us to calculate the oxidation index (I\u003csub\u003eox\u003c/sub\u003e) (Vicente et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${I}_{ox}=\\frac{\\int {Abs\\left(t\\right)}_{C=O}}{\\int {Abs\\left(t\\right)}_{ref}}-\\frac{\\int {Abs\\left(0\\right)}_{C=O}}{\\int {Abs\\left(0\\right)}_{ref}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003eAbs(t)\u003csub\u003eC=O\u003c/sub\u003e absorbance of C\u0026thinsp;=\u0026thinsp;O (1,615\u0026ndash;1,840 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at reaction time t\u003c/p\u003e \u003cp\u003eAbs(t)\u003csub\u003eref\u003c/sub\u003e absorbance of reference peaks (1,471\u0026ndash;1,522 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at reaction time t\u003c/p\u003e \u003cp\u003eAbs(0)\u003csub\u003eC=O\u003c/sub\u003e absorbance of C\u0026thinsp;=\u0026thinsp;O (1,615\u0026ndash;1,840 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for unmodified polystyrene\u003c/p\u003e \u003cp\u003eAbs(0)\u003csub\u003eref\u003c/sub\u003e absorbance of reference peaks (1,471\u0026ndash;1,522 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for unmodified polystyrene.\u003c/p\u003e \u003cp\u003eThis parameter can be used as a reference for the ageing of the material under study and quantifies this process as a function of the oxidised groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eTHP-1 cell line was obtained from the European Collection of Cell Cultures (ECACC). Monocytes derived from peripheral blood patients with acute monocytic leukaemia were used to mimic the toxicological responses of systemic monocytes/macrophages. Briefly, the THP-1 cell was maintained in a complete culture medium (RPMI, 10% FBS, 10% Glutamine and 1% Penicillin/Streptomycin) and split every two days or used for different assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eMTT assay measures the insoluble formazan derived from tetrazolium salt and generated by the activity of mitochondria of viable cells. The assay was performed in cells cultured for 24 h in 96-well microplates (1.5 x 10\u003csup\u003e4\u003c/sup\u003e). Eight wells were inoculated with oxidated or virgin microplastics at sizes of 5 and 1 \u0026micro;m at different concentrations (25, 50, 100, 250, and 500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with two different exposure times (24 and 48h).\u003c/p\u003e \u003cp\u003eAfter exposure, 0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of MTT was added to each well, and the microplates were reincubated at 37\u0026deg;C for 3 h. Dimetil sulfoxide was used to solubilise the purple-coloured formazan crystals, proportional to the number of metabolic active cells. Measurement was performed at 570 nm using a microplate spectro-photometer reader (Tecan Italia, Milan, Italy). The values obtained were compared to the negative control, assigned 100% vitality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eComet assay\u003c/h2\u003e \u003cp\u003eThe alkaline comet assay was performed using a protocol suggested by Singh et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) with some modifications (La Maestra \u003cem\u003eet al.\u003c/em\u003e, 2020\u003cem\u003e)\u003c/em\u003e. Briefly, the THP-1 cells were seeded in 96 well plates at the density of 1.5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well and exposed to microplastic, both oxidised and non-oxidised at two different sizes (1 and 5 \u0026micro;m), and two concentrations (25 and 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) per 24 h. After incubation, the viability of cells was checked by trypan blue exclusion test. Viability of less than 80% was considered an exclusion factor of the sample. After that, about 20,000 cells were embedded in 75 \u0026micro;L of 0.5% low melting-point agarose, coated onto slides, covered with a coverslip, and allowed to solidify at 4\u0026deg;C, followed by a second layer of low melting-point agarose.\u003c/p\u003e \u003cp\u003eThe slides were immersed in cold lysis solutions (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid, 10 mM Tris, pH 10, 1% Triton X-100, and 10% dimethyl sulfoxide) overnight, rinsed in alkaline solution (0.3 M NaOH, 1 mM ethylenediaminetetraacetic acid, pH 13) and placed horizontally in an electrophoresis chamber in which it was performed, in fresh alkaline solution, electrophoresis (30 min at 25 V (0.66 V/cm), adjusted to 300 mA). After electrophoresis, the slides were gently removed and washed in a neutralisation buffer (0.4 M Tris-HCl, pH 7.5) for 5 min and stained with propidium iodide (2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). One hundred random nuclei were acquired with a fluorescence microscope and a digital camera at a magnification of 200\u0026times;. The analysis was conducted using CASP (Comet assay software project, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.casp.sourceforge.net\u003c/span\u003e\u003cspan address=\"http://www.casp.sourceforge.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the results were expressed in terms of the percentage of DNA in the tail (TDNA %).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytokinesis-block micronucleus assay\u003c/h2\u003e \u003cp\u003eCytokinesis-block micronucleus assay (CBMN) was performed in duplicate to highlight breakage or loss of chromosomes following exposure to microplastic, as described by Fenech (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Briefly, 10 x 10\u003csup\u003e4\u003c/sup\u003e THP-1 cells were seeded onto 15 ml tubes and exposed to equal microplastics and concentrations used for the comet assay. After 24 h exposure, cells added with 4 \u0026micro;g/mL cytochalasin B. THP-1 cells were maintained in culture for an additional 28 h and then washed and resuspended in 0.075 M KCl hypotonic solution for 2 min, centrifugate 10 min at 1,200 rpm and prefixed in 3:5 methanol/acetic acid, and washed twice with a 6:1 methanol/acetic acid fixative solution.\u003c/p\u003e \u003cp\u003eMicroscopic slides were obtained by smearing each sample. Subsequently, the slides underwent acid hydrolysis for 1 h ( HCl 5 N), rinsed in distilled water and deoxyribonucleic acid specifically stained with Schiff\u0026rsquo;s reagent (Sigma Chemical Co., St. Louis, MO) for 30 min, washed in distilled water, and left for 5 min in running tap water in order to intensify the pink colour. Finally, the samples were washed, blotted dry, and mounted. One thousand cells from each sample were examined under an optical microscope at 1000\u0026times; magnification to score the presence of micronucleated cells and binucleate cells. Fisher\u0026rsquo;s exact test was performed to determine a statistically significant difference between different treatments and the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA)\u003c/h2\u003e \u003cp\u003eBriefly, 15 x 10\u003csup\u003e4\u003c/sup\u003e THP-1 cells were seeded onto 15 ml tubes and exposed to concentrations MPs reported above. After 24 h exposure, the cells were collected by centrifuging 10 min at 2,200 rpm, and each cell aliquot was tested for lipid peroxidation by concentration of TBARS as described by Ohkawa et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). Therefore, 200 \u0026micro;l of 8.1% sodium dodecyl sulfate, 1,500 \u0026micro;l of 20% acetic acid solution (pH 3.5), and 1,500 \u0026micro;l of 0.8% aqueous solution thiobarbituric acid were added to the cells. Distilled water was added to reach a volume of 4 ml, and the samples were heated at 95\u0026deg;C for 60 min. After, 4 ml of n-butanol and pyridine (15:1 v/v) were added, shaken vigorously, and centrifugated at 4,000 rpm for 10 min. The organic layer was taken, and a fluorometric measurement was made (Ex 533 nm; Em 553 nm). Moreover, an external standard, MDA, was used, and the results were expressed as nanomoles MDA equivalent per cell number.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe analyses were performed by JMP software. The results regarding multiple individual experiments were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and data were analysed by one-way analysis of variance (ANOVA) with \u003cem\u003epost hoc\u003c/em\u003e testing using the Bonferroni test. A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered as statistically significant. Fisher\u0026rsquo;s exact test was performed with a statistically significant difference between each treatment and the control with respect to the frequency of micronucleated cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (A, B) reports FTIR spectra obtained for virgin and oxidated MPs of both dimensions, 1 \u0026micro;m and 5 \u0026micro;m MPs, respectively. Differently, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC reports the comparison of virgin samples with PS standard reference spectrum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analyses of the MPs highlighted a broad band centred around 3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, only in the photo-aged PS, typical of the stretching vibration of the O-H groups. This band most likely expresses the increase in the number of oxygenated features. Furthermore, at the wavelength of 1650\u0026ndash;1750 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1100\u0026ndash;1350 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e it is possible to report peaks attributable to C\u0026thinsp;=\u0026thinsp;O vibrations, as reported by (Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll spectra detect signals around 3020 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, due to the stretching vibration of the C-H bond of the aromatic groups, along with additional signals at 2910 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, due to the stretching of the C-H bonds of the methylene groups. The presence of aromatic groups is further highlighted with the set of low but typical signals between 1750\u0026ndash;2100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which are also slightly affected by the ageing process, likely due to the oxidation of the aromatic group.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the evolution of CI, highlighting an increase in the index value as the ageing process progressed for both PS sizes. This result provides further evidence of the oxidation process, particularly of the mechanisms that consider the presence of oxygen-containing functional groups due to the UV ageing action.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs further evidence of the oxidation process, the I\u003csub\u003eox\u003c/sub\u003e, as suggested by Vicente et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), was roughly calculated for aged samples and compared with a standard PS sample (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Even though an absolute range of I\u003csub\u003eox\u003c/sub\u003e values is unavailable (I\u003csub\u003eox\u003c/sub\u003e values are reported between 0,0 and 2,0), we can see the difference between the aged samples and the standard PS.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eI\u003csub\u003eox\u003c/sub\u003e value in UVB ageing MPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003csub\u003eox\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePS 1 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePS 5 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard PS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eζ-potential\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEventually, Z-potential values found for investigated samples are reported in Fig.\u0026nbsp;3. Even though values do not agree with the ones reported by the manufacturer (specifications about the experimental set-up were not reported, so the measurement conditions are unknown), Z-pot values are negative for all samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3Z\u003c/b\u003e-potential measurement in investigated samples.\u003c/p\u003e \u003cp\u003eAged samples present a much negative value of Z-pot, which can be safely attributed to more oxygen-containing functional groups that make the surface of the MPs more negative. This outcome confirms the presence of a different surface condition characterising aged MPs concerning the bare samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eMTT assays performed on THP-1 cells at increasing doses of microplastics (25, 50, 100, 250 and 500 ug/ml), both oxidised and virgin, with contact times of 24H and 48h, did not show any effect on viability, even at higher doses and at longer exposure times. For this reason, the results were not reported in the text.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDNA Damage\u003c/h2\u003e \u003cp\u003eComet assay was performed to evaluate genotoxic damage induced by exposition of THP-1 cells at two different sizes of MPs (1 and 5 \u0026micro;m) and concentrations (25 and 50 \u0026micro;g/ml) when oxidated by UVB exposition or in their native state. Genotoxic damage in THP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was expressed as a percentage of DNA in the tail (%TailDNA) after 24h or 48h of exposure, and the results were compared with untreated cells (Ctrl).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe assay showed a significant increase in %TailDNA when the cells were exposed to MPs. In particular, after 24h, cells exposed to 1 \u0026micro;m MPs\u003csub\u003eox\u003c/sub\u003e showed 5.9-fold DNA damage in the samples treated with 25 \u0026micro;g/ml and 7.2 in the samples treated with 50 \u0026micro;g/ml (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A moderate increase in damage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was reported in all cells exposed to 1 \u0026micro;m MPs\u003csub\u003ev\u003c/sub\u003e, with fold variations of 2.4 at 25 \u0026micro;g/ml and 2.2 at 50 \u0026micro;g/ml. When the cells were exposed to 5 \u0026micro;m MPs, an increase in DNA damage was observed but with a lower magnitude. In general, the DNA damage riched a fold change around 2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all samples tested. Forty-eight hours of exposition showed a similar DNA damage trend, albeit the magnitude was higher in all tested samples, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMicronuclei\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe photograph in Fig.\u0026nbsp;5 provides examples of internalisation MPs in THP-1 cells and the same example of micronucleus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Photomicrographs obtained under an optical microscope representative of THP-1 cells exposed to 5 \u0026micro;m MPs (right panel) and 1 \u0026micro;m MPs (left panel), respectively. In the image, the intracellular MPs are indicated by dashed arrows, while solid arrows indicate the presence of micronuclei. 100X magnification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e Frequency of micronuclei (MN), cytokinesis-block proliferation index (CBPI) in THP-1 cell lines either untreated (CTRL-) or exposed to methanesulfonate (CTRL+) or to MPs of varying size (1 or 5 \u0026micro;m) when oxidate or virgin.\u003c/p\u003e \u003cp\u003eTreatment with MPs elicited increased MN formation, although not all differences reached statistical significance. A higher statistically significant change was observed in THP-1 cells exposed to 1\u0026micro;m MPs\u003csub\u003eV\u003c/sub\u003e with a dose-dependent response. In particular, cells treated with 1\u0026micro;m MPs\u003csub\u003eV\u003c/sub\u003e at 25 \u0026micro;g/ml reported a fold change of 3.1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when at 50 \u0026micro;g/ml of 5-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Micronucleus formation increased in cells in contact with 1\u0026micro;m MPs\u003csub\u003eox\u003c/sub\u003e at higher concentration as 4 fold change (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMDA\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;7, thiobarbituric acid reactive substances (TBARS) generation significantly increased in THP-1 when exposed to 1\u0026micro;m MPs\u003csub\u003eV\u003c/sub\u003e at concentrations of 25 \u0026micro;g (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 50 \u0026micro;g (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Differently, a significant increase was only at the concentration of 50 \u0026micro;g for MPsOx 5\u0026micro;m (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;7.\u003c/b\u003e TBARS levels, expressed as nmol/mg protein, in THP-1 cells after contact for 24 h with two concentrations of MPs (25 and 50 \u0026micro;g/ml) of different size (5 or 1 \u0026micro;m) and status (virgin or oxidate). The columns report the means\u0026thinsp;+\u0026thinsp;SD of triplicate analyses. Statistical analysis: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. controls.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the present study show that UVB triggers oxidative processes that alter MPs surface structure. Ageing changes their morphology, colour, size, and surface charge. (Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe lab-based model for UVB ageing simulated the solar irradiation of plastic particles dispersed in the environmental water. UVB ageing process can increase the release of toxic components such as phthalates and bisphenol-A (Weis and Alava, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), or increased absorption of environmental organic pollutants enhances the toxicity of MPs dispersed in different environmental matrices (Bhagat et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePS is reported to have a negative surface charge, which increases after ageing (Pelley \u003cem\u003eet al.\u003c/em\u003e, 2008), as obtained by our measure. This behaviour may be explained by the oxidation of the carbon atoms induced by hydroxyl groups to carbonyl, carboxylic, and carbon dioxide (Zhang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe particle surface charge is one of the critical determinants of biological injury that influences cellular uptake efficiency through other factors such as aggregation/agglomeration, protein corona formation, and composition (Jeon et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e study evaluated the effects of both oxidate and virgin MPs at two different sizes in THP-1 cell line to reproduce the systemic effect when the particles are taken by ingestion or inhalation. The results indicate the fact that MPs are able to generate widespread damage in the cellular environment. The action of MPs depends mainly on size, surface features, and dose. In fact, although our viability test has not highlighted a significant mortality increase at the different concentrations tested, an impairment of oxidative stress and genotoxic damage was identified after MPs cell exposure.\u003c/p\u003e \u003cp\u003eIn particular, ingested or inhaled MPs can cross the epithelial barrier, diffuse into the circulatory or lymphatic systems, and, by macrophages, deposit in different organs such as the intestine, liver, and kidney (Barcel\u0026oacute; et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). MPs can cross membrane cells, triggering negative consequences in different cellular structures. The observed redox imbalance, expressed as MDA generation, reflects the ability of small-size MPs\u003csub\u003ev\u003c/sub\u003e to generate ROS. In this case, the generation of ROS in the cellular environment can be due to mitochondrial membrane damage. In fact, it was observed that MDA increases as the MPs sizes decrease (Jeong et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, ROS generation can be attributed to direct lysosomal damage induced by attempts of these organelles to digest the foreign body or to the excessive production of intracellular ROS that can denature lysosome membranes.\u003c/p\u003e \u003cp\u003ePS is a stable, inert material that has a slight negative charge. Following oxidative processes, the surface charge acquires a more negative potential. The enrichment of charges determines more electrochemical interactions, facilitating bonds with serum proteins and triggering protein corona formation. Corona structures critically impact biological systems, offering new identities to MPs, such as cellular internalisation and interaction targets (Cao et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Indeed, genotoxic tests indicate that the deleterious effects of MPs are mainly due to their smaller size and ageing state. Specifically, the comet test results show that oxidised particles are more capable of causing DNA damage. This could be related to the more significant negative surface charges on MPs ox, which promote greater interaction with serum proteins, increasing their bio-availability. Indeed, it is believed that both the primary physicochemical properties of MPs and those acquired in biological systems play a predominant role in cellular pathogenesis.\u003c/p\u003e \u003cp\u003eOn the other hand, the aneugenic and clastogenic effects observed in THP-1 cells after MPs exposition highlight that the particles of 1 \u0026micro;m increase the genomic instability. The segregation error can be most likely attributable to the disturbance that the microparticles cause in the organisation of the mitotic spindle. These results are supported by \u0026Ccedil;obanoğlu et al., (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), reporting an MN frequency increase in human peripheral blood lymphocytes exposed to PE \u003cem\u003ein vitro\u003c/em\u003e, concluding that MPs can cause genetic instabilities after chronic exposure. Nevertheless, the mechanisms by which MPs cause MN formation have yet to be investigated (Shamy et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Somorovsk\u0026aacute; et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ballesteros et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Laffon et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMPs represent a ubiquitous pollutant of significant importance for health. The increase in plastic waste inevitably determines its dispersion into the environment, where it undergoes various degradation processes, which lead to continuous fragmentation. UVB irradiation is one of the first causes of the agng of these materials, which inevitably alters their structure. Particles that are increasingly smaller and have an increasingly negative surface charge can trigger biochemical and structural alterations within the cellular compartment, laying the foundations for establishing even important pathological events.\u003c/p\u003e \u003cp\u003eIn the general population, exposure to MPs can occur directly (contact, inhalation, or ingestion) or indirectly (ascending of MPs in the trophic chain), and these particles can be excreted or accumulate in various tissues, increasing the toxicological risk dictated by exposure to different xenobiotics environmental. This phenomenon may cause different diseases, considering current estimates suggest that an individual ingests between 0.1 and 5 grams of plastic material per week.\u003c/p\u003e \u003cp\u003eIn conclusion, further studies would be necessary to evaluate the effects of MPs \u003cem\u003ein vivo\u003c/em\u003e and also through biomonitoring studies to be performed on the general population in order to identify the long-term effects of this recent class of persistent pollutants.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.L.M. designed and wrote the manuscript. M.B., S.L.M., and L.F. performed \u003cem\u003ein vitro\u003c/em\u003e experiments. S.A. performed chemical evaluation and characterization. S.L.M. and F.D., analyzed the data. S.L.M., S.A. and F.D. assisted with the manuscript revision. S.L.M. supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interests\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNo funding was received for conducting this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlomar C, Sureda A, Cap\u0026oacute; X, Guijarro B, Tejada S, Deudero S (2017) Microplastic ingestion by Mullus surmuletus Linnaeus, 1758 fish and its potential for causing oxidative stress. 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Environ Sci Technol 55:13802\u0026ndash;13811. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.est.1c02772\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.1c02772\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microplastics, UVB aging, DNA damage, oxidative stress, aneuploidy","lastPublishedDoi":"10.21203/rs.3.rs-3951751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3951751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlastics are synthetic organic compounds whose widespread use generates enormous waste. Different processes, such as mechanical abrasion, microbiological activity, and UVB irradiation, can fragment the plastic material and generate microplastics (MPs). MPs are ubiquitous, and various organisms, including humans, can ingest or inhale them, with potential adverse health effects.\u003c/p\u003e \u003cp\u003eThe differences between UV-aged and virgin particles were studied to evaluate the genotoxic damage and oxidative stress induced by polystyrene MPs with 1 and 5 \u0026micro;m sizes on the monocyte-like cell line (THP-1). Fourier transform infrared spectroscopy and Ζ-potential measurements were used to characterise MP particles after UVB exposure.\u003c/p\u003e \u003cp\u003eCells exposed to MPs show a widespread change in the cellular environment with the generation of Reactive Oxidative Species (ROS), as indicated by the increased malondialdehyde (MDA) level. The occurrence of genotoxic damage is correlated to the smaller size and ageing state of the MPs. The biochemical and genomic alterations observed in this in vitro study suggest that MPs, ubiquitous pollutants, following natural degradation and oxidation processes can cause various adverse effects on the health of the exposed population, making it necessary to carry out further studies to better define the real risk.\u003c/p\u003e","manuscriptTitle":"UVB-aged microplastics and cellular damage : An in vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 09:32:09","doi":"10.21203/rs.3.rs-3951751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-06T22:20:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-06T22:02:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-12T13:28:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Environmental Contamination and Toxicology","date":"2024-02-12T05:51:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9f4a97c2-f3e3-497f-bcc6-b8d5dc92a30c","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:46:54+00:00","versionOfRecord":{"articleIdentity":"rs-3951751","link":"https://doi.org/10.1007/s00244-024-01073-x","journal":{"identity":"archives-of-environmental-contamination-and-toxicology","isVorOnly":false,"title":"Archives of Environmental Contamination and Toxicology"},"publishedOn":"2024-06-19 15:46:54","publishedOnDateReadable":"June 19th, 2024"},"versionCreatedAt":"2024-02-14 09:32:09","video":"","vorDoi":"10.1007/s00244-024-01073-x","vorDoiUrl":"https://doi.org/10.1007/s00244-024-01073-x","workflowStages":[]},"version":"v1","identity":"rs-3951751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3951751","identity":"rs-3951751","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}