Xture was dropped on pre-cooled GelBonds (GelBondfilm, GBF, Lonza, Bend, OR, USA) and they have been left dry at four C. All samples had been dripped in two separate GBFs, one to assess oxidative DNA damage as well as the other for genotoxic damage. Right after drying, GBFs were submerged in lysis 1-Ethynylpyrene Protocol buffer (NaCl 2.five M, EDTA 0.1 M, Tris 0.01 M, NaOH 0.2 M) and incubated overnight at four C. The following day, GBFs have been washed in enzyme buffer twice (HEPES 0.04 M, KCl 0.1 M, EDTA 0.0005 M, BSA 0.two mg/mL) for 10 and 50 min. Samples have been then incubated in enzyme buffer at 37 C for 30 min, using the addition of formamidopyrimidine-DNA glycosylase (FPG) in the case from the GBFs employed for oxidative damage evaluation. Subsequently, GBFs have been submerged in electrophoresis option (NaOH 0.3M, EDTA 0.001 M) at 4 C for 35 min and subjected to electrophoresis at 20 V and 300 mA for 20 min at 4 C. Samples had been then washed twice with PBS and after with water, and GBFs had been fixed in pure ethanol for 1 h at room temperature. Ethanol was then removed and GBFs had been air-dried. To dye samples, GBFs were submerged in SYBR Gold and left in agitation for 20 min. Following that time, GBFs had been Dihydroactinidiolide supplier rinsed with MilliQ water, mounted on slides, and visualized working with an epifluorescence microscope (Olympus BX50F, Olympus Optical Co. Ltd., Tokyo, Japan). Comet counting and evaluation were carried out using the Komet 5.5 software (Kinetic Imaging, Liverpool, UK). one hundred nuclei per sample have been counted. The application supplied the percentages of DNA in comet tails for every single with the counted nuclei. Oxidative DNA harm values were calculated by subtracting the percentages of total genotoxic damage per sample in the damage measured in samples treated with FPG. two.ten. Oxidative Tension Assessment with the DCFH-DA Process Intracellular reactive oxygen species (ROS) production was evaluated right after the exposure of Caco-2 cells to PSNPs for 24 h and 8 weeks. After the exposure time, cells were incubated with 20 dichloro-dihydro-fluorescein diacetate (DCFH-DA) in serum-free DMEM for 1 h at 37 C. In both experimental approaches, positive control cells have been treated with one hundred mM H2 O2 for 1 h before incubation with DCFH-DA. Cell fluorescence was then measured at 490/530 nm employing the Victor 1420 Multilabel Counter fluorimeter (PerkinElmer, Waltham, MA, USA). For statistical evaluation, the readings for each dose have been averaged and normalized against the values for positive handle samples. two.11. Statistical Analysis All experiments were carried out in triplicates and one-way ANOVA was carried out with the information from every from the experiments described above, to analyze their statistical significance, unless stated otherwise. To this finish, GraphPad Prism 5 computer software (GraphPad Computer software, Inc., San Diego, CA, USA) was utilised. When easy, Dunnett’s various comparison test was subsequently conducted. Statistical significance was set as p 0.05, p 0.01, p 0.001. three. Final results three.1. Nanoplastic Particles Characterization The shape and size of PSNPs and y-PSNPs were assessed by TEM. As shown in Figure 1, each nanoparticles are round-shaped when diluted in distilled water or DMEM. Table 1 summarizes the outcomes obtained for the nanoparticles’ characterization. TEM sizes have been consistent with all the ones indicated by the manufacturer, at about 50 nm diameter. On the other hand, the hydrodynamic radius, measured by DLS, showed bigger particle sizes, particularly for particles diluted in DMEM. The obtained polydispersity index (PdI) values indicate differences.