nanomaterials Article The Comet Assay as a Tool to Detect the Genotoxic Potential of Nanomaterials Alba García-Rodríguez 1 , Laura Rubio 2 , Laura Vila 1 , Noel Xamena 1,3 , Antonia Velázquez 1,3 , Ricard Marcos 1,3, * and Alba Hernández 1,3, * 1 Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain; albagr.garcia@gmail.com (A.G.-R.); lauravilavecilla@hotmail.com (L.V.); noel.xamena@uab.cat (N.X.); antonia.velazquez@uab.cat (A.V.) 2 Nanobiology Laboratory, Department of Natural and Exact Sciences, Pontificia Universidad Católica Madre y Maestra, PUCMM, Santiago de los Caballeros 50000, Dominican Republic; l.rubio@ce.pucmm.edu.do 3 Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Carlos III Institute of Health, 28029 Madrid, Spain * Correspondence: ricard.marcos@uab.es (R.M.); alba.hernandez@uab.es (A.H.); Tel.: +34-93-581-2052 (R.M.); +34-93-581-8048 (A.H.); Fax: +34-93-581-2387 (R.M & A.H)  Received: 6 August 2019; Accepted: 18 September 2019; Published: 27 September 2019  Abstract: The interesting physicochemical characteristics of nanomaterials (NMs) has brought about their increasing use and, consequently, their increasing presence in the environment. As emergent contaminants, there is an urgent need for new data about their potential side-effects on human health. Among their potential effects, the potential for DNA damage is of paramount relevance. Thus, in the context of the EU project NANoREG, the establishment of common robust protocols for detecting genotoxicity of NMs became an important aim. One of the developed protocols refers to the use of the comet assay, as a tool to detect the induction of DNA strand breaks. In this study, eight different NMs—TiO2 NP (2), SiO2 NP (2), ZnONP, CeO2 NP, AgNP, and multi-walled carbon nanotubes (MWCNT)—were tested using two different human lung epithelial cell lines (A549 and BEAS-2B). The comet assay was carried out with and without the use of the formamidopyrimidine glycosylase (FPG) enzyme to detect the induction of oxidatively damaged DNA bases. As a high throughput approach, we have used GelBond films (GBF) instead of glass slides, allowing the fitting of 48 microgels on the same GBF. The results confirmed the suitability of the comet assay as a powerful tool to detect the genotoxic potential of NMs. Specifically, our results indicate that most of the selected nanomaterials showed mild to significant genotoxic effects, at least in the A549 cell line, reflecting the relevance of the cell line used to determine the genotoxic ability of a defined NM. Keywords: comet assay; FPG enzyme; TiO2 NP; SiO2 NP; ZnONP; CeO2 NP; AgNP; multi-walled carbon nanotubes (MWCNT) 1. Introduction Nanomaterials (NMs) are being increasingly used in many fields, due to their new physicochemical properties at the nanometric scale [1]. In this context, the evidence seems to indicate that the use of such NMs will continue experiencing an exponential increase. This opens a new scenario where people will certainly be exposed to such NMs. Since human health risks associated with such exposures are uncertain, new information on their potentially harmful effects is urgently required. Among the different health effects that NMs can cause, those related to their potential interaction with DNA requires special attention. Thus, the detection of the genotoxic effects induced by NMs is emerging as a specific research field covering such demands [2,3]. Since different NMs can react with Nanomaterials 2019, 9, 1385; doi:10.3390/nano9101385 www.mdpi.com/journal/nanomaterials Nanomaterials 2019, 9, 1385 2 of 20 DNA following different mechanisms, a wide range of tools have been proposed to detect and quantify different genotoxic effects; among them, the comet assay stands out. The comet assay measures the induction of DNA breaks as well as oxidatively damaged DNA bases by using single-cell gel electrophoresis [4,5]. The simplicity of the assay and its potential use in any type of eukaryotic cell has expanded its use in many fields, and this assay is successfully used in in vitro and in vivo testing, including human biomonitoring studies [6]. The comet assay has already been used to test different NMs as it has been summarized in different reviews [7–9]. Several criticisms have been raised about the use of the comet assay to test NMs arguing that their presence in the head, or in the tail of the comet, can interfere with the scoring [7]. This means that the scoring of NMs using the comet assay is under discussion. At present, there is no official guideline for using the comet assay in in vitro studies. In this frame, the EU project NANoREG has aimed to establish strong protocols to be used in the testing of NMs, mainly from a regulatory point of view. According to its simplicity, sensitivity, and speed, the comet assay was one of the proposed test protocols. To reach that objective, we here present the results obtained with the comet assay testing eight different NMs namely NM100 and NM101 (TiO2 NPs), NM110 (ZnONPs), NM200 and NM203 (SiO2 NPs), NM212 (CeO2 NPs), NM300K (AgNPs), and NM401 (MWCNT, multiwalled carbon nanotubes). Taking into account that inhalation is one of the main routes of exposure to NMs, two different human cell lines (A549 and BEAS-2B) have been used as a model of human bronchial epithelial cells. To cover one of the aims of the NANoREG project a high throughput approach was used. Thus, GelBond films (GBF) instead of glass slides were used, allowing the fitting of 48 microgels on the same GBF, in comparison with the 1–2 samples contained in a glass slide. 2. Material and Methods 2.1. Selected NMs The tested NMs were: 100 and 6 nm anatase TiO2 NPs (NM100 and NM101), 18.3 and 24.7 nm amorphous SiO2 NPs (NM200 and NM203), 147 nm ZnONPs (NM110), 33 nm CeO2 NPs (NM212), AgNPs (NM300K), and 64.2 nm MWCNT (NM401). All eight NMs were supplied by the NANoREG Consortium, and the EU Joint Research Centre at Ispra (Italy) prepared and sent aliquots to all the participants. Although these NMs were well characterized in the frame of the project, we confirmed nanoparticle size and morphology by transmission electron microscopy (TEM) using a JEOL 1400 instrument (JEOL Ltd., Tokyo, Japan). In addition, the hydrodynamic size in whole-cell culture medium was measured by using dynamic light scattering (DLS). This analysis was performed on a Malvern Zetasizer Nano-ZS zen3600 (Malvern Panalytical, Malvern, UK) instrument. 2.2. Cell Culture The transformed human bronchial epithelial BEAS-2B cell line was kindly provided by Dr. H. Karlson, from the Swedish Karolinska Institute (Stockholm, Sweden). The adenocarcinomic human alveolar basal epithelial A540 cell line was kindly provided by Dr. G. Linsel, from the Federal Institute for Occupational Safety and Health of Germany (BAuA, Berlin, Germany), BEAS-2B cells were cultured as a monolayer in 75 cm2 culture flasks coated with 0.03% collagen in serum-free bronchial epithelial cell growth medium (BEGM, Lonza, CA, USA) and passaged weekly. A549 cells were also cultured in 75 cm2 culture flasks with DMEM high glucose (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; PAA, Pasching, Austria), 1% of non-essential amino acids (NEAA; PAA, Pasching, Austria) and 2.5 µg/mL Plasmocin™ (InvivoGen, San Diego, CA, USA). Log-phase cells were grown on 12 well plates without 0.03% collagen coating, to determine, cytotoxicity and DNA damage levels. Cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦ C. We selected epithelial lung cells because inhalation is considered one of the most relevant exposure routes for humans. In addition, both BEAS-2B cells have been extensively used in the testing of nanomaterials. Nanomaterials 2019, 9, 1385 3 of 20 2.3. Cell Viability BEAS-2B and A549 cells were exposed to different concentrations of the selected NMs, according to previous toxicity data, and exposures lasting for 24 h. The highest tested dose ranged from 10 µg/mL, for the most toxic NMs (ZnONP), to 200 µg/mL for the least toxic NMs. According to the obtained toxicities, up to 4 different concentrations were selected to determine cell viability. The 4 appropriate subtoxic concentrations were chosen to work with the comet assay enabling the establishment of dose/response relationships. Untreated cells, just with cell culture medium, were used as a negative control. Toxicity was measured directly counting the number of cells surviving the 24 h exposure treatments. The procedure was as follows: after exposure, cells were washed three times with 0.5 mL of PBS (1%), to eliminate dead cells, and incubated 3 min at 37 ◦ C with 0.25 mL of trypsin-EDTA (Ethylenediaminetetraacetic acid) (1%) to detach and individualize them. After these initial steps, cells were diluted (1/10) in ISOTON™ (isotonic buffer) and counted with a ZTM Series colter-counter (Beckman Coulter Inc., Brea, CA, USA) [10]. The final viability values were determined by averaging three independent viability experiments, each containing three replicates per each concentration. 2.4. The Comet Assay The alkaline comet assay facilitates the determining of the levels of both the genotoxic (DNA breaks) and the oxidative DNA damage (ODD). The detection of ODD can be easily detected when the comet assay is complemented with the use of formamidopyrimidine DNA glycosylase (FPG) enzyme. This enzyme detects oxidized DNA bases, cut them and, consequently, transient DNA breaks are generated. The net oxidative effect is determined by subtracting the breaks induced in normal conditions from those obtained when FPG enzyme is used. The used protocol is as follows: exposed/control cells were washed with PBS 1X thrice, detached with trypsin-EDTA 1%, incubated at 37 ◦ C for 5 min, and centrifuged at 130 g for 8 min. After that, cells were resuspended in PBS 1X to obtain the concentration of 1 × 106 cells/mL. The obtained cells were mixed 1:10 with 0.75% low melting point agarose at 37 ◦ C, dropped on GelBond® films (GBF) (Life Sciences, Vilnius, Lithuania) in triplicates, and lysed in cold lysis buffer overnight at 4 ◦ C. As a high throughput approach, the use of GBF instead of glass slides allowed for the fitting of 48 microgels on the same GBF. After that, the GBF were gently washed twice with enzyme buffer for 5 and 50 min respectively at 4 ◦ C. Then, the GBF were incubated with enzyme buffer containing, or not, FPG enzyme 1:25,000 for 30 min at 37 ◦ C, followed by a washing step with electrophoresis buffer for 5 min. A second incubation in electrophoresis buffer was followed, to allow DNA unwinding and expression of alkali-labile sites, for 25 min at 4 ◦ C. Subsequently, electrophoresis was carried out at 20 V and 300 mA at 4 ◦ C. GBF were washed twice with cold PBS 1X for 5 and 10 min respectively, cells were fixed in absolute ethanol for at least 1 h and air-dried overnight at room temperature. Cells were stained with 1:10,000 SYBR Gold in TE buffer for 20 min at room temperature. GBF were mounted and visualized in an epifluorescent microscope (Olympus BX50, Hamburg, Germany) at 20× magnification. The DNA damage was quantified with the Komet 5.5 Image analysis system (Kinetic Imaging Ltd, Liverpool, UK) as the percentage of DNA in the tail. A total of 100 comet images randomly selected were analyzed per sample. Two different samples were analyzed for each condition, in each one of the three experiments performed. 2.5. Statistical Analysis All measurements were made in triplicate, at least for two separate experiments. Results are expressed in mean ± standard error. The one-way ANOVA with Tukey’s post-test and an unpaired Student’s t-test were used to compare differences between means. Data were analyzed with GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA, http://graphpad.com). Differences between means were considered significant at p < 0.05. Nanomaterials 2019, 9, 1385 4 of 20 Nanomaterials 3. Results 2019, 9, x FOR PEER REVIEW and Discussion 4 of 20 3. Results andCharacterization 3.1. Nanoparticles Discussion First of all, we wish 3.1. Nanoparticles to point out that the nanomaterials used were supplied by the UE JRC at Ispra, Characterization and are considered as reference First of all, we wish to point nanomaterials, and used in many out that the nanomaterials different used were EU projects. supplied by the UEThis JRC atmeans that they Ispra, and are considered as reference nanomaterials, and used in many different EU projects. This to have been extensively characterized. To gather sound genotoxicity data, it is necessary ensure that nanoparticles means that they have are beenwell dispersed extensively [11]. In our To characterized. case, we have gather soundused the dispersion genotoxicity data, itprotocol is generated in thetoframe necessary ensureof that the NanoGenotox nanoparticles areEU well project [12]. As[11]. dispersed observed In ourincase, Figure we1,have TEMusedimages theshow dispersion that each NMs is protocol dispersedgenerated in thedepending distinctly, frame of the onNanoGenotox EU projectFor its primary structure. [12]. As observed example, AgNPs in and Figure 1, TEM images show that each NMs is dispersed distinctly, depending TiO2 NPs (NM101) are clearly not or less agglomerated than the rest of the NPs. Then, by selecting over on its primary structure.inFor 100 particles example, random AgNPs fields and TiO of view, we2NPs (NM101) the determined are TEM clearlydiameters not or less in agglomerated dry conditions, than the and the rest of the NPs. Then, by selecting over 100 particles in random fields of view, we determined the obtained results are indicated in Table 1. The same table also shows the DLS results, that measured the TEM diameters in dry conditions, and the obtained results are indicated in Table 1. The same table average of the NPs hydrodynamic size when were dispersed and suspended in a liquid. As expected, also shows the DLS results, that measured the average of the NPs hydrodynamic size when were the average dispersed of and the hydrodynamic suspended in a sizes, liquid.once dispersed As expected, theinaverage the culture of the medium, are higher hydrodynamic sizes,than oncethose reported by TEM dispersed in the in the dried culture form, due medium, to theirthan are higher interactions with the those reported by TEMproteins. in theSince driedexposures form, due lastto for 24 h, their we aimed to detect interactions withifthetime has important proteins. effectslast Since exposures on dispersion for 24 h, weby inducing aimed to detectNMs aggregation. if time has Thus,important our results summarized effects on dispersionin Table 1 do not by inducing NMs seem to indicate aggregation. that Thus, ourthe incubation results summarizedtimeinhas a direct Table 1 do not seem to indicate that the incubation time has a direct effect effect on the NMs agglomeration. Concretely, only NM200, NM203, and NM300K showed apparent on the NMs agglomeration. Concretely, increases in the only NM200, NM203, hydrodynamic size and afterNM300K showed apparent 24 h, regarding the valuesincreases in thejust observed hydrodynamic size after the dispersion after 24 h, regarding the values observed just after the dispersion procedure procedure (0 h). It should be indicated that, due to the fibrillary characteristics of MWCNT NM401, (0 h). It should be indicated that, due to the fibrillary characteristics of MWCNT NM401, we do not report DLS values we do not report DLS values for this NM (ND). Nevertheless, we indicated their average length and for this NM (ND). Nevertheless, we indicated their average length and thickness, as evaluated by thickness, as evaluated by TEM. Although some of the values, observed mainly after 48 h, were difficult TEM. Although some of the values, observed mainly after 48 h, were difficult to interpret, these to interpret, these over-time discrepancies discrepancies over-time are not important, are not important, because because only exposures only lasting forexposures 24 h were usedlasting for 24 h in the were comet used in the assay. comet assay. NM100 (TiO2NPs) NM101 (TiO2NPs) NM110 (ZnONPs) NM200 (SiO2NPs) NM203 (SiO2NPs) NM212 (CeO2NPs) NM300K (AgNPs) NM401 (MWCNTs) NM401 (MWCNTs) Figure Figure 1. RepresentativeTEM 1. Representative TEMimages images of of the theeight eightused usednanoparticles (NPs). nanoparticles Figures (NPs). correspond Figures to correspond TiO 2NPs (NM100), TiO2NPs (NM101), ZnONPs (NM110), SiO2NPs (NM200), SiO2NPs (NM203), to TiO2 NPs (NM100), TiO2 NPs (NM101), ZnONPs (NM110), SiO2 NPs (NM200), SiO2 NPs (NM203), CeO2 NPs (NM212), AgNPs (NM300K), and MWCNT (NM401). For NM401 two images are include to visualize their length and thickness. Nanomaterials 2019, 9, 1385 5 of 20 Table 1. Comparison of pristine sizes (transmission electron microscopy, TEM) and 10 and 100 µL dispersions (dynamic light scattering, DLS). Sizes were evaluated just after the dispersion procedure and 24 h later. NM100 (TiO2 NPs) NM101 (TiO2 NPs) TEM 104.01 ± 39.42 nm TEM 54.69 ± 35.39 nm 10 µg/mL 194.6 ± 8.49 10 µg/mL 152,2 ± 62.47 0h 0h 100 µg/mL 166.2 ± 1.01 100 µg/mL 166.1 ± 12.55 DLS DLS 10 µg/mL 151.6 ± 6.30 10 µg/mL 265.1 ± 10.50 24 h 24 h 100 µg/mL 141.2 ± 2.23 100 µg/mL 141.5 ± 84.88 NM110 (ZnONPs) NM200 (SiO2 NPs) TEM 132.37 ± 69.53 nm TEM 16.5 ± 4.18 nm 10 µg/mL 260.3 ± 88.63 10 µg/mL 67.76 ± 0.10 0h 0h 100 µg/mL 213.9 ± 92.48 100 µg/mL 70.59 ± 1.43 DLS DLS 10 µg/mL 117.2 ± 37.71 10 µg/mL 75.84 ± 3.62 24 h 24 h 100 µg/mL 114.7 ± 0.86 100 µg/mL 147.8 ± 13.70 NM203 (SiO2 NPs) NM212 (CeO2 NPs) TEM 24.26 ± 9.38 nm TEM 70.33 ± 49.61 nm 10 µg/mL 69.56 ± 9.50 10 µg/mL 229.9 ± 10.13 0h 0h 100 µg/mL 86.86 ± 68.08 100 µg/mL 230.5 ± 4.05 DLS DLS 10 µg/mL 121.4 ± 10.02 10 µg/mL 117.1 ± 4.70 24 h 24 h 100 µg/mL 342 ± 76.05 100 µg/mL 124.0 ± 1.51 NM300K (AgNPs) NM401 (MWCNTs) TEM 7.75 ± 2.48 nm TEM 4.23 ± 1.01 (diameter), 6012.09 ± 4091.45 (length) 10 µg/mL 28.71 ± 17.67 10 µg/mL ND 0h 0h 100 µg/mL 38.46 ± 16.31 100 µg/mL ND DLS DLS 10 µg/mL 81.37 ± 4.63 10 µg/mL ND 24 h 24 h 100 µg/mL 97.23 ± 6.22 100 µg/mL ND 3.2. Toxic/Genotoxic Effects of NM100 and NM101 (TiO2 NPs) TiO2 NPs are one of the most produced NMs. They are included in many products, such as paints, coatings, plastics, papers, inks, medicines, pharmaceuticals, food products, cosmetics, sunscreens, and toothpaste [13]. This extensive use makes it necessary to evaluate their potential risk for human health. Toxicity data are required to determine the range of concentrations to be used in genotoxicity studies. In our case, we have not detected relevant toxic effects for the two selected TiO2 NPs (Figures 2A and 3A). Although TiO2 NPs have largely been evaluated from the genotoxic point of view, the already reported results are not sufficiently clear to reach a convincing conclusion [14]. Nevertheless, in our study, we have demonstrated that both TiO2 NPs (NM100 and NM101) are able to induce direct DNA strand breaks, as detected by the comet assay (Figures 2 and 3), although no oxidative DNA damage induction was detected on the DNA bases. Overall, we can say that A540 cells are more sensitive than BEAS-2B cells and, consequently, exposures induced higher levels of damage in that cell line. Furthermore, NM100 showed to be slightly more genotoxic than NM101. From the literature, positive DNA damage induction for both used cell lines has been reported. Nevertheless, some studies failed to detect DNA damage induction. Thus, negative results in BEAS-2B cells were obtained under different exposure scenarios [15–17]. In addition, negative findings in the comet assay were also reported in the A549 cells [18]. From the different positive reports in BEAS-2B cells, nanosized anatase and fine rutile produced a concentration and time-dependent effect in exposures lasting up to 72 h [19]. On the other hand, using the NM100 and NM101 nanomaterials (as in our study) Di Bucchiano et al. demonstrated a weak but positive induction of genotoxic effects, NM100 being the most dangerous [20]. A recent study in BEAS-2B indicated that the induced effects were associated with the shape. In this way, only those forms that were clearly internalized (food grade, P25, and platelets) were able to induce genotoxicity [21]. As in our case, A549 cells showed a higher sensitivity to TiO2 NPs and, Nanomaterials 2019, 9, x FOR PEER REVIEW 6 of 20 Nanomaterials 2019, 9, 1385 6 of 20 being the most dangerous [20]. A recent study in BEAS-2B indicated that the induced effects were associated with the shape. In this way, only those forms that were clearly internalized (food grade, P25, and platelets) consequently, wereeffects positive able towere induce genotoxicity reported for both[21]. As and small in our case, A549 spherical cells and anatase showed a higher rutile forms sensitivity after to TiOexposures short-term 2NPs and, consequently, [22], and also positive effects were after long-term reported exposures for both lasting up tosmall twoand spherical months [23]. anatase and rutile forms after short-term exposures [22], and also after long-term In addition, the positive induction of DNA damage was reported at non-toxic concentrations, but exposures lasting up to twoDNA induced months [23]. In damage addition,when decreased the positive induction exposures extendedof DNA fromdamage 3 to 24 hwas reported [24]. at non-toxic Interestingly, there concentrations, is but induced one study comparing DNA damage the effects decreased of TiO2 NPs whenand in BEAS-2B exposures extended A549 cells. from In that 3 todirect study, 24 h [24]. and Interestingly, there is one study comparing the effects of TiO oxidative DNA damage was only observed in A549 cells [16], confirming the highest sensitivity of that 2 NPs in BEAS-2B and A549 cells. In this study, cell direct and oxidative DNA damage was only observed in A549 cells [16], confirming the highest line. sensitivity of this cell line. (A) (B) (C) Figure 2. Results from the TiO22NPs (NM100) on A549 and BEAS-2B cells. (A) (A) Toxicity Toxicity curves for both cell lines. lines. (B) (B) Genotoxicity Genotoxicityresults resultsobtained obtainedininA549 A549cells. cells.(C) (C)Genotoxicity Genotoxicityresults obtained results obtainedin in BEAS-2B BEAS- cells. Genotoxicity 2B cells. Genotoxicitydata dataare areplotted plottedas mean ±± SEM as mean SEMofoftwo twoindependent independent experiments. experiments. **p ** p ≤ 0.01, ≤ 0.01, ***p *** p ≤ 0.001 (one way-ANOVA). ≤ 0.001 (one way-ANOVA). CT, control.CT, control. Nanomaterials 2019, 9, 1385 7 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 7 of 20 (A) (B) (C) Results from Figure 3. Results from the the TiO TiO22NPs NPs (NM101) (NM101) onon A549 A549 and and BEAS-2B BEAS-2B cells. cells. (A) (A)Toxicity Toxicity curves curves for both cell lines. (B) Genotoxicity Genotoxicity results resultsobtained obtainedininA549 A549cells. cells.(C) (C)Genotoxicity Genotoxicityresults obtained results obtainedin in BEAS-2B BEAS- cells. 2B Genotoxicity cells. Genotoxicitydata dataare areplotted mean±± SEM of plottedasasmean of two two independent independentexperiments. p ≤ 0.05, experiments.*p*≤ 0.05, **p ** p ≤ 0.01, *** p ≤ 0.001 (one way-ANOVA). ≤ 0.01, ***p ≤ 0.001 (one way-ANOVA). CT, control. CT, control. ZnONPs, together ZnONPs, together with with TiO TiO22NPs, NPs, are are among among thethe most most produced produced NMs.NMs. ZnONPs ZnONPs are are widely widely used used in personal in personal care care products, products, sensors, sensors, antibacterial antibacterial creams, creams, and and biomedical biomedical applications. Their broad applications. Their broad range of applications raises concerns regarding their potential health effects [25]. In our range of applications raises concerns regarding their potential health effects [25]. In our study, study, ZnONPs (NM110) (NM110) ZnONPs exerted aexerted very important toxic effect a very important in effect toxic both cell lines, in both with cell thewith lines, concentration of 10 µg/mL the concentration of 10 being completely μg/mL toxic to toxic being completely BEAS-2B cells. However, to BEAS-2B ZnONPsZnONPs cells. However, were unable wereto cause to unable direct cause DNA damage direct DNA in A549 and damage BEAS-2B in A549 cells. It should and BEAS-2B cells. Itbe indicated should that some be indicated thatgenotoxic effects were some genotoxic observed effects in A549 were observed cells when the FPG enzyme was used. Thus, significant increases in the levels in A549 cells when the FPG enzyme was used. Thus, significant increases in the levels of oxidativeof oxidative DNA damage DNA were observed damage in cellsinexposed were observed to 0.2 and cells exposed to 0.26 and µg/mL, as indicated 6 μg/mL, in Figure as indicated 4. in Figure 4. x FOR PEER REVIEW Nanomaterials 2019, 9, 1385 88 of of 20 (A) (B) (C) Figure 4. Results from the ZnONPs (NM110) on A549 and BEAS-2B cells. (A) Toxicity curves for both cell lines. lines. (B) (B) Genotoxicity Genotoxicityresults resultsobtained obtainedininA549 A549cells. cells.(C) (C)Genotoxicity Genotoxicityresults obtained results in in obtained BEAS-2B BEAS- cells. Genotoxicity 2B cells. Genotoxicitydata dataare areplotted plottedas mean ±± SEM of asmean of two twoindependent experiments.**p**≤ p0.01, independentexperiments. ≤ 0.01, *** *** p ≤ 0.001 (one way-ANOVA). CT, p ≤ 0.001 (one way-ANOVA). CT, control. control. ZnONPs ZnONPs are are easily easily taken taken up up byby cells, cells, and and quickly quickly dissolved intracellularly to dissolved intracellularly to their their ionic ionic form, form, which which can cause the strongly observed toxicity. The use of ICP-MS techniques demonstrated after can cause the strongly observed toxicity. The use of ICP-MS techniques demonstrated that, that, 48 h in culture media, more than 80% of the initial ZnONPs dissolved to their ionic after 48 h in culture media, more than 80% of the initial ZnONPs dissolved to their ionic form [26]. form [26]. In that In study, although the authors found that exposures lasting for 24 h induced significant that study, although the authors found that exposures lasting for 24 h induced significant levels of levels of DNA damage DNA damagein mouse fibroblast in mouse cells, this fibroblast DNA cells, thisdamage induction DNA damage was not was induction evident not when evidentcells were when exposed cells were long-term (up 12 weeks). Similar effects were observed in BEAS-2B cells, where exposed long-term (up 12 weeks). Similar effects were observed in BEAS-2B cells, where short-term short-term exposures lasting exposuresfor 3–6 h were lasting forable 3–6 toh induce were ableDNA to damage induce DNAin the damage absence ofinBSAthe [27]. Positive absence genotoxic of BSA effects [27]. Positive genotoxic effects were also obtained in A549 cells at short exposure times (4 h). Nevertheless, this Nanomaterials 2019, 9, 1385 9 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 20 were also obtained in A549 cells at short exposure times (4 h). Nevertheless, this genotoxicity was not associated genotoxicity with was oxidative DNA damage, not associated nor the induction with oxidative DNA damage, of intracellular Reactive Oxygen nor the induction Species of intracellular (ROS) Reactivelevels [28]. Species Oxygen More recently, ZnONPs (ROS) levels [28].were Morealso evaluated recently, in A549 ZnONPs cellsalso were affecting cell survival, evaluated and in A549 cells inducing high levels of DNA damage at cytotoxic concentrations. Lower affecting cell survival, and inducing high levels of DNA damage at cytotoxic concentrations. Lowerconcentrations also were able to induce aalso concentrations genotoxic response were able and the to induce induced response a genotoxic damage persisted over 24 h and the induced [24]. The damage disparity persisted of over reported 24 h [24].data, mainly related The disparity to the exposure of reported data, mainly times, reflects related to the the relevance exposure of the intracellular times, solubility reflects the relevance as a modulator of the observed effects. of the intracellular solubility as a modulator of the observed effects. Due to their their appealing appealing properties, SiO2NPs are extensive and increasingly used in agriculture, food, and consumer consumer products products including including cosmetics. Accordingly, large amounts cosmetics. Accordingly, amounts are placed in the global market global market and, consequently, consequently, into the environment. Many products containing SiO into the environment. Many products containing SiO22NPs are listed in a consumer consumer product inventory and placed in the third third position position among among the most most produced produced NMs worldwide worldwide[20]. [20].Although Although manymanystudies have been studies havecarried been out, to identify carried out, totheidentify toxicological mechanisms the toxicological of action of SiO NPs, no conclusive positions have been established linking mechanisms of 2 action of SiO2NPs, no conclusive positions have been established linking their their physicochemical properties to toxicity, physicochemical bioavailability, properties or human to toxicity, health effects bioavailability, [29]. health or human In a similar effects way, although [29]. potential In a similar way, biomarkers of genotoxicity have been suggested, the obtained experimental although potential biomarkers of genotoxicity have been suggested, the obtained experimental results are not conclusive enough, due results are notto conclusive a variety ofenough, factors [30]. dueOur to a findings variety ofshowed factorssimilar results [30]. Our for both findings evaluated showed SiO similar 2 NPs, results with BEAS-2B for both evaluatedbeingSiO more 2NPs,sensitive than A549 with BEAS-2B beingcells to their more toxicthan sensitive effects. A549Regarding their cells to their genotoxic toxic effects. potential, Regardingboth theirSiO 2 NPs were genotoxic able toboth potential, induce SiO2direct NPs weregenotoxicity in both able to induce cellgenotoxicity direct lines. Nevertheless, in in both cell A549 lines. cells both SiO2 NPs Nevertheless, werecells in A549 ableboth to induce SiO2NPsoxidative were ableDNA todamage induce at the concentrations oxidative DNA damage of 15 atand the 30 µg/mL (Figures concentrations of 155 and and 30 6). μg/mL (Figures 5 and 6). (A) (B) Figure 5. Cont. Nanomaterials 2019, 9, 1385 10 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 10 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 10 of 20 (C) (C) Figure Resultsfrom Figure 5. Results fromthe theSiO SiO 2 NPs 2NPs (NM200), (NM200), onon A549 A549 andand BEAS-2B BEAS-2B cells. cells. (A) Toxicity (A) Toxicity curves curves for for both Figure both 5. cell lines. Results cell lines. from the (B) Genotoxicity SiO 2NPs (NM200), on A549 and BEAS-2B cells. (A) Toxicity curves for both (B) Genotoxicity resultsresults obtained obtained in A549incells. A549 (C)cells. (C) Genotoxicity Genotoxicity results obtained results obtained in BEAS- cell in lines.Genotoxicity 2BBEAS-2B cells. (B)cells. Genotoxicity results Genotoxicity data are obtained data plotted are in A549 plotted as mean as cells. ± SEM mean (C) of two Genotoxicity ± independent SEM results obtained of two independent experiments. ≤ in BEAS- experiments. *p 0.05, ***p 2B cells. *≤p0.001 ≤ 0.05, Genotoxicity (one data are plotted p ≤ 0.001 (one way-ANOVA). ***way-ANOVA). as mean ± SEM CT, control. CT, control. of two independent experiments. *p ≤ 0.05, ***p ≤ 0.001 (one way-ANOVA). CT, control. (A) (A) (B) (B) Figure 6. Cont. Nanomaterials 2019, 9, 1385 11 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 11 of 20 (C) Figure Figure 6. Resultsfrom 6. Results fromthe theSiO SiO 2 NPs 2NPs (NM203), (NM203), onon A549 A549 andand BEAS-2B BEAS-2B cells. cells. (A) Toxicity (A) Toxicity curves curves for for both both cell lines. cell lines. (B) Genotoxicity (B) Genotoxicity resultsresults obtainedobtained in A549incells. A549 (C)cells. (C) Genotoxicity Genotoxicity results obtained results obtained in BEAS- in 2BBEAS-2B cells. Genotoxicity cells. Genotoxicity data are data are plotted plotted as mean as mean ± SEM of two± independent SEM of two independent experiments. experiments. *p ≤ 0.05, ***p *≤p0.001 ≤ 0.05, (one p ≤ 0.001 (one way-ANOVA). ***way-ANOVA). CT, control. CT, control. No No studies studies using using BEAS-2B BEAS-2B cellscells have have been been found found testing testing the the genotoxicity genotoxicityof ofSiO SiO22NPs NPs and and using using the comet assay. Nevertheless, two studies reported data using the A549 the comet assay. Nevertheless, two studies reported data using the A549 cell line. In the first study,cell line. In the first study, short short periods periods of of incubation incubation (15 (15 min min andand 44 h) h) did not induce did not induce DNADNA strand strand breaks breaks or or FPG FPG sensitive sensitive sites, sites, when testing two different when testing two different SiO SiO NPs sizes (16 and 60 nm) [31]. The second study evaluated 2 2NPs sizes (16 and 60 nm) [31]. The second study evaluated the effects the effects of non-cytotoxic concentration of non-cytotoxic concentration of SiO of SiO NPs, with an average diameter of 39.6 nm, in treatments 2 2NPs, with an average diameter of 39.6 nm, in treatments lasting lasting for 24 h. Results indicated that the exposure for 24 h. Results indicated that the exposure did not increase did the intracellular not increase levels oflevels the intracellular ROS or of the ROS primary or the DNA damage, as measured with the comet assay [32]. primary DNA damage, as measured with the comet assay [32]. Interestingly, Interestingly, SiO 2 NPs exerted a synergistic SiO2NPs exerted a action on the synergistic effects action onofthelead, amplifying effects of lead,the levels of the amplifying ROSlevels and DNA of ROS damage and DNAinduced by lead damage [32]. by induced Regarding lead [32]. CeO 2 NPs, they have widespread use in industry, cosmetic and consumer products. Its use as automobile Regarding CeO2NPs,exhaust theycatalysts draws special have widespread useattention, in industry,duecosmetic to their extensive and consumer releaseproducts. into the environment. At present, Its use as automobile therecatalysts exhaust is a growingdraws interest specialinattention, this compound, sinceextensive due to their it has been proposed release for into the biomedical use, due to the potent regenerative antioxidant properties. Although environment. At present, there is a growing interest in this compound, since it has been proposed for in vivo exposures to CeO 2 NPs biomedical can exert respiratory tract adverse effects, such as sensory irritation and use, due to the potent regenerative antioxidant properties. Although in vivo exposures to airflow limitation [33], its CeO2NPs can activity analogous to two key exert respiratory antioxidant tract enzymes, adverse effects, suchsuch as superoxide as sensory irritationdismutase and airflow andlimitation catalase, explains its role as a potent free radical scavenger. Among the proposed [33], its analogous activity to two key antioxidant enzymes, such as superoxide dismutase and health effects, the potential application in neurodegenerative catalase, explains its role as a potent pathologies free radicalstands out [34].Among the proposed health effects, the scavenger. potential application in neurodegenerative pathologiesnot Our results indicate that CeO 2 NPs (NM212) does exertout stands any[34]. toxic effect in the used cell lines. In addition, non-genotoxic effects were observed in both A549 Our results indicate that CeO2NPs (NM212) does not exert any toxic effect and BEAS-2B (Figure 7). It in the is interesting used cell lines. to note the observed reduction of oxidative DNA damage observed In addition, non-genotoxic effects were observed in both A549 and BEAS-2B (Figure 7). in treated A549 cells, which would It is support the antioxidant potential interesting to note the observed reduction of CeO NPs. 2 of oxidative DNA damage observed in treated A549 cells, which Although some authors would support have reported the antioxidant potential theofgenotoxic CeO2NPs.effects of CeO2 NPs, there is no general agreement about this point [35]. It has been proposed that the apparent discordant reported data are the result of both the pro-oxidant and anti-oxidant role of CeO2 NPs. Nevertheless, in a recent study using a wide set of cell lines, from both tumoral and non-tumoral origin, only anti-oxidant effects were observed [36]. Regarding the use of BEAS-2B and A549 cells to evaluate the genotoxic effects of CeO2 NPs, only three studies have been found in the literature using the comet assay. In BEAS-2B cells, no induction of intracellular ROS of DNA damage was observed [37], but positive induction of DNA damage was observed in A549 cells [24]. In that study, the effects were mainly observed after short exposures (3 h), while only a slight effect at the highest tested dose (42 µg/mL) was observed. Conversely, Frieke-Kuper et al. used both cell lines and demonstrated that in both cases exposures lasting for 24 h of CeO2 NPs induced significant levels of DNA damage, as evaluated using the comet (A) environment. At present, there is a growing interest in this compound, since it has been proposed for biomedical use, due to the potent regenerative antioxidant properties. Although in vivo exposures to CeO2NPs can exert respiratory tract adverse effects, such as sensory irritation and airflow limitation [33], its analogous activity to two key antioxidant enzymes, such as superoxide dismutase and catalase, explains its role as a potent free radical scavenger. Among the proposed health effects, the Nanomaterials 2019, 9, 1385 12 of 20 potential application in neurodegenerative pathologies stands out [34]. Our results indicate that CeO2NPs (NM212) does not exert any toxic effect in the used cell lines. In addition, assay non-genotoxic [38]. Nevertheless, effects the use of anwere observed in vitro in both 3D human A549epithelial bronchial and BEAS-2B (Figure 7). model indicated thatIt the is interesting mucociliarytodefense note theprevents observed CeOreduction 2 NPs of from oxidative reaching DNA the damage respiratoryobserved epithelialin treated cells [38]. A549 cells, which would support the antioxidant potential of CeO2NPs. Nanomaterials 2019, 9, x FOR PEER REVIEW 12 of 20 (A) (B) (C) Figure 7.7. Results Resultsfrom fromthetheCeO CeO2 NPs 2NPs(NM212), (NM212), onon A549 A549 andand BEAS-2B BEAS-2Bcells. (A) Toxicity cells. curves (A) Toxicity for both curves for cell bothlines. (B) Genotoxicity cell lines. results results (B) Genotoxicity obtained in A549in obtained cells. A549 (C)cells. Genotoxicity results obtained (C) Genotoxicity in BEAS-2B results obtained in cells. Genotoxicity data are plotted as mean ± SEM of two independent experiments. BEAS-2B cells. Genotoxicity data are plotted as mean ± SEM of two independent experiments. It It should be noted shouldthat there isthat be noted a significant there is a reduction significantinreduction the levelsinofthe oxidative levels ofDNA damage oxidative DNAobserved damagein observed the A549 cells. in theCT, A549control. cells. CT, control. The wide spectrum Although of applications some authors of AgNPs have reported in biomedicine, the genotoxic effects and in related of CeO fields, explain their 2NPs, there is no general extended use [39]. Due to their proved antimicrobial efficacy, AgNPs are incorporated agreement about this point [35]. It has been proposed that the apparent discordant reported into different data are the result of both the pro-oxidant and anti-oxidant role of CeO2NPs. Nevertheless, in a recent study using a wide set of cell lines, from both tumoral and non-tumoral origin, only anti-oxidant effects were observed [36]. Regarding the use of BEAS-2B and A549 cells to evaluate the genotoxic effects of CeO2NPs, only three studies have been found in the literature using the comet assay. In BEAS-2B cells, no induction of intracellular ROS of DNA damage was observed [37], but positive induction of Nanomaterials 2019, 9, 1385 13 of 20 materials such as textile fibers and wound dressings. This explains that, although the production of AgNPs is significantly lower than that of other NMs, they constitute the most popular advertised nanomaterial in the Nanotechnology Consumer Products Inventory [40]. Among the different harmful effects associated Nanomaterials 2019, 9, with x FORAgNPs exposure, it has been extensively reported that may cause genotoxicity, PEER REVIEW 13 of 20 although additional data are required to assess its carcinogenic potential [41]. study, AgNPs In our study, AgNPs induced induced significant significant cytotoxicity cytotoxicity in in both both cell cell lines, lines, BEAS-2B BEAS-2B cells cells showing showing a higher sensitivity higher sensitivity (Figure (Figure 8). 8). In addition, AgNPs exposure was also able to induce genotoxic damage in both cell lines. Nevertheless, when the FPG enzyme was used to detect the induction of oxidatively in damaged DNA, damaged DNA, onlyonly A549 cells showed showed this type of damage (Figure (Figure 8). Until now, four studies have published testing AgNPs in BEAS-2B using the comet been published comet assay assay [42–45]. [42–45]. Interestingly, completely completely different genotoxicity data were reported in the different genotoxicity data were reported in the indicated studies. indicated studies. Thus, AgNPs with a size ranging a size ranging from 20–200 from 20–200nm, nm, werewere unable unable to induce to induce DNADNA strandstrand breaksbreaks in treatments in treatments lasting for lasting forContrarily, 4 h [44]. 4 h [44]. Contrarily, positive DNA positive strandDNA breaks strand were breaks observed were observed using different using types different of AgNPs types of AgNPs as citrate coatedas (10, citrate 40 coated (10, 40 and 75 nm), PVP coated (10 nm), and uncoated (50 and 75 nm), PVP coated (10 nm), and uncoated (50 nm). Positive induction of primary DNA damage nm). Positive induction of primary DNAobserved was damage for was allobserved forms, but foronly all forms, but only after after treatments lasting treatments for 24 h. lasting for 24negative In contrast, h. In contrast, results negative results were obtained for short exposures (4 h), and no induction were obtained for short exposures (4 h), and no induction of intracellular ROS was detected, assuming of intracellular ROS was detected, that assuming the observed DNA that damage the observedwas not DNA a asdamage result of wasthenot a as result oxidative of the stress oxidative status stressresults [45]. These status [45].opposed are These results to thoseare reported opposed to bythose Nymark reported et al.,bywho Nymark et al.,DNA indicated who indicated strand breaks,DNA strand together breaks, with together with intracellular ROS induction, when BEAS-2B cells were intracellular ROS induction, when BEAS-2B cells were exposed to PVP-coated AgNPs (average diameter exposed to PVP-coated AgNPs (average 42.5 ± 14.5 diameter nm) for 42.5 ± 14.5 nm) treatments lasting for4treatments and 24 h [43].lasting The4genotoxic and 24 h potential [43]. The genotoxic of AgNPs potential using A549 of AgNPs using A549 cells has also reported variability of data, cells has also reported variability of data, with negative results using nanoparticles sized 20–200 nm with negative results using nanoparticles and exposuressizedlasting 20–200 for 4 nm andand h [44], exposures negative lasting resultsfor in4PVK-coated h [44], and negative AgNPs sized results 20in nm,PVK-coated although AgNPs small sized sizes (2020nm) nm, although induced small sizes a significant (20 nm) of induction induced DNA damagea significant induction [46]. This positive of induction DNA damage was [46]. This positive induction was also observed in cells exposed for also observed in cells exposed for 24 h to 20–50 nm diameter nanoparticles [47]. The size of AgNPs has 24 h to 20–50 nm diameter nanoparticles been considered [47].as The one size of theofcharacteristics AgNPs has been considered modulating as one of the genotoxicity. Highcharacteristics levels of DNA modulating damage genotoxicity. were observed High levels for the of DNA smaller sizeddamage AgNPs were (50 observed nm) when forthree the smaller differentsized sizesAgNPs (50, 80(50andnm)200when nm) three different sizes (50, 80 and 200 nm) were evaluated [48]. Similar were evaluated [48]. Similar results were reported when 20 and 200 nm were compared. In that case, results were reported when 20 and 200levels higher nm were of DNA compared. damage In were that case, higher observed whenlevels of DNA cells were damage exposed to 20were observed nm AgNPs, inwhen cells exposures were exposed to 20 nm AgNPs, in exposures lasting for 2 and lasting for 2 and 24 h. The use of the FPG enzyme demonstrated that most of the damage was caused24 h. The use of the FPG enzyme demonstrated by oxidation atthat DNA mostbasesof the [49].damage Finally,was caused it should beby oxidation indicated at DNA that, usingbases [49].type the same Finally, it should of AgNPs as beour in indicated that, using the study (NM300K), same type El Yamani of reported et al. AgNPs aspositive in our study induction(NM300K), of DNAEldamage, Yamaniindependently et al. reported positive of induction the exposure timeof(3 DNA damage, or 24 h), where the independently observed damage of thewas exposure mainlytime due to(3 oxidative or 24 h), damage, where the as observed damage measured combining wasthe mainly comet dueassayto oxidative with the damage, use of FPG as enzyme measured combining the comet assay with [24]. the use of FPG enzyme [24]. (A) Figure 8. Cont. Nanomaterials 2019, 9, 1385 14 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 14 of 20 (B) (C) Figure 8. Resultsfrom 8. Results fromthe theAgNPs AgNPs (NM300K), (NM300K), onon A549 A549 andand BEAS-2B BEAS-2B cells. cells. (A) Toxicity (A) Toxicity curves curves for for both both cell lines. (B) Genotoxicity results obtained in A549 cells. (C) Genotoxicity results cell lines. (B) Genotoxicity results obtained in A549 cells. (C) Genotoxicity results obtained in BEAS-obtained in BEAS-2B 2B cells. Genotoxicity cells. Genotoxicity data are data are plotted plotted as mean as mean ± SEM ± SEM of two of two independent independent experiments. experiments. *p ≤ 0.05, **p *≤ p0.01, ***p**≤ p0.001 ≤ 0.05, ≤ 0.01, (one p ≤ 0.001 (one way-ANOVA). ***way-ANOVA). CT, control. CT, control. MWCNTs are MWCNTs arefibrous fibrous materials materialsformed fromfrom formed honeycomb honeycombcrystalcrystal lattice layers latticeoflayers graphite of wrapped graphite into a multiple tube shape. They are applied in multiple fields, including in their wrapped into a multiple tube shape. They are applied in multiple fields, including in their use as use as semiconductors, solar cell mobiles,solar semiconductors, and optical instruments. cell mobiles, From the and optical toxicological instruments. point From theoftoxicological view, they arepointconsidered of view, to have carcinogenic they are considered potential, to havecausing lung tumors carcinogenic [50]. However, potential, causing the lungunderlying tumors [50]. mechanisms However, are not the well understood and, in particular, the reported genotoxicity data are conflicting underlying mechanisms are not well understood and, in particular, the reported genotoxicity data [51]. In our study, are conflicting [51].MWCNT (NM401) did not exert toxic effects on A549 cells, but toxicities around 50% wereIn observed our study,the in BEAS-2B MWCNT cell exposed (NM401) did not toexert the whole range of toxic effects ontested A549 concentrations (Figure cells, but toxicities 9A). around Regarding the ability of this NM to induce DNA damage, our results indicate 50% were observed in the BEAS-2B cell exposed to the whole range of tested concentrations (Figure that direct genotoxicity was 9A). not observed Regarding theat ability any of ofthethis tested NMconcentrations, to induce DNA in damage, any of the ourused cell lines. results indicateNevertheless, that direct oxidative DNA damage was observed in the A549 cell line, but only at the genotoxicity was not observed at any of the tested concentrations, in any of the used cell lines. lowest concentrations (15 and 30 µg/mL) (Figure 9). Using BEAS-2B cells, the literature mainly reports Nevertheless, oxidative DNA damage was observed in the A549 cell line, but only at the lowest negative results [52–54]. Although the comparison concentrations (15 and 30ofμg/mL) straight (Figure and tangled MWCNTs 9). Using demonstrated BEAS-2B cells, thethat the straight literature forms mainly were reports positive at low concentrations, tangled MWCNTs showed to be only positive negative results [52–54]. Although the comparison of straight and tangled MWCNTs demonstrated at the highest tested dose the that [55].straight The A549 cell were forms line has also been positive used at low in studies testing concentrations, the genotoxic tangled MWCNTs potential showed oftoMWCNTs be only in the comet assay, with contradictory results. Negative results were reported positive at the highest tested dose [55]. The A549 cell line has also been used in studies testing thein exposures lasting for 24–72 h [54,56], although a positive response in the comet assay without FPG genotoxic potential of MWCNTs in the comet assay, with contradictory results. Negative results were was also observed [57,58]. Nevertheless, reported both studies in exposures got negative lasting for 24–72results whenalthough h [54,56], the comet a assay wasresponse positive complemented with the in the comet use assay of the FPG enzyme. The role of modifications in the MWCNTs structure has without FPG was also observed [57,58]. Nevertheless, both studies got negative results when the been evaluated, comparing comet assay was complemented with the use of the FPG enzyme. The role of modifications in the MWCNTs structure has been evaluated, comparing the pristine versus the functionalized forms. Nanomaterials 2019, 9, 1385 15 of 20 Nanomaterials 2019, 9, x FOR PEER REVIEW 15 of 20 the pristine Positive versus effects werethereported, functionalized forms. Positive independently effects were if the pristine fromreported, independently was compared with theifOH-the pristine from was compared with the OH-functionalized form [58] or with the acid-treated functionalized form [58] or with the acid-treated forms [59]. However, when A549 cells were used to forms [59]. However, when test pristine A549carboxyl versus cells were used to test MWCNTs, pristine only versus positive carboxyl results wereMWCNTs, obtained only positive in the cometresults assay. were obtained in the comet assay. Interestingly, both MWCNTs forms gave positive Interestingly, both MWCNTs forms gave positive induction of strand breaks when BEAS-2B cellsinduction of strand breaks when were used BEAS-2B cells were used [53]. [53]. (A) (B) (C) Figure 9. Figure 9. Results Results from from the the MWCNTs MWCNTs (NM401), (NM401), onon A549 A549 and and BEAS-2B BEAS-2B cells. cells. (A) Toxicity Toxicity curves curves for for both cell both cell lines. lines. (B) (B) Genotoxicity Genotoxicityresults resultsobtained obtainedininA549 A549cells. cells.(C) (C)Genotoxicity Genotoxicity results obtained results obtainedin BEAS-2B in BEAS-2B cells. Genotoxicity cells. datadata Genotoxicity are are plotted as mean plotted ± SEM as mean of two ± SEM independent of two independentexperiments. *p ≤ experiments. p ≤ 0.05, *0.05, **p ≤** p ≤(one 0.01 0.01way-ANOVA). (one way-ANOVA). CT, control. CT, control. 4. Conclusions Nanomaterials 2019, 9, 1385 16 of 20 4.Nanomaterials Conclusions 2019, 9, x FOR PEER REVIEW 16 of 20 A summary of the results reported above is shown in Figure 10. From the reported data, we A summary of the results reported above is shown in Figure 10. From the reported data, we can can conclude that the comet assay is a sensitive tool to determine the ability of NMs to produce conclude that the comet assay is a sensitive tool to determine the ability of NMs to produce DNA DNA damage. According to the obtained results, the tested nanomaterials can be classified as weak damage. According to the obtained results, the tested nanomaterials can be classified as weak genotoxic agents and, in such cases, most of the obtained results do not follow a clear dose–response genotoxic agents and, in such cases, most of the obtained results do not follow a clear dose–response pattern. This can result from the inherent variability associated with sampling size, an important factor pattern. This can result from the inherent variability associated with sampling size, an important when the increases in the levels of genetic damage are small. Alternatively, it is feasible that the cell factor when the increases in the levels of genetic damage are small. Alternatively, it is feasible that uptake of nanomaterials is not always associated with their concentration in the culture media. At high the cell uptake of nanomaterials is not always associated with their concentration in the culture concentrations, aggregation can occur, reducing the cell uptake. Furthermore, the observed differences media. At high concentrations, aggregation can occur, reducing the cell uptake. Furthermore, the between cell lines indicate that the selection of the cell-type is an important factor when the comet observed differences between cell lines indicate that the selection of the cell-type is an important assay is used for testing NMs. In our case, we have demonstrated the highest sensitivity of the A549 factor when the comet assay is used for testing NMs. In our case, we have demonstrated the highest cell line, regarding BEAS-2B cells. This is especially true when the induction of oxidative damage on sensitivity of the A549 cell line, regarding BEAS-2B cells. This is especially true when the induction DNA bases was evaluated. Thus, the positive induction of oxidative damage in A549 cells exposed to of oxidative damage on DNA bases was evaluated. Thus, the positive induction of oxidative damage ZnONPs, SiO2 NPs, AgNPs, and MWCNT was not obtained in BEAS-2B cells. This could be due to in A549 cells exposed to ZnONPs, SiO2NPs, AgNPs, and MWCNT was not obtained in BEAS-2B cells. the important antioxidant capacity of this cell line [60]. In fact, different behaviors of both cell lines This could be due to the important antioxidant capacity of this cell line [60]. In fact, different have also been observed by other authors regarding different genetic endpoints. Thus, A549 cells were behaviors of both cell lines have also been observed by other authors regarding different genetic more sensitive when exposed to cadmium than BEAS-2B cells, the higher sensitivity of A549 cells endpoints. Thus, A549 cells were more sensitive when exposed to cadmium than BEAS-2B cells, the being associated to changes in cell type-specific gene expression patterns, including the induction of higher sensitivity of A549 cells being associated to changes in cell type-specific gene expression genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling, patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, apoptosis and DNA repair pathways [61]. Similarly, A549 cells were more sensitive when exposed to cell cycle control, mitotic signaling, apoptosis and DNA repair pathways [61]. Similarly, A549 cells TiO2 NPs than BEAS-2B cells, and this sensitivity was associated with changes in the expression of DNA were more sensitive when exposed to TiO2NPs than BEAS-2B cells, and this sensitivity was associated repair genes [62]. Finally, the use of hydrophobic plastic supports (HBF) permits an easy throughput with changes in the expression of DNA repair genes [62]. Finally, the use of hydrophobic plastic approach of the comet assay and, in addition, the use of FPG enzyme has become a powerful tool to supports (HBF) permits an easy throughput approach of the comet assay and, in addition, the use of understand the mechanism of action of the tested nanomaterials, detecting those inducing oxidative FPG enzyme has become a powerful tool to understand the mechanism of action of the tested DNA damage on the DNA bases. nanomaterials, detecting those inducing oxidative DNA damage on the DNA bases. Figure 10. Heat map summarizing the overall results of testing the eight nanomaterials in the two cell Figure 10. Heat map summarizing the overall results of testing the eight nanomaterials in the two cell lines. Toxicity, genotoxicity and oxidative damage induction are schematically represented. lines. Toxicity, genotoxicity and oxidative damage induction are schematically represented. Nanomaterials 2019, 9, 1385 17 of 20 Author Contributions: R.M. and A.H. planned the experiments. A.G.-R., L.R., L.V., N.X. and A.V. carried out the experimental part. A.G.-R., L.R. and L.V. analysed the data, carried out the statistical analysis, and prepared tables/figures. A.H. and R.M. wrote the final manuscript. Funding: This investigation was supported by the EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584). A. García-Rodríguez was funded by a postgraduate fellowship (PIF) from the Universitat Autònoma de Barcelona. L. Vila was funded by a postgraduate fellowship from the Generalitat de Catalunya. 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