Cytotoxic and Genotoxic Assessment of Zinc Oxide, Copper Oxide and Graphene Nanoparticles on A549 cell line

Cytotoxic and Genotoxic Assessment of Zinc Oxide, Copper Oxide and Graphene Nanoparticles on A549 cell line | 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 Cytotoxic and Genotoxic Assessment of Zinc Oxide, Copper Oxide and Graphene Nanoparticles on A549 cell line Borja Mercado-Casares, Carlos Fito-López, Luis Roca-Pérez, Rafael Boluda-Hernández, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4730027/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The utilization of Zinc oxide (ZnO), Copper oxide (CuO), and graphene nanoparticles has attracted substantial interest within the industrial sector, notably in the realm of inkjet printing. Consequently, the primary aim of this research was to evaluate the cytotoxicity and genotoxicity of these nanoparticles using the MTT assay and the Comet assay on the A459 cell line. In this context, it is necessary to assess the environmental and human health implications of these novel materials, with the intention of categorizing them as emerging contaminants if it was necessary. The characterization of ZnO, CuO, and graphene nanoparticles revealed particle sizes in the range of 10–70 nm for the metal oxides, and multi-layer graphene platelets with lateral size < 0.6 µm. Our findings demonstrated a concentration-dependent relationship between increasing nanoparticle concentration and both cytotoxicity and DNA damage. Specifically, MTT assay results indicated a higher level of toxicity associated with ZnO nanoparticles, whereas genotoxicity was more pronounced with CuO nanoparticles. Furthermore, all nanoparticles exhibited lower EC50 values in the Comet assay. In summary, the current study unveils the cytotoxic and genotoxic effects of ZnO, CuO, and graphene nanoparticles on the A549 cell line. Genotoxicity cytotoxicity nanomaterial comet assay graphene ZnO CuO Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The increasingly common use of nanoparticles (NPs) in the industry has generated the need for studies that provide more information about their environmental impact. Nanotechnology can be defined as: the manipulation, precision, placement, measurement, modelling, or manufacture of sub-100 nanometer scale matter (Meyer et al. 2001 ). These particles exhibit different properties compared to the same materials on a macro or micrometric scale. When a material reduces its size to below 100 nm, its characteristics can be altered (Bondarenko et al. 2013 ). Among these nanoparticles there are copper oxide (CuO), zinc oxide (ZnO) and graphene. The main goal of this study is to evaluate if there are effects of cytotoxicity and genotoxicity of the mentioned molecules. The selected molecules have been used in the industry in several ways and during the last years tones of them have been produced. Therefore, it is crucial to rule out potential damage caused by these molecules The exploration of potential hazardous effects associated with CuO NPs remains limited in comparison to other nanoparticle counterparts, likely due to their relatively lower usage quantities compared with other NPs (Kahru and Savolainen 2010 ). It is reasonable to infer that the production volume of CuO NPs is high but also comparatively modest. The predominant and distinct application niche for CuO NPs revolves around electronics and technology, encompassing semiconductors, electronic chips, and heat transfer nanofluids, owing to the exceptional thermophysical properties of CuO (Ebrahimnia-Bajestan et al. 2011 ; Bondarenko et al. 2013 ). Furthermore, CuO NPs have been proposed for diverse applications, including gas sensors (Li et al. 2008 ), catalytic processes (Carnes and Klabunde 2003 ), solar cells, and lithium batteries (Guo et al. 2009 ; Sau et al. 2010 ). Research indicates that CuO NPs exhibit inhibitory effects on microorganism growth and demonstrate antiviral properties (Borkow and Gabbay 2004 ). Consequently, the integration of CuO NPs into face masks, wound dressings, and socks has been explored to confer biocidal properties (Borkow et al. 2009 ). The ZnO, the chemical formula for zinc oxide, is an inorganic substance presented in the form of a white powder that is insoluble in water (Ahmad et al. 2020 ). This versatile material finds application in various industries, including paints, adhesives, plastics, sealants, pigments, food, ointments, batteries, ferrites, and fire retardants. While zincite mineral in the Earth's crust contains ZnO, the majority used in commercial applications is synthesized. Zinc and oxygen correspond to the second and sixth groups in the periodic table. In materials science, ZnO is commonly referred to as a II-VI semiconductor. With unique optical characteristics, it possesses a large bandgap of 3.3 eV in the ultraviolet spectrum, exhibits high binding energy at room temperature, and demonstrates high electrical conductivity of n-type (Al Jabri et al. 2022 ). It is well known that Graphene has found extensive applications in various nanobiotechnological fields, including environmental applications, biomedicine, and biotechnology (Bitounis et al. 2013 ). The scientific literature on graphene has witnessed a significant surge since its discovery in 2004, with the number of papers surpassing 8,500 in 2012 according to a topic search on the ISI Web of Science. In comparison to other carbon materials, graphene-based systems, though relatively young in development, exhibit substantial potential for numerous biomedical applications. Notably, the evaluation of the in vitro and in vivo toxicity of graphenes in recent studies has yielded conflicting results, with both toxic and non-toxic effects observed simultaneously. This divergence highlights the necessity of avoiding broad generalizations, as the safety risks associated with graphenes are contingent upon the specific type of material under analysis. It is imperative to recognize that conclusive assessments must be approached cautiously, considering the nuanced nature of graphene's impact (Sreeprasad and Pradeep 2012 ; Seabra et al. 2014 ). The scientific community currently faces a substantial gap in understanding the potential adverse effects of NPs, which significantly lags behind the advancements in nanotechnology development (Shvedova et al. 2010 ; Kahru and Ivask 2013 ). Moreover, the available data on nanotoxicity lack consistency due to variations in experimental approaches across different articles, hindering the comparability of results. To tackle these issues, there is an ongoing discourse within the nanotoxicology community regarding the need for comprehensive guidelines in nanotoxicology research and the establishment of common parameters to be addressed in all nanotoxicological articles (Schrurs and Lison, 2012 ). For all these reasons, this article will contribute new information on the toxicity of CuO, ZnO, and Graphene nanoparticles. It is well accepted that further studies are needed to extend our toxicological knowledge on the newly developed nanoparticles or nanoenabled products. Accordingly, we aimed to investigate in vitro toxicology of various NPs, which may have catalytic or biological applications. Nanoparticles possess a heightened ability to traverse through an organism via inhalation compared to larger particles, potentially exhibiting increased toxicity due to their expanded surface area and distinct structural/chemical properties. Gradoń et al. ( 2000 ) established a correlation between the presence of nanoparticles in workplace air, inhaled by individuals, and instances of acute morbidity and even mortality in the elderly. Furthermore, NPs, following inhalation exposure, have been documented to travel through the nasal nerves to reach the brain (Kreuter et al. 2002 ; Oberdörster et al. 2005 ). In a separate investigation, it was revealed that inhaled ultrafine 99mtechnetium-labeled carbon particles rapidly diffused into the systemic circulation, peaking between 10 and 20 minutes and maintaining this level for up to 60 minutes (Nemmar et al.). In addition reports indicate that NPs present in food can traverse gut lymphatics and redistribute to other organs more readily than larger particles (Jani et al. 1990 ; Hillery et al. 1994 ). In essence, NPs may be deposited into human cells through nasal inhalation or digestion pathways, exerting effects on various organs and tissues. Another study emphasized that the observed biological effects were influenced by the size and composition of NPs (Wottrich et al. 2004 ). Materials and Methods Stock solutions of 400 mg L -1 for each NP and Graphene were made in cell culture medium free of fetal bovine serum (FBS) and sonicated at 37 °C for 1 h in an Elmasonic® S30h (Elma™, Singen, Germany) ultrasonic bath. The stocks were kept at 4 °C and before each use were sonicate at 40°C and then diluted to the desired working concentration. Cell culture A549 cells (ATCC - American Type Culture Collection: CL-185) are originated from a human pulmonary adenocarcinoma. Because of their phenotypic resemblance to type II alveolar epithelial cells, this cell line has been extensively employed in studies related to the function of alveolar epithelium. The adenocarcinomic human alveolar basal epithelial A549 cells (passage 10) from own stock were subcultured as a monolayer in T-75 flasks with Dulbecco’s Modified Eagle’s Medium high glucose (DMEM GlutaMAX™, Gibco, Gaithersburg, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA), 100 units/mL of penicillin and 100 µg/mL of streptomycin, 1% of Fungizone (Life Technologies, Carlsbad, USA) and 2.5 g/mL Plasmocin™ (InvivoGen, San Diego, USA). Cells were maintained in a humidified atmosphere containing 5% CO 2 and 95% air at 37°C. Log-phase cells were seeded at 2.5 × 10 5 cells/well in polystyrene 6-well plate (genotoxicity evaluation) or 2.0 × 10 4 cells/well in 96-well plate (cytotoxicity determination), incubated for 24 h, resulting in approximately 75% confluence prior to dosing with NPs. Particles The particle size characterization was assessed using a Hitachi S-4800 scanning electron microscope (SEM), TECNAI G2 F20 of FEI high resolution transmission electron microscope (HRTEM). Stability and dispersion state were explored using Zeta potential and Dynamic light scattering, using a NanoZS (Malvern) device. The structures were determined by X ray diffraction. The SEM and HRTEM analyses confirmed that the CuO nanoparticles predominantly exhibited a spherical morphology, with occasional observations of slightly more cubic shapes. The primary diameter of the CuO nanoparticles ranged from 40 to 60 nm. In contrast, the ZnO nanoparticles were identified as hexagonal nanocrystals with varying sizes falling within the range of 10 to 70 nm. Examination of graphene through SEM indicated predominantly multi-layer graphene platelets, with a typical lateral size distribution ranging from 0.5 to 1 µm (Fig. 1 ). All three molecules were purchased from different companies: ZnO from Tec Star S.r:l. (Bomporto, Italy), and CuO nanoparticles from Plasmachem GmbH (Adlershof, Germany) and graphene from Thomas Swan and Co. Ltd (Consett, UK). Table 1 Characteristics of the studied particles. NPs Size Shape Purity (%) ZnO 10 ~ 70 nm Hexagonal-Zincite > 99 CuO 40 ~ 60 nm Spherical > 99 Graphene 0.5 ~ 1µm 2D > 95 MTT Assay The cytotoxicity assessment of nanoparticles (NPs) was conducted using the MTT assay, following the method outlined (Cree, 2011 ). Initially, 2 10 4 cells/well were seeded in 96-well plates and incubated for 24 hours. Subsequently, the cells were exposed to varying concentrations of NPs (200 − 10 µg/mL) during 24 hours. After discarding the exposure medium containing NPs, each well was filled with 110 µL of MTT solution (100 µL PBS: 10 µL of 5 mg/mL of MTT) and incubated at 37 °C for 3 hours. Following incubation, the MTT solution was aspirated, and cell lysis was initiated by adding 100 µL of a mixture of isopropanol: 37% HCl (99:1 v/v) to each well. The absorbance of the resulting formazan violet was measured using a multiwell plate spectrophotometer (MultiskanGo™, Thermo Fisher Scientific, Finland) at 570 nm (background value) and 690 nm. Comet assay The comet assay, initially developed Östling and Johanson (1984), assesses DNA strand breaks under neutral conditions. Singh et al. ( 1988 ) introduced an alkaline version (pH > 13) that detects frank single-strand breaks (SSB), SSBs related to incomplete excision repair, and alkali-labile sites (ALSs) (Singh et al. 1988 ). Comet assay was performed according to the protocol described by Singh et al. ( 1988 ) with some modifications. 25 µl of cell suspension (~ 1x10 6 cells/mL) was added to vial containing 225 µL of liquified low-melting agarose and mixed by pipetting. Slides of 7 x 8 cm of GelBond® (GB) film (FMC Bioproducts Inc) were cut and 7 µL (in triplicate) of each cells mix were dropped in the GB and placed in fridge at 4°C until solidification. GB slides were placed in cold freshly prepared lysis solution [2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.2, to which 1% Triton X-100 supplemented with 10% dimethyl sulfoxide (DMSO)] overnight. After that, GB were allocated in a single cell gel electrophoresis tank (Cleaver Scientific™) at 4°C. The tank was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) at 4°C for 30 min to allow DNA unwinding. After that, the electrophoresis was performed at 20 V (0.75 V/cm) and 300 mA for 20 min. The steps previously described were all performed in subdued light to minimize induction of DNA damage. After electrophoresis, GB were neutralized with 0.4 M Tris (pH 7.5) twice and rinsed with Milli-Q water, fixed in absolute ethanol (2x) one hour at room temperature and air dried at this point the GB can be stored (light protected) until stained with SYBR Gold®. The levels of DNA damage were evaluated in randomly selected cells under fluorescence microscopy (100 cells per concentration in each NP were checked) by means of the ImageJ plugin OpenComet, an open-source software tool providing automated analysis of comet assay images (Gyori et al, 2014). According to comet-tail DNA (%) the damage scale developed by Mitchelmore and Chipman ( 1998 ) was used. Five-classes: from 1, no or minimal damage (10%); 2, low damage (10–20%); 3, mid damage (20–40%); 4, high damage (40–80%); 5, extreme damage (> 80%). A common artificial culture medium for primary and diploid cultures is used for cell testing: DMEM (Dulbecco modified Eagle culture medium), which contains, among others, a high concentration of amino acids, vitamins, ferric nitrate, sodium pyruvate and supplemental amino acids. This culture medium is used both supplemented with bovine fetal serum, and without supplementation, depending on the phase of the trial being carried out. Statistical analyses Statistical analyses were executed using the SPSS™ v22 (IBM) statistical package. To identify significant differences among treatments, a one-way ANOVA test and Dunnett’s Multiple Comparison, followed by a post hoc test, were employed (p values < 0.05) all of them presented as means ± standard deviation. The IC 20,50 values, representing the concentration causing 20 and 50% inhibition of viability in the MTT assay compared or the to the control, or the EC 10,50 values, representing the effective concentration causing 10% and 50% of cell damage in the Comet assay were determined using probit regression (Litchfield and Wilcoxon, 1949 ). An ANOVA statistical test (p < 0.05) was applied to assess damages in water samples relative to the negative control. Results and Discussion Scientists, building upon prior research on the inhalation of particulate matter and its association with respiratory diseases, have directed their attention to engineered nanoparticles and their analogous effects. Over the last decade, investigations have explored the potential of NPs, inhaled under diverse conditions, to induce oxidative stress in the respiratory system, triggering inflammation and initiating lung carcinogenesis. Recent experimental data offer limited evidence linking NPs to respiratory diseases and lung cancer in humans. Despite the longstanding production of manufactured microparticles and ultrafine particles, epidemiological evidence supporting an increased cancer risk from human exposure to these particles is scarce. Toxicologists have explored mechanistic pathways, drawing insights from conventional particle toxicology, indicating that harmful nanoparticles generally exhibit similar cellular effects as coarse particles. Scientists emphasize the heterogeneous nature of NPs and their potential to cause damage to the respiratory system, revealing their capability to increase the formation of reactive oxygen/nitrogen species oxidants (ROS/RNS), and induce oxidative stress, inflammation, and the initiation (and promotion) of carcinogenic mechanisms and/or genotoxicity (Valavanidis et al. 2013 ). Our study underscores the genotoxic and cytotoxic effects of ZnO, CuO, and graphene nanoparticles on lung cells. This emphasizes the imperative to further investigate these molecules to enhance the precision of understanding their impact on human health. As mentioned earlier two distinct methodologies were employed, the MTT assay and the comet assay, to comprehensively assess the cytotoxic effects and DNA damage induced by the experimental exposure to NPs. The MTT assay enabled the quantification of cell viability, providing insights into the potential cytotoxicity of the tested compounds. The results of this assay were represented in Fig. 2 were direct and proportional association between increasing NPs concentrations and the inhibition of cell viability was showed. As NP concentrations rose, a corresponding decline in cellular survival became evident. This fact suggests a dose-dependent impact of NPs on cellular health. Upon closer examination of the individual nanoparticles, it became apparent that ZnO exhibited the highest toxicity followed by CuO and graphene, respectively. This ranking highlights the varying degrees of harm induced by different NP types, with ZnO emerging as the most potent in compromising cell viability among the tested compounds. Interestingly, at higher concentrations, CuO demonstrated a more pronounced effect on cells compared to ZnO and graphene. Moreover, at lower doses, ZnO exhibited heightened toxicity, except at the 12.5 mg \(\:\bullet\:\) l -1 concentration where CuO surpassed its impact. Notably, across all concentrations studied, graphene consistently exhibited lower toxicity compared to the other two compounds. Our results underscore a direct relationship between NP concentration and cellular response, with ZnO displaying the highest toxicity overall. This is well supported by other works were Zn NPs had significant cytotoxic effect toward A549 cells (Park et al. 2007 ). Furthermore, the concentration-dependent variations in toxicity profiles emphasize the importance of considering both NP type and concentration in assessing their impact on cellular viability. While this study refrains from venturing into the classification of toxicity levels, it can be asserted that toxicity is indeed present within the three molecules under examination and according to Bondarenko et al. ( 2013 ), it can be confirmed that for pulmonary cells, characterized as mammalian cells, both ZnO and CuO as well as graphene, induce any damage at concentrations from 6.25 to 100 mg∙l -1 . Consequently, their non-classified status requires an update within the authors' review for ZnO and CuO ( see Table 2 . Table 2 Results of MTT probit regression for IC 50 and IC 20 and confidence limits (C.L. 95%) after 24 h of exposure. NP 24 h-IC 50 95%C.L. 24 h-IC 20 95%C.L. CuO 35.9 11.7–80.9 7 15-17.7 ZnO 21.5 18.3–24.9 3.9 2.7–5.3 Graphene 44.1 39.3–49.5 13.9 11.4–16.3 As we mentioned above, it is important to highlight that according to other authors studied particles showed different degrees of toxicity, in this case for lung cells (Renwick 2004 ). Simultaneously, the comet assay, with its ability to reveal DNA strand breaks under neutral and alkaline conditions, allowed for a detailed evaluation of genotoxic effects (Fig. 3 ). The comet assay is a simple and reliable test especially useful for nanomaterial testing due to its robustness and it is the most used assay in nanogenotoxicology studies (Magdolenova et al. 2014 ). Alkaline conditions provide heightened sensitivity for investigating alkali-labile DNA lesions. Over recent decades, the alkaline comet assay has become the preferred method, particularly for detecting low levels of DNA damage in human lymphocyte samples and in vitro and in vivo genotoxicity testing (Karlsson 2010 ). Our assay results demonstrated genotoxic effects within the studied concentration range (0.31, 0.62, 1.25, 2.5, and 5 mg∙l -1 ), revealing again a dose-dependent relationship in the observed damage. Notably, CuO exhibited the most substantial impact in this context, contrasting with the MTT analysis where ZnO demonstrated the highest efficacy. The genotoxic damage induced by the CuO molecule surpassed that of ZnO, with graphene registering the least impact, consistent with the MTT findings. Statistical confirmation of these differences was obtained through analysis of variance, revealing significant distinctions from the control at a 95% confidence level (Fig. 4 ). Comparing both analyses, cytotoxic effects were predominantly observed at higher concentrations, while genotoxic effects manifested at lower concentrations. This aligns with findings from studies such as El Yamani et al. ( 2017 ), suggesting the continued exploration of genotoxic effects at concentrations below the cytotoxic threshold. Considering the EC 50 during the first 24 h of exposition for the MTT and Comet assay, which are supported by the concentration range also registered for TiO 2 , CeO 2 and Ag NPs (El Yamani et al. 2017 ), we can suggest different degrees of toxicity toward de human cell. This could be related with the different size of surface interaction, Renwick et al. (2004) reported that NPs with large surface area could induce more inflammatory reactions. There is some evidence in vitro that NPs can penetrate cellular membranes and gain access to the genetic material of the nucleus (Renwick 2004 ; El Yamani et al. 2017 ). But however, some authors do not reveal penetration into de cell of metal particles and thus, it is suspected that extracellular generation of ROS could be one of the reasons for the toxicity. Another hypothesis posits that, during mitosis when the nuclear membrane dissolves, larger nanoparticles (15–60 nm) gain access to the DNA (Magdolenova et al. 2014 ). In our study the particle size of the molecules was between 10–100 nm and thus a percentage of them could reach nuclear DNA as Hackenberg et al. ( 2011 ) reported for ZnO. Supporting our results, Park et al. in 2007 confirm that Zn NPs could induce variable extents of cellular toxicity in a dose-dependent manner and at least, the toxic effect might be caused by generation of ROS (Park et al. 2007 ). It is important to highlight that derived from ROS, the apoptosis could be the main cellular dead mechanism. Among the different pathways, mitochondrial apoptosis could have an important role in this process derived from the effect of NPs (Kumaran et al. 2015 ). Our results reported effects for both cytotoxic and genotoxic levels. However, we should notice that concentrations for genotoxicity studies should be realistic to possible exposures. It is recommended for comet assay that concentrations 60%-80%viability, since DNA breaks can be secondary effects of cytotoxicity an so could give false positives (Ogris and Oupicky, 2013 ). Thus, following the recommendations and in order to reinforce our analysis we have done in conjunction with the comet assay, cytotoxicity assessment utilizing the same cell lines within the same experimental setting (Cowie et al. 2015 ). Table 3 Results of comet assay probit regression to calculate EC50 NP 24h-IC50 95%CI CuO 1.28 1.09–1.47 ZnO 2.16 1.86–2.49 Graphene 3.14 2.64–3.82 Conclusions The cytotoxicity and genotoxicity assays served as complementary assessments, revealing deleterious effects on A549 lung cells, in contrast to controls, for the three substances investigated: ZnO, CuO, and graphene. This study enabled the determination of effective concentrations causing high DNA damage (genotoxicity) in cells exposed to NPs for 24 hours (24 h-EC 50 ), as well as safety concentrations (24 h-EC 10 ) that do not induce damage or induce minimal damage in exposed cells. This allows for the establishment of thresholds or safety limits for exposure to these substances. The observed toxicity in both assays ranked ZnO as the most cytotoxic, followed by CuO, and graphene as the least. However, in the genotoxicity assay, CuO emerged as the most detrimental, followed by ZnO, and graphene as the least. Thus, it is established that particles ranging from 10 to 90 nm of the three studied substances induce cellular and genetic damage at concentrations starting from 0.62 mg∙l − 1 . Consequently, ongoing analysis of the damage inflicted by nanoparticles of different molecules in various tissues is recommended, to assess potential variability across different organs in humans. Declarations Conflict of Interests The authors have no relevant financial or non-financial interests to disclose. Funding Interreg SUDOE NanoDESK SOE1/P1/E02015 Author Contribution OA-S and CF-L: Conceptualization and Methodology. OA-S, BM-C and LR-P: Material preparation, data collection, analysis were performed and data curation. BM-C: Writing—Original Draft . CF-L: Project administration, Funding acquisition. BM-C, OA-S, LR-P, RB-H, CF-L: Review, Editing and Approved the final manuscript. Acknowledgement Authors thank Nuria Ruiz Costa for her assistance in the laboratory work References Ahmad P, Alyemeni MN, Al-Huqail AA, Alqahtani MA, Wijaya L, Ashraf M, Kaya C, Bajguz A (2020) Zinc Oxide Nanoparticles Application Alleviates Arsenic (As) Toxicity in Soybean Plants by Restricting the Uptake of as and Modulating Key Biochemical Attributes, Antioxidant Enzymes, Ascorbate-Glutathione Cycle and Glyoxalase System. Plants 9(7):825. https://doi.org/10.3390/plants9070825 Al Jabri H, Saleem MH, Rizwan M, Hussain I, Usman K, Alsafran M (2022) Zinc Oxide Nanoparticles and Their Biosynthesis: Overview. Life 12(4):594. https://doi.org/10.3390/life12040594 Bitounis D, Ali-Boucetta H, Hong BH, Min D, Kostarelos K (2013) Prospects and Challenges of Graphene in Biomedical Applications. Advanced Materials 25(16):2258–2268. https://doi.org/10.1002/adma.201203700 Bondarenko O, Juganson K, Ivask A, Kasemets K, Mortimer M, Kahru A (2013) Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review. Arch Toxicol 87(7):1181–1200. https://doi.org/10.1007/s00204-013-1079-4 Borkow G, Gabbay J (2004) Putting copper into action: copper-impregnated products with potent biocidal activities. FASEB j 18(14):1728–1730. https://doi.org/10.1096/fj.04-2029fje Borkow G, Zatcoff RC, Gabbay J (2009) Reducing the risk of skin pathologies in diabetics by using copper impregnated socks. Medical Hypotheses 73(6):883–886. https://doi.org/10.1016/j.mehy.2009.02.050 Carnes CL, Klabunde KJ (2003) The catalytic methanol synthesis over nanoparticle metal oxide catalysts. Journal of Molecular Catalysis A: Chemical 194(1–2):227–236. https://doi.org/10.1016/S1381-1169(02)00525-3 Cowie H, Magdolenova Z, Saunders M, Drlickova M, Correia Carreira S, Halamoda Kenzaoi B, Gombau L, Guadagnini R, Lorenzo Y, Walker L, Fjellsbø LM, Huk A, Rinna A, Tran L, Volkovova K, Boland S, Juillerat-Jeanneret L, Marano F, Collins AR, Dusinska M (2015) Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles. Nanotoxicology 9(sup1):57–65. https://doi.org/10.3109/17435390.2014.940407 Cree IA (ed) (2011) Cancer Cell Culture: Methods and Protocols. Humana Press, Totowa, NJ Ebrahimnia-Bajestan E, Niazmand H, Duangthongsuk W, Wongwises S (2011) Numerical investigation of effective parameters in convective heat transfer of nanofluids flowing under a laminar flow regime. International Journal of Heat and Mass Transfer 54(19–20):4376–4388. https://doi.org/10.1016/j.ijheatmasstransfer.2011.05.006 El Yamani N, Collins AR, Rundén-Pran E, Fjellsbø LM, Shaposhnikov S, Zielonddiny S, Dusinska M (2017) In vitro genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment. MUTAGE 32(1):117–126. https://doi.org/10.1093/mutage/gew060 Gradoń L, Orlicki D, Podgórski A (2000) Deposition and Retention of Ultrafine Aerosol Particles in the Human Respiratory System. Normal and Pathological Cases. International Journal of Occupational Safety and Ergonomics 6(2):189–207. https://doi.org/10.1080/10803548.2000.11076451 Guo Z, Ng HW, Yee GaryL, Hahn HT (2009) Differential Scanning Calorimetry Investigation on Vinyl Ester Resin Curing Process for Polymer Nanocomposite Fabrication. j nanosci nanotechnol 9(5):3278–3285. https://doi.org/10.1166/jnn.2009.VC06 Hackenberg S, Zimmermann F, Scherzed A, Friehs G, Froelich K, Ginzkey C, Koehler C, Burghartz M, Hagen R, Kleinsasser N (2011) Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures. Environ and Mol Mutagen 52(7):582–589. https://doi.org/10.1002/em.20661 Hillery A, Jani P, Florence A (1994) Comparative, Quantitative Study of Lymphoid and Non-Lymphoid Uptake of 60 nm Polystyrene Particles. Journal of Drug Targeting 2(2):151–156. https://doi.org/10.3109/10611869409015904 Jani P, Halbert GW, Langridge J, Florence AT (1990) Nanoparticle Uptake by the Rat Gastrointestinal Mucosa: Quantitation and Particle Size Dependency. Journal of Pharmacy and Pharmacology 42(12):821–826. https://doi.org/10.1111/j.2042-7158.1990.tb07033.x Kahru A, Ivask A (2013) Mapping the Dawn of Nanoecotoxicological Research. Acc Chem Res 46(3):823–833. https://doi.org/10.1021/ar3000212 Kahru A, Savolainen K (2010) Potential hazard of nanoparticles: From properties to biological and environmental effects. Toxicology 269(2–3):89–91. https://doi.org/10.1016/j.tox.2010.02.012 Karlsson HL (2010) The comet assay in nanotoxicology research. Anal Bioanal Chem 398(2):651–666. https://doi.org/10.1007/s00216-010-3977-0 Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, Alyautdin R (2002) Apolipoprotein-mediated Transport of Nanoparticle-bound Drugs Across the Blood-Brain Barrier. Journal of Drug Targeting 10(4):317–325. https://doi.org/10.1080/10611860290031877 Kumaran R, Choi Y-K, Singh V, Song H-J, Song K-G, Kim K, Kim H (2015) In Vitro Cytotoxic Evaluation of MgO Nanoparticles and Their Effect on the Expression of ROS Genes. IJMS 16(12):7551–7564. https://doi.org/10.3390/ijms16047551 Li Y, Liang J, Tao Z, Chen J (2008) CuO particles and plates: Synthesis and gas-sensor application. Materials Research Bulletin 43(8–9):2380–2385. https://doi.org/10.1016/j.materresbull.2007.07.045 Litchfield JR JT, Wilcoxon F (1949) A SIMPLIFIED METHOD OF EVALUATING DOSE-EFFECT EXPERIMENTS. The Journal of pharmacology and experimental therapeutics 96(2):99–113 Magdolenova Z, Collins A, Kumar A, Dhawan A, Stone V, Dusinska M (2014) Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles. Nanotoxicology 8(3):233–278. https://doi.org/10.3109/17435390.2013.773464 Meyer M, Persson O, Power Y (2001) Mapping excellence in nanotechnologies:Preparatory study (Nanotechnology expert group and Eurotech data). European Comission Mitchelmore CL, Chipman JK (1998) DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutation Research/Fundamental and molecular mechanisms of mutagenesis 399(2):135–147 Nemmar A, Hoet PHM, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, Vanbilloen H, Mortelmans L, Nemery B Passage of Inhaled Particles Into the Blood Circulation in Humans Oberdörster G, Oberdörster E, Oberdörster J (2005) Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environ Health Perspect 113(7):823–839. https://doi.org/10.1289/ehp.7339 Ogris M, Oupicky D (eds) (2013) Nanotechnology for Nucleic Acid Delivery: Methods and Protocols. Humana Press, Totowa, NJ Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammal cells. Biochemical and biophysical communications 123(1):291–298 Park S, Lee YK, Jung M, Kim KH, Chung N, Ahn E-K, Lim Y, Lee K-H (2007) Cellular Toxicity of Various Inhalable Metal Nanoparticles on Human Alveolar Epithelial Cells. Inhalation Toxicology 19(sup1):59–65. https://doi.org/10.1080/08958370701493282 Renwick LC (2004) Increased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types. Occupational and Environmental Medicine 61(5):442–447. https://doi.org/10.1136/oem.2003.008227 Sau TK, Rogach AL, Jäckel F, Klar TA, Feldmann J (2010) Properties and Applications of Colloidal Nonspherical Noble Metal Nanoparticles. Advanced Materials 22(16):1805–1825. https://doi.org/10.1002/adma.200902557 Schrurs F, Lison D (2012) Focusing the research efforts. Nature Nanotech 7(9):546–548. https://doi.org/10.1038/nnano.2012.148 Seabra AB, Paula AJ, De Lima R, Alves OL, Durán N (2014) Nanotoxicity of Graphene and Graphene Oxide. Chem Res Toxicol 27(2):159–168. https://doi.org/10.1021/tx400385x Shvedova AA, Kagan VE, Fadeel B (2010) Close Encounters of the Small Kind: Adverse Effects of Man-Made Materials Interfacing with the Nano-Cosmos of Biological Systems. Annu Rev Pharmacol Toxicol 50(1):63–88. https://doi.org/10.1146/annurev.pharmtox.010909.105819 Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175(1):184–191. https://doi.org/10.1016/0014-4827(88)90265-0 Sreeprasad TS, Pradeep T (2012) GRAPHENE FOR ENVIRONMENTAL AND BIOLOGICAL APPLICATIONS. Int J Mod Phys B 26(21):1242001. https://doi.org/10.1142/S0217979212420015 Valavanidis A, Vlachogianni T, Fiotakis K, Loridas S, Perdicaris S (2013) Potential toxicity and safety evaluation of nanomaterials for the respiratory system and lung cancer. LCTT:71. https://doi.org/10.2147/LCTT.S23216 Wottrich R, Diabaté S, Krug HF (2004) Biological effects of ultrafine model particles in human macrophages and epithelial cells in mono- and co-culture. International Journal of Hygiene and Environmental Health 207(4):353–361. https://doi.org/10.1078/1438-4639-00300 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4730027","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329724785,"identity":"19b1baab-65b6-4c39-8c68-b93da419c623","order_by":0,"name":"Borja Mercado-Casares","email":"","orcid":"","institution":"European University of València","correspondingAuthor":false,"prefix":"","firstName":"Borja","middleName":"","lastName":"Mercado-Casares","suffix":""},{"id":329724786,"identity":"5dfb1619-0181-47fe-8b86-8e12003461ab","order_by":1,"name":"Carlos Fito-López","email":"","orcid":"","institution":"ITENE, Technological Institute of Packaging, Transport and Logistics","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Fito-López","suffix":""},{"id":329724787,"identity":"83f12211-55bc-4334-bdac-7479ed8a67f0","order_by":2,"name":"Luis Roca-Pérez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACPgbGBgiLvYFILWxwLTwHiNYCAxIJxGqRSG7+wLjHRp5/5hszCYaKOmK0JLZJMDxLM5xxOweo5cxh4rQwMBw4nMBwO8fYgLHtAFFamj+AtMjfPAPU8o84hzVIgLQY3OAxfMDYwEyEFp6HbRIJB9IMN55JK3yQcIwIv/Czpz/+8OGAjbzc8cMbDnyoIcJhYJCAwRgFo2AUjIJRQBkAAEEaNJkk36FSAAAAAElFTkSuQmCC","orcid":"","institution":"University of Valencia","correspondingAuthor":true,"prefix":"","firstName":"Luis","middleName":"","lastName":"Roca-Pérez","suffix":""},{"id":329724788,"identity":"f6d4886f-7f95-4267-9a0a-ba7ab7bcfdab","order_by":3,"name":"Rafael Boluda-Hernández","email":"","orcid":"","institution":"University of Valencia","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Boluda-Hernández","suffix":""},{"id":329724789,"identity":"7848b0e4-3d16-4291-a3c6-8d6ee44f857a","order_by":4,"name":"Oscar Andreu-Sánchez","email":"","orcid":"","institution":"University of Valencia","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Andreu-Sánchez","suffix":""}],"badges":[],"createdAt":"2024-07-12 11:45:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4730027/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4730027/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61935170,"identity":"9f82163e-13ad-47d0-ad21-1061d14aefe6","added_by":"auto","created_at":"2024-08-07 09:05:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78443,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscope images (a) ZnO (b) CuO (c) Graphene.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4730027/v1/fb5319c27a83e54e8391ca8e.png"},{"id":61935168,"identity":"c1852951-8330-44c7-b177-089cd5bba923","added_by":"auto","created_at":"2024-08-07 09:05:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6651,"visible":true,"origin":"","legend":"\u003cp\u003eDoses response evaluated by de MTT assay for the three NP’s.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4730027/v1/cfe50bcffd5ba00e1d03b88a.png"},{"id":61935169,"identity":"d103d10f-fe2b-41c1-80be-ab42212df1ad","added_by":"auto","created_at":"2024-08-07 09:05:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11702,"visible":true,"origin":"","legend":"\u003cp\u003eImages from the comet assay results with three levels of classification (a) level 1 with no damage. (b) level 3 some damage detected and (c) level 5 total cell damage.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4730027/v1/2a1318207bb77b8af2bec142.png"},{"id":61935171,"identity":"32a13f30-332f-49a2-9dca-dd5fe82dfbec","added_by":"auto","created_at":"2024-08-07 09:05:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4586,"visible":true,"origin":"","legend":"\u003cp\u003eComet assay results at different concentrations for the tested molecules. Asterisks highlights differences with the blank control.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4730027/v1/1609d43ddb06f54ba55df7c0.png"},{"id":62408685,"identity":"c8376e14-c12b-4b8c-84bd-3ded028b20ae","added_by":"auto","created_at":"2024-08-13 22:46:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":526364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4730027/v1/0eb9224e-4498-49c8-aefd-dbc2428cd5bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic and Genotoxic Assessment of Zinc Oxide, Copper Oxide and Graphene Nanoparticles on A549 cell line","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe increasingly common use of nanoparticles (NPs) in the industry has generated the need for studies that provide more information about their environmental impact. Nanotechnology can be defined as: the manipulation, precision, placement, measurement, modelling, or manufacture of sub-100 nanometer scale matter (Meyer et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). These particles exhibit different properties compared to the same materials on a macro or micrometric scale. When a material reduces its size to below 100 nm, its characteristics can be altered (Bondarenko et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong these nanoparticles there are copper oxide (CuO), zinc oxide (ZnO) and graphene. The main goal of this study is to evaluate if there are effects of cytotoxicity and genotoxicity of the mentioned molecules. The selected molecules have been used in the industry in several ways and during the last years tones of them have been produced. Therefore, it is crucial to rule out potential damage caused by these molecules\u003c/p\u003e \u003cp\u003eThe exploration of potential hazardous effects associated with CuO NPs remains limited in comparison to other nanoparticle counterparts, likely due to their relatively lower usage quantities compared with other NPs (Kahru and Savolainen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is reasonable to infer that the production volume of CuO NPs is high but also comparatively modest. The predominant and distinct application niche for CuO NPs revolves around electronics and technology, encompassing semiconductors, electronic chips, and heat transfer nanofluids, owing to the exceptional thermophysical properties of CuO (Ebrahimnia-Bajestan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bondarenko et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, CuO NPs have been proposed for diverse applications, including gas sensors (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), catalytic processes (Carnes and Klabunde \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), solar cells, and lithium batteries (Guo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sau et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Research indicates that CuO NPs exhibit inhibitory effects on microorganism growth and demonstrate antiviral properties (Borkow and Gabbay \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Consequently, the integration of CuO NPs into face masks, wound dressings, and socks has been explored to confer biocidal properties (Borkow et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ZnO, the chemical formula for zinc oxide, is an inorganic substance presented in the form of a white powder that is insoluble in water (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This versatile material finds application in various industries, including paints, adhesives, plastics, sealants, pigments, food, ointments, batteries, ferrites, and fire retardants. While zincite mineral in the Earth's crust contains ZnO, the majority used in commercial applications is synthesized. Zinc and oxygen correspond to the second and sixth groups in the periodic table. In materials science, ZnO is commonly referred to as a II-VI semiconductor. With unique optical characteristics, it possesses a large bandgap of 3.3 eV in the ultraviolet spectrum, exhibits high binding energy at room temperature, and demonstrates high electrical conductivity of n-type (Al Jabri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well known that Graphene has found extensive applications in various nanobiotechnological fields, including environmental applications, biomedicine, and biotechnology (Bitounis et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The scientific literature on graphene has witnessed a significant surge since its discovery in 2004, with the number of papers surpassing 8,500 in 2012 according to a topic search on the ISI Web of Science. In comparison to other carbon materials, graphene-based systems, though relatively young in development, exhibit substantial potential for numerous biomedical applications. Notably, the evaluation of the in vitro and in vivo toxicity of graphenes in recent studies has yielded conflicting results, with both toxic and non-toxic effects observed simultaneously. This divergence highlights the necessity of avoiding broad generalizations, as the safety risks associated with graphenes are contingent upon the specific type of material under analysis. It is imperative to recognize that conclusive assessments must be approached cautiously, considering the nuanced nature of graphene's impact (Sreeprasad and Pradeep \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Seabra et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe scientific community currently faces a substantial gap in understanding the potential adverse effects of NPs, which significantly lags behind the advancements in nanotechnology development (Shvedova et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kahru and Ivask \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Moreover, the available data on nanotoxicity lack consistency due to variations in experimental approaches across different articles, hindering the comparability of results. To tackle these issues, there is an ongoing discourse within the nanotoxicology community regarding the need for comprehensive guidelines in nanotoxicology research and the establishment of common parameters to be addressed in all nanotoxicological articles (Schrurs and Lison, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For all these reasons, this article will contribute new information on the toxicity of CuO, ZnO, and Graphene nanoparticles.\u003c/p\u003e \u003cp\u003eIt is well accepted that further studies are needed to extend our toxicological knowledge on the newly developed nanoparticles or nanoenabled products. Accordingly, we aimed to investigate in vitro toxicology of various NPs, which may have catalytic or biological applications. Nanoparticles possess a heightened ability to traverse through an organism via inhalation compared to larger particles, potentially exhibiting increased toxicity due to their expanded surface area and distinct structural/chemical properties. Gradoń et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) established a correlation between the presence of nanoparticles in workplace air, inhaled by individuals, and instances of acute morbidity and even mortality in the elderly.\u003c/p\u003e \u003cp\u003eFurthermore, NPs, following inhalation exposure, have been documented to travel through the nasal nerves to reach the brain (Kreuter et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Oberd\u0026ouml;rster et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In a separate investigation, it was revealed that inhaled ultrafine 99mtechnetium-labeled carbon particles rapidly diffused into the systemic circulation, peaking between 10 and 20 minutes and maintaining this level for up to 60 minutes (Nemmar et al.). In addition reports indicate that NPs present in food can traverse gut lymphatics and redistribute to other organs more readily than larger particles (Jani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Hillery et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). In essence, NPs may be deposited into human cells through nasal inhalation or digestion pathways, exerting effects on various organs and tissues. Another study emphasized that the observed biological effects were influenced by the size and composition of NPs (Wottrich et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStock solutions of 400 mg L\u003csup\u003e-1\u003c/sup\u003e for each NP and Graphene were made in cell culture medium free of fetal bovine serum (FBS) and sonicated at 37 \u0026deg;C for 1 h in an Elmasonic\u0026reg; S30h (Elma\u0026trade;, Singen, Germany) ultrasonic bath. The stocks were kept at 4 \u0026deg;C and before each use were sonicate at 40\u0026deg;C and then diluted to the desired working concentration.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA549 cells (ATCC - American Type Culture Collection: CL-185) are originated from a human pulmonary adenocarcinoma. Because of their phenotypic resemblance to type II alveolar epithelial cells, this cell line has been extensively employed in studies related to the function of alveolar epithelium. The adenocarcinomic human alveolar basal epithelial A549 cells (passage 10) from own stock were subcultured as a monolayer in T-75 flasks with Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium high glucose (DMEM GlutaMAX\u0026trade;, Gibco, Gaithersburg, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA), 100 units/mL of penicillin and 100 \u0026micro;g/mL of streptomycin, 1% of Fungizone (Life Technologies, Carlsbad, USA) and 2.5 g/mL Plasmocin\u0026trade; (InvivoGen, San Diego, USA). Cells were maintained in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air at 37\u0026deg;C. Log-phase cells were seeded at 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in polystyrene 6-well plate (genotoxicity evaluation) or 2.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plate (cytotoxicity determination), incubated for 24 h, resulting in approximately 75% confluence prior to dosing with NPs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eParticles\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe particle size characterization was assessed using a Hitachi S-4800 scanning electron microscope (SEM), TECNAI G2 F20 of FEI high resolution transmission electron microscope (HRTEM). Stability and dispersion state were explored using Zeta potential and Dynamic light scattering, using a NanoZS (Malvern) device. The structures were determined by X ray diffraction.\u003c/p\u003e \u003cp\u003eThe SEM and HRTEM analyses confirmed that the CuO nanoparticles predominantly exhibited a spherical morphology, with occasional observations of slightly more cubic shapes. The primary diameter of the CuO nanoparticles ranged from 40 to 60 nm. In contrast, the ZnO nanoparticles were identified as hexagonal nanocrystals with varying sizes falling within the range of 10 to 70 nm. Examination of graphene through SEM indicated predominantly multi-layer graphene platelets, with a typical lateral size distribution ranging from 0.5 to 1 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll three molecules were purchased from different companies: ZnO from Tec Star S.r:l. (Bomporto, Italy), and CuO nanoparticles from Plasmachem GmbH (Adlershof, Germany) and graphene from Thomas Swan and Co. Ltd (Consett, UK).\u003c/p\u003e \u003c/div\u003e \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\u003eCharacteristics of the studied particles.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eShape\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePurity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZnO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u0026thinsp;~\u0026thinsp;70 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHexagonal-Zincite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u0026thinsp;~\u0026thinsp;60 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpherical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGraphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u0026thinsp;~\u0026thinsp;1\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95\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=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMTT Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cytotoxicity assessment of nanoparticles (NPs) was conducted using the MTT assay, following the method outlined (Cree, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Initially, 2 10\u003csup\u003e4\u003c/sup\u003e cells/well were seeded in 96-well plates and incubated for 24 hours. Subsequently, the cells were exposed to varying concentrations of NPs (200\u0026thinsp;\u0026minus;\u0026thinsp;10 \u0026micro;g/mL) during 24 hours. After discarding the exposure medium containing NPs, each well was filled with 110 \u0026micro;L of MTT solution (100 \u0026micro;L PBS: 10 \u0026micro;L of 5 mg/mL of MTT) and incubated at 37 \u0026deg;C for 3 hours. Following incubation, the MTT solution was aspirated, and cell lysis was initiated by adding 100 \u0026micro;L of a mixture of isopropanol: 37% HCl (99:1 v/v) to each well. The absorbance of the resulting formazan violet was measured using a multiwell plate spectrophotometer (MultiskanGo\u0026trade;, Thermo Fisher Scientific, Finland) at 570 nm (background value) and 690 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eComet assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe comet assay, initially developed \u0026Ouml;stling and Johanson (1984), assesses DNA strand breaks under neutral conditions. Singh et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) introduced an alkaline version (pH\u0026thinsp;\u0026gt;\u0026thinsp;13) that detects frank single-strand breaks (SSB), SSBs related to incomplete excision repair, and alkali-labile sites (ALSs) (Singh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Comet assay was performed according to the protocol described by Singh et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) with some modifications. 25 \u0026micro;l of cell suspension (~\u0026thinsp;1x10\u003csup\u003e6\u003c/sup\u003e cells/mL) was added to vial containing 225 \u0026micro;L of liquified low-melting agarose and mixed by pipetting. Slides of 7 x 8 cm of GelBond\u0026reg; (GB) film (FMC Bioproducts Inc) were cut and 7 \u0026micro;L (in triplicate) of each cells mix were dropped in the GB and placed in fridge at 4\u0026deg;C until solidification. GB slides were placed in cold freshly prepared lysis solution [2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.2, to which 1% Triton X-100 supplemented with 10% dimethyl sulfoxide (DMSO)] overnight. After that, GB were allocated in a single cell gel electrophoresis tank (Cleaver Scientific\u0026trade;) at 4\u0026deg;C. The tank was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH\u0026thinsp;\u0026gt;\u0026thinsp;13) at 4\u0026deg;C for 30 min to allow DNA unwinding. After that, the electrophoresis was performed at 20 V (0.75 V/cm) and 300 mA for 20 min. The steps previously described were all performed in subdued light to minimize induction of DNA damage. After electrophoresis, GB were neutralized with 0.4 M Tris (pH 7.5) twice and rinsed with Milli-Q water, fixed in absolute ethanol (2x) one hour at room temperature and air dried at this point the GB can be stored (light protected) until stained with SYBR Gold\u0026reg;. The levels of DNA damage were evaluated in randomly selected cells under fluorescence microscopy (100 cells per concentration in each NP were checked) by means of the ImageJ plugin OpenComet, an open-source software tool providing automated analysis of comet assay images (Gyori et al, 2014). According to comet-tail DNA (%) the damage scale developed by Mitchelmore and Chipman (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) was used. Five-classes: from 1, no or minimal damage (10%); 2, low damage (10\u0026ndash;20%); 3, mid damage (20\u0026ndash;40%); 4, high damage (40\u0026ndash;80%); 5, extreme damage (\u0026gt;\u0026thinsp;80%).\u003c/p\u003e \u003cp\u003eA common artificial culture medium for primary and diploid cultures is used for cell testing: DMEM (Dulbecco modified Eagle culture medium), which contains, among others, a high concentration of amino acids, vitamins, ferric nitrate, sodium pyruvate and supplemental amino acids. This culture medium is used both supplemented with bovine fetal serum, and without supplementation, depending on the phase of the trial being carried out.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStatistical analyses were executed using the SPSS\u0026trade; v22 (IBM) statistical package. To identify significant differences among treatments, a one-way ANOVA test and Dunnett\u0026rsquo;s Multiple Comparison, followed by a \u003cem\u003epost hoc\u003c/em\u003e test, were employed (p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05) all of them presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The IC\u003csub\u003e20,50\u003c/sub\u003e values, representing the concentration causing 20 and 50% inhibition of viability in the MTT assay compared or the to the control, or the EC\u003csub\u003e10,50\u003c/sub\u003e values, representing the effective concentration causing 10% and 50% of cell damage in the Comet assay were determined using probit regression (Litchfield and Wilcoxon, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1949\u003c/span\u003e). An ANOVA statistical test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was applied to assess damages in water samples relative to the negative control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eScientists, building upon prior research on the inhalation of particulate matter and its association with respiratory diseases, have directed their attention to engineered nanoparticles and their analogous effects. Over the last decade, investigations have explored the potential of NPs, inhaled under diverse conditions, to induce oxidative stress in the respiratory system, triggering inflammation and initiating lung carcinogenesis. Recent experimental data offer limited evidence linking NPs to respiratory diseases and lung cancer in humans. Despite the longstanding production of manufactured microparticles and ultrafine particles, epidemiological evidence supporting an increased cancer risk from human exposure to these particles is scarce. Toxicologists have explored mechanistic pathways, drawing insights from conventional particle toxicology, indicating that harmful nanoparticles generally exhibit similar cellular effects as coarse particles. Scientists emphasize the heterogeneous nature of NPs and their potential to cause damage to the respiratory system, revealing their capability to increase the formation of reactive oxygen/nitrogen species oxidants (ROS/RNS), and induce oxidative stress, inflammation, and the initiation (and promotion) of carcinogenic mechanisms and/or genotoxicity (Valavanidis et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our study underscores the genotoxic and cytotoxic effects of ZnO, CuO, and graphene nanoparticles on lung cells. This emphasizes the imperative to further investigate these molecules to enhance the precision of understanding their impact on human health.\u003c/p\u003e \u003cp\u003eAs mentioned earlier two distinct methodologies were employed, the MTT assay and the comet assay, to comprehensively assess the cytotoxic effects and DNA damage induced by the experimental exposure to NPs. The MTT assay enabled the quantification of cell viability, providing insights into the potential cytotoxicity of the tested compounds.\u003c/p\u003e \u003cp\u003eThe results of this assay were represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e were direct and proportional association between increasing NPs concentrations and the inhibition of cell viability was showed. As NP concentrations rose, a corresponding decline in cellular survival became evident. This fact suggests a dose-dependent impact of NPs on cellular health. Upon closer examination of the individual nanoparticles, it became apparent that ZnO exhibited the highest toxicity followed by CuO and graphene, respectively. This ranking highlights the varying degrees of harm induced by different NP types, with ZnO emerging as the most potent in compromising cell viability among the tested compounds. Interestingly, at higher concentrations, CuO demonstrated a more pronounced effect on cells compared to ZnO and graphene. Moreover, at lower doses, ZnO exhibited heightened toxicity, except at the 12.5 mg\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\bullet\\:\\)\u003c/span\u003e\u003c/span\u003el\u003csup\u003e-1\u003c/sup\u003e concentration where CuO surpassed its impact. Notably, across all concentrations studied, graphene consistently exhibited lower toxicity compared to the other two compounds. Our results underscore a direct relationship between NP concentration and cellular response, with ZnO displaying the highest toxicity overall. This is well supported by other works were Zn NPs had significant cytotoxic effect toward A549 cells (Park et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Furthermore, the concentration-dependent variations in toxicity profiles emphasize the importance of considering both NP type and concentration in assessing their impact on cellular viability.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhile this study refrains from venturing into the classification of toxicity levels, it can be asserted that toxicity is indeed present within the three molecules under examination and according to Bondarenko et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), it can be confirmed that for pulmonary cells, characterized as mammalian cells, both ZnO and CuO as well as graphene, induce any damage at concentrations from 6.25 to 100 mg∙l\u003csup\u003e-1\u003c/sup\u003e. Consequently, their non-classified status requires an update within the authors' review for ZnO and CuO ( see Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of MTT probit regression for IC\u003csub\u003e50\u003c/sub\u003e and IC\u003csub\u003e20\u003c/sub\u003e and confidence limits (C.L. 95%) after 24 h of exposure.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 h-IC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95%C.L.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 h-IC\u003csub\u003e20\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95%C.L.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.7\u0026ndash;80.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15-17.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZnO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.3\u0026ndash;24.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.7\u0026ndash;5.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGraphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.3\u0026ndash;49.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.4\u0026ndash;16.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs we mentioned above, it is important to highlight that according to other authors studied particles showed different degrees of toxicity, in this case for lung cells (Renwick \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Simultaneously, the comet assay, with its ability to reveal DNA strand breaks under neutral and alkaline conditions, allowed for a detailed evaluation of genotoxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The comet assay is a simple and reliable test especially useful for nanomaterial testing due to its robustness and it is the most used assay in nanogenotoxicology studies (Magdolenova et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Alkaline conditions provide heightened sensitivity for investigating alkali-labile DNA lesions. Over recent decades, the alkaline comet assay has become the preferred method, particularly for detecting low levels of DNA damage in human lymphocyte samples and \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e genotoxicity testing (Karlsson \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our assay results demonstrated genotoxic effects within the studied concentration range (0.31, 0.62, 1.25, 2.5, and 5 mg∙l\u003csup\u003e-1\u003c/sup\u003e), revealing again a dose-dependent relationship in the observed damage. Notably, CuO exhibited the most substantial impact in this context, contrasting with the MTT analysis where ZnO demonstrated the highest efficacy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe genotoxic damage induced by the CuO molecule surpassed that of ZnO, with graphene registering the least impact, consistent with the MTT findings. Statistical confirmation of these differences was obtained through analysis of variance, revealing significant distinctions from the control at a 95% confidence level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Comparing both analyses, cytotoxic effects were predominantly observed at higher concentrations, while genotoxic effects manifested at lower concentrations. This aligns with findings from studies such as El Yamani et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), suggesting the continued exploration of genotoxic effects at concentrations below the cytotoxic threshold.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConsidering the EC\u003csub\u003e50\u003c/sub\u003e during the first 24 h of exposition for the MTT and Comet assay, which are supported by the concentration range also registered for TiO\u003csub\u003e2\u003c/sub\u003e, CeO\u003csub\u003e2\u003c/sub\u003e and Ag NPs (El Yamani et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), we can suggest different degrees of toxicity toward de human cell. This could be related with the different size of surface interaction, Renwick et al. (2004) reported that NPs with large surface area could induce more inflammatory reactions. There is some evidence in vitro that NPs can penetrate cellular membranes and gain access to the genetic material of the nucleus (Renwick \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; El Yamani et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). But however, some authors do not reveal penetration into de cell of metal particles and thus, it is suspected that extracellular generation of ROS could be one of the reasons for the toxicity. Another hypothesis posits that, during mitosis when the nuclear membrane dissolves, larger nanoparticles (15\u0026ndash;60 nm) gain access to the DNA (Magdolenova et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In our study the particle size of the molecules was between 10\u0026ndash;100 nm and thus a percentage of them could reach nuclear DNA as Hackenberg et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) reported for ZnO. Supporting our results, Park et al. in 2007 confirm that Zn NPs could induce variable extents of cellular toxicity in a dose-dependent manner and at least, the toxic effect might be caused by generation of ROS (Park et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It is important to highlight that derived from ROS, the apoptosis could be the main cellular dead mechanism. Among the different pathways, mitochondrial apoptosis could have an important role in this process derived from the effect of NPs (Kumaran et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results reported effects for both cytotoxic and genotoxic levels. However, we should notice that concentrations for genotoxicity studies should be realistic to possible exposures. It is recommended for comet assay that concentrations 60%-80%viability, since DNA breaks can be secondary effects of cytotoxicity an so could give false positives (Ogris and Oupicky, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, following the recommendations and in order to reinforce our analysis we have done in conjunction with the comet assay, cytotoxicity assessment utilizing the same cell lines within the same experimental setting (Cowie et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of comet assay probit regression to calculate EC50\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24h-IC50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95%CI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.09\u0026ndash;1.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZnO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.86\u0026ndash;2.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGraphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.64\u0026ndash;3.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe cytotoxicity and genotoxicity assays served as complementary assessments, revealing deleterious effects on A549 lung cells, in contrast to controls, for the three substances investigated: ZnO, CuO, and graphene. This study enabled the determination of effective concentrations causing high DNA damage (genotoxicity) in cells exposed to NPs for 24 hours (24 h-EC\u003csub\u003e50\u003c/sub\u003e), as well as safety concentrations (24 h-EC\u003csub\u003e10\u003c/sub\u003e) that do not induce damage or induce minimal damage in exposed cells. This allows for the establishment of thresholds or safety limits for exposure to these substances. The observed toxicity in both assays ranked ZnO as the most cytotoxic, followed by CuO, and graphene as the least. However, in the genotoxicity assay, CuO emerged as the most detrimental, followed by ZnO, and graphene as the least. Thus, it is established that particles ranging from 10 to 90 nm of the three studied substances induce cellular and genetic damage at concentrations starting from 0.62 mg∙l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Consequently, ongoing analysis of the damage inflicted by nanoparticles of different molecules in various tissues is recommended, to assess potential variability across different organs in humans.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eInterreg SUDOE NanoDESK SOE1/P1/E02015\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOA-S and CF-L: Conceptualization and Methodology. OA-S, BM-C and LR-P: Material preparation, data collection, analysis were performed and data curation. BM-C: Writing\u0026mdash;Original Draft . CF-L: Project administration, Funding acquisition. BM-C, OA-S, LR-P, RB-H, CF-L: Review, Editing and Approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors thank Nuria Ruiz Costa for her assistance in the laboratory work\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad P, Alyemeni MN, Al-Huqail AA, Alqahtani MA, Wijaya L, Ashraf M, Kaya C, Bajguz A (2020) Zinc Oxide Nanoparticles Application Alleviates Arsenic (As) Toxicity in Soybean Plants by Restricting the Uptake of as and Modulating Key Biochemical Attributes, Antioxidant Enzymes, Ascorbate-Glutathione Cycle and Glyoxalase System. Plants 9(7):825. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants9070825\u003c/span\u003e\u003cspan address=\"10.3390/plants9070825\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Jabri H, Saleem MH, Rizwan M, Hussain I, Usman K, Alsafran M (2022) Zinc Oxide Nanoparticles and Their Biosynthesis: Overview. Life 12(4):594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/life12040594\u003c/span\u003e\u003cspan address=\"10.3390/life12040594\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBitounis D, Ali-Boucetta H, Hong BH, Min D, Kostarelos K (2013) Prospects and Challenges of Graphene in Biomedical Applications. Advanced Materials 25(16):2258\u0026ndash;2268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.201203700\u003c/span\u003e\u003cspan address=\"10.1002/adma.201203700\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBondarenko O, Juganson K, Ivask A, Kasemets K, Mortimer M, Kahru A (2013) Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review. Arch Toxicol 87(7):1181\u0026ndash;1200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00204-013-1079-4\u003c/span\u003e\u003cspan address=\"10.1007/s00204-013-1079-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorkow G, Gabbay J (2004) Putting copper into action: copper-impregnated products with potent biocidal activities. FASEB j 18(14):1728\u0026ndash;1730. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1096/fj.04-2029fje\u003c/span\u003e\u003cspan address=\"10.1096/fj.04-2029fje\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorkow G, Zatcoff RC, Gabbay J (2009) Reducing the risk of skin pathologies in diabetics by using copper impregnated socks. Medical Hypotheses 73(6):883\u0026ndash;886. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mehy.2009.02.050\u003c/span\u003e\u003cspan address=\"10.1016/j.mehy.2009.02.050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarnes CL, Klabunde KJ (2003) The catalytic methanol synthesis over nanoparticle metal oxide catalysts. Journal of Molecular Catalysis A: Chemical 194(1\u0026ndash;2):227\u0026ndash;236. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1381-1169(02)00525-3\u003c/span\u003e\u003cspan address=\"10.1016/S1381-1169(02)00525-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCowie H, Magdolenova Z, Saunders M, Drlickova M, Correia Carreira S, Halamoda Kenzaoi B, Gombau L, Guadagnini R, Lorenzo Y, Walker L, Fjellsb\u0026oslash; LM, Huk A, Rinna A, Tran L, Volkovova K, Boland S, Juillerat-Jeanneret L, Marano F, Collins AR, Dusinska M (2015) Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles. Nanotoxicology 9(sup1):57\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/17435390.2014.940407\u003c/span\u003e\u003cspan address=\"10.3109/17435390.2014.940407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCree IA (ed) (2011) Cancer Cell Culture: Methods and Protocols. Humana Press, Totowa, NJ\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahimnia-Bajestan E, Niazmand H, Duangthongsuk W, Wongwises S (2011) Numerical investigation of effective parameters in convective heat transfer of nanofluids flowing under a laminar flow regime. International Journal of Heat and Mass Transfer 54(19\u0026ndash;20):4376\u0026ndash;4388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijheatmasstransfer.2011.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ijheatmasstransfer.2011.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl Yamani N, Collins AR, Rund\u0026eacute;n-Pran E, Fjellsb\u0026oslash; LM, Shaposhnikov S, Zielonddiny S, Dusinska M (2017) \u003cem\u003eIn vitro\u003c/em\u003e genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment. MUTAGE 32(1):117\u0026ndash;126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/mutage/gew060\u003c/span\u003e\u003cspan address=\"10.1093/mutage/gew060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGradoń L, Orlicki D, Podg\u0026oacute;rski A (2000) Deposition and Retention of Ultrafine Aerosol Particles in the Human Respiratory System. Normal and Pathological Cases. International Journal of Occupational Safety and Ergonomics 6(2):189\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10803548.2000.11076451\u003c/span\u003e\u003cspan address=\"10.1080/10803548.2000.11076451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Z, Ng HW, Yee GaryL, Hahn HT (2009) Differential Scanning Calorimetry Investigation on Vinyl Ester Resin Curing Process for Polymer Nanocomposite Fabrication. j nanosci nanotechnol 9(5):3278\u0026ndash;3285. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1166/jnn.2009.VC06\u003c/span\u003e\u003cspan address=\"10.1166/jnn.2009.VC06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHackenberg S, Zimmermann F, Scherzed A, Friehs G, Froelich K, Ginzkey C, Koehler C, Burghartz M, Hagen R, Kleinsasser N (2011) Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures. Environ and Mol Mutagen 52(7):582\u0026ndash;589. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/em.20661\u003c/span\u003e\u003cspan address=\"10.1002/em.20661\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHillery A, Jani P, Florence A (1994) Comparative, Quantitative Study of Lymphoid and Non-Lymphoid Uptake of 60 nm Polystyrene Particles. Journal of Drug Targeting 2(2):151\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/10611869409015904\u003c/span\u003e\u003cspan address=\"10.3109/10611869409015904\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJani P, Halbert GW, Langridge J, Florence AT (1990) Nanoparticle Uptake by the Rat Gastrointestinal Mucosa: Quantitation and Particle Size Dependency. Journal of Pharmacy and Pharmacology 42(12):821\u0026ndash;826. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.2042-7158.1990.tb07033.x\u003c/span\u003e\u003cspan address=\"10.1111/j.2042-7158.1990.tb07033.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKahru A, Ivask A (2013) Mapping the Dawn of Nanoecotoxicological Research. Acc Chem Res 46(3):823\u0026ndash;833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ar3000212\u003c/span\u003e\u003cspan address=\"10.1021/ar3000212\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKahru A, Savolainen K (2010) Potential hazard of nanoparticles: From properties to biological and environmental effects. Toxicology 269(2\u0026ndash;3):89\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tox.2010.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.tox.2010.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlsson HL (2010) The comet assay in nanotoxicology research. Anal Bioanal Chem 398(2):651\u0026ndash;666. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-010-3977-0\u003c/span\u003e\u003cspan address=\"10.1007/s00216-010-3977-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, Alyautdin R (2002) Apolipoprotein-mediated Transport of Nanoparticle-bound Drugs Across the Blood-Brain Barrier. Journal of Drug Targeting 10(4):317\u0026ndash;325. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10611860290031877\u003c/span\u003e\u003cspan address=\"10.1080/10611860290031877\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumaran R, Choi Y-K, Singh V, Song H-J, Song K-G, Kim K, Kim H (2015) In Vitro Cytotoxic Evaluation of MgO Nanoparticles and Their Effect on the Expression of ROS Genes. IJMS 16(12):7551\u0026ndash;7564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms16047551\u003c/span\u003e\u003cspan address=\"10.3390/ijms16047551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Liang J, Tao Z, Chen J (2008) CuO particles and plates: Synthesis and gas-sensor application. Materials Research Bulletin 43(8\u0026ndash;9):2380\u0026ndash;2385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.materresbull.2007.07.045\u003c/span\u003e\u003cspan address=\"10.1016/j.materresbull.2007.07.045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLitchfield JR JT, Wilcoxon F (1949) A SIMPLIFIED METHOD OF EVALUATING DOSE-EFFECT EXPERIMENTS. The Journal of pharmacology and experimental therapeutics 96(2):99\u0026ndash;113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagdolenova Z, Collins A, Kumar A, Dhawan A, Stone V, Dusinska M (2014) Mechanisms of genotoxicity. A review of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies with engineered nanoparticles. Nanotoxicology 8(3):233\u0026ndash;278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/17435390.2013.773464\u003c/span\u003e\u003cspan address=\"10.3109/17435390.2013.773464\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer M, Persson O, Power Y (2001) Mapping excellence in nanotechnologies:Preparatory study (Nanotechnology expert group and Eurotech data). European Comission\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchelmore CL, Chipman JK (1998) DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutation Research/Fundamental and molecular mechanisms of mutagenesis 399(2):135\u0026ndash;147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNemmar A, Hoet PHM, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, Vanbilloen H, Mortelmans L, Nemery B Passage of Inhaled Particles Into the Blood Circulation in Humans\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOberd\u0026ouml;rster G, Oberd\u0026ouml;rster E, Oberd\u0026ouml;rster J (2005) Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environ Health Perspect 113(7):823\u0026ndash;839. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1289/ehp.7339\u003c/span\u003e\u003cspan address=\"10.1289/ehp.7339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgris M, Oupicky D (eds) (2013) Nanotechnology for Nucleic Acid Delivery: Methods and Protocols. Humana Press, Totowa, NJ\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOstling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammal cells. Biochemical and biophysical communications 123(1):291\u0026ndash;298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark S, Lee YK, Jung M, Kim KH, Chung N, Ahn E-K, Lim Y, Lee K-H (2007) Cellular Toxicity of Various Inhalable Metal Nanoparticles on Human Alveolar Epithelial Cells. Inhalation Toxicology 19(sup1):59\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/08958370701493282\u003c/span\u003e\u003cspan address=\"10.1080/08958370701493282\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRenwick LC (2004) Increased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types. Occupational and Environmental Medicine 61(5):442\u0026ndash;447. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/oem.2003.008227\u003c/span\u003e\u003cspan address=\"10.1136/oem.2003.008227\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSau TK, Rogach AL, J\u0026auml;ckel F, Klar TA, Feldmann J (2010) Properties and Applications of Colloidal Nonspherical Noble Metal Nanoparticles. Advanced Materials 22(16):1805\u0026ndash;1825. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adma.200902557\u003c/span\u003e\u003cspan address=\"10.1002/adma.200902557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrurs F, Lison D (2012) Focusing the research efforts. Nature Nanotech 7(9):546\u0026ndash;548. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nnano.2012.148\u003c/span\u003e\u003cspan address=\"10.1038/nnano.2012.148\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeabra AB, Paula AJ, De Lima R, Alves OL, Dur\u0026aacute;n N (2014) Nanotoxicity of Graphene and Graphene Oxide. Chem Res Toxicol 27(2):159\u0026ndash;168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/tx400385x\u003c/span\u003e\u003cspan address=\"10.1021/tx400385x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShvedova AA, Kagan VE, Fadeel B (2010) Close Encounters of the Small Kind: Adverse Effects of Man-Made Materials Interfacing with the Nano-Cosmos of Biological Systems. Annu Rev Pharmacol Toxicol 50(1):63\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.pharmtox.010909.105819\u003c/span\u003e\u003cspan address=\"10.1146/annurev.pharmtox.010909.105819\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175(1):184\u0026ndash;191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0014-4827(88)90265-0\u003c/span\u003e\u003cspan address=\"10.1016/0014-4827(88)90265-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSreeprasad TS, Pradeep T (2012) GRAPHENE FOR ENVIRONMENTAL AND BIOLOGICAL APPLICATIONS. Int J Mod Phys B 26(21):1242001. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1142/S0217979212420015\u003c/span\u003e\u003cspan address=\"10.1142/S0217979212420015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValavanidis A, Vlachogianni T, Fiotakis K, Loridas S, Perdicaris S (2013) Potential toxicity and safety evaluation of nanomaterials for the respiratory system and lung cancer. LCTT:71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/LCTT.S23216\u003c/span\u003e\u003cspan address=\"10.2147/LCTT.S23216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWottrich R, Diabat\u0026eacute; S, Krug HF (2004) Biological effects of ultrafine model particles in human macrophages and epithelial cells in mono- and co-culture. International Journal of Hygiene and Environmental Health 207(4):353\u0026ndash;361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1078/1438-4639-00300\u003c/span\u003e\u003cspan address=\"10.1078/1438-4639-00300\" 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":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Genotoxicity, cytotoxicity, nanomaterial, comet assay, graphene, ZnO, CuO","lastPublishedDoi":"10.21203/rs.3.rs-4730027/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4730027/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe utilization of Zinc oxide (ZnO), Copper oxide (CuO), and graphene nanoparticles has attracted substantial interest within the industrial sector, notably in the realm of inkjet printing. Consequently, the primary aim of this research was to evaluate the cytotoxicity and genotoxicity of these nanoparticles using the MTT assay and the Comet assay on the A459 cell line. In this context, it is necessary to assess the environmental and human health implications of these novel materials, with the intention of categorizing them as emerging contaminants if it was necessary. The characterization of ZnO, CuO, and graphene nanoparticles revealed particle sizes in the range of 10\u0026ndash;70 nm for the metal oxides, and multi-layer graphene platelets with lateral size\u0026thinsp;\u0026lt;\u0026thinsp;0.6 \u0026micro;m. Our findings demonstrated a concentration-dependent relationship between increasing nanoparticle concentration and both cytotoxicity and DNA damage. Specifically, MTT assay results indicated a higher level of toxicity associated with ZnO nanoparticles, whereas genotoxicity was more pronounced with CuO nanoparticles. Furthermore, all nanoparticles exhibited lower EC50 values in the Comet assay. In summary, the current study unveils the cytotoxic and genotoxic effects of ZnO, CuO, and graphene nanoparticles on the A549 cell line.\u003c/p\u003e","manuscriptTitle":"Cytotoxic and Genotoxic Assessment of Zinc Oxide, Copper Oxide and Graphene Nanoparticles on A549 cell line","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 09:05:04","doi":"10.21203/rs.3.rs-4730027/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91d66a85-f72e-4d4a-bbd6-63c7fcb95d95","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T05:47:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-07 09:05:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4730027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4730027","identity":"rs-4730027","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}