de Luna et al. Particle and Fibre Toxicology (2022) 19:62 https://doi.org/10.1186/s12989-022-00502-w RESEARCH Open Access Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile Luis Augusto Visani de Luna1,2,3† , Thomas Loret1,2,3† , Alexander Fordham1,2,3 , Atta Arshad1,2,3, Matthew Drummond1,2 , Abbie Dodd1,2, Neus Lozano4 , Kostas Kostarelos1,2,4   and Cyrill Bussy1,2,3*    Abstract Background: A key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, has a role in the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to measure DNA damages in both epithelial and immune cells, after either single or repeated exposure. Results: GO induced DNA damages were size and dose dependent, in both exposure scenario. After single exposure to a high dose, both USGO and LGO induced significant DNA damage in the lung parenchyma, but only during the acute phase response (p < 0.05 for USGO; p < 0.01 for LGO). This was followed by a fast lung recovery at day 7 and 28 for both GOs. When evaluating the chronic impact of GO after repeated exposure, only a high dose of LGO induced long-term DNA damages in lung alveolar epithelia (at 84 days, p < 0.05). Regardless of size, low dose GO did not induce any significant DNA damage after repeated exposure. A multiparametric correlation analysis of our repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damage was correlated to efficient recovery from acute inflammation (i.e., significant secretion of SAA3, p < 0.001; infiltration of neutrophils, p < 0.01). In contrast, the persistence of LGO in lungs was associated to a long-lasting presence of multinucleated macrophages (up to 84 days, p < 0.05), an underlying inflammation (IL-1α secretion up to 28 days, p < 0.05) and the presence of persistent DNA damages at 84 days. Conclusions: Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace. † Luis Augusto Visani de Luna and Thomas Loret contributed equally to this work. *Correspondence: cyrill.bussy@manchester.ac.uk 1 Nanomedicine Lab 2.0, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester M13 9PT, UK Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 2 of 21 Keywords: Lungs, Graphene oxide, Genotoxicity, γ-H2AX, Inflammation, Toxicology Introduction investigations on this topic remain scarce [7]. Our group Inhalation of airborne particulate matter has been linked has previously shown that a high dose of micrometric to long-term pulmonary adverse effects and diseases, GO delivered to the lungs of mice by single intranasal including asthma, susceptibility to infections, chronic aspiration can lead to the activation of molecular path- obstructive disease, genotoxicity and cancer [1–4]. For ways associated to genotoxicity or tumor development the latter, it has been established that any particle or sub- [27]. In a study directly investigating the genotoxicity of stance that has the ability to cause increased and long- GO after single pulmonary exposure in mice, Bengtson lasting DNA damages with inefficient repair could induce et al. have reported that significant DNA damages could genomic instability, mutation hotspots and finally tumor be found in cells from the broncho-alveolar fluid (BALF), [5]. More recently, both air pollution and occupational but not in lung or liver tissue sections [28]. In a follow-up health studies have stressed the potential of airborne study, the same authors confirmed the outcomes in lung carbon-based particles to give rise to genotoxicity and and liver at mRNA level [29]. carcinogenicity [6]. And with the expansion of nano- Although these different studies demonstrated that technologies and advanced materials, similar concerns GO may have a genotoxic impact on some lung cells have been raised for engineered carbon nanomaterials after inhalation, they remain limited in their design by (CNMs) [7, 8]. the use of single/bolus exposure. Chronic exposure and Among those CNMs, carbon nanotubes (CNTs) are by long-term assessment represent a more realistic scenario far the most studied. While some CNTs were not found to evaluate persistent DNA damages that may lead to to cause any toxicity, others were reported to induce carcinogenesis events [16]. In this respect, we recently a large range of adverse effects, including cytotoxicity, demonstrated that a repeated pulmonary exposure to inflammation, genotoxicity and cancer [9, 10]. In the GO via oro-pharyngeal aspiration induced a size, dose lungs, some CNTs were shown to elicit frustrated phago- and time dependent lung inflammation without tissue cytosis in macrophages [11], to have poor biodegradabil- remodeling in mice [26]. Importantly, we determined ity and high tissue biopersistence [11, 12], or to induce that these effects and their progressive disappearance fibrosis [13, 14], mutagenesis [10] and carcinogenesis [15, were associated with a size-dependent lung clearance of 16]. When comparing these effects with those of asbestos the GO materials [26]. However, long-lasting inflamma- fibers, it became apparent that shape and size along with tion such as the one observed with micrometric GO [26] other variables (i.e., diameter, rigidity, composition, sur- has been previously associated with an increased risk of face chemistry, or metal impurities) were key drivers of developing cancer [31]. There is therefore a need to eval- CNT toxicity [12, 17]. uate the potential of these micrometric materials to cause In the last 15 years, another type of CNMs with large genotoxicity after chronic exposure, and the possible link industrial potential has appeared, namely graphene based between inflammation and DNA damage. materials. In particular, graphene oxide (GO), the oxi- In pulmonary hazard assessment studies, achieving an dized form of graphene, or its reduced form (rGO), have even distribution and deposition of nanomaterials in the potential application in different sectors of the economy lungs is known to be challenging. This is primarily due to including the biomedical industry [18–21]. GO can be the tendency of nanomaterials, including GO sheets, to formulated either as powder or as aqueous suspension agglomerate while progressing through the airways, from of two dimensional sheets, in a wide range of sizes (from the upper bronchial ramifications down to the alveolar micrometers to nanometers) and with varied degrees cavities [10, 32, 33]. As a consequence of agglomeration, of surface oxidation. Interestingly, due to their size and nanomaterials accumulate in greater amount in various shape, two dimensional GO sheets can easily form but discrete parts of the lungs (in both lung parenchyma aerosols [22–25]. For this reason and considering the and airways) where they trigger region-specific adverse toxicological profile of others CNMs, in particular the effects, as previously described for silica particles, asbes- genotoxicity and carcinogenicity of some CNTs, efforts tos fibers or carbon nanotubes [14, 34]. Adverse effects have been made to evaluate the impact of GO or rGO to initiated from these local areas of greater accumulation the lungs after inhalation [7, 26–30]. then spread across the whole tissue via cell signaling While there are several toxicology studies reporting and/or the secretome. At tissue level, the overall/aver- the genotoxic potential of GO on lung cells using dif- age effect of such nanomaterials is therefore dominated ferent models and in vitro settings, in vivo pulmonary by how intense the effects were in regions of greater de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 3 of 21 accumulation and balanced out by the limited to negligi- (i.e., epithelial or immune cells), and correlative imag- ble response from the remaining tissue, where materials ing to identify the location of DNA damages in respect accumulated less. From this it follows that whole tissue to the cell mediated immune response within the lungs. analysis, which is often the default approach to probe To complement these image-based techniques, we finally the effects of nanomaterials, tends to provide a skewed used a multi-endpoint approach to correlate new data on picture and underestimates how adverse the effects of DNA repair generated by RT-qPCR with our published nanomaterials can be locally (i.e., dilution of the local data on lung inflammation and oxidative stress induced effect in an average tissue level assessment). This is par- by repeated exposure to GO [26]. ticularly true for genotoxicity that may lead to malignant transformation from events happening in a single/small Results and discussion group of cells and not in the whole tissue. Moreover, even In the first part of the present study (Fig. 1), we evalu- for whole tissue analysis, there is still debate about the ated the potential genotoxicity of GO sheets after single applicability of current OECD recommended methods (30 µg) or repeated (3 × 1 µg or 3 × 10 µg) pulmonary (i.e., TG 489, comet assay or TG 488, transgenic rodents) exposure. The low dose of 3 × 1 µg represented a realistic to determine DNA damages elicited by nanomaterials dose of exposure at the workplace when limited mitigat- in vivo [35, 36]. Conversely, we argue that methods based ing measures are applied, whereas 30 µg (or 3 × 10 µg) on local analysis of specific lung regions could be advan- was used here as worst-case exposure scenario (accident) tageous compared to whole organ analysis for the assess- that may happen during specific tasks such as dry pow- ment of nanomaterial genotoxicity, as they provide highly der handling without protective measures [39, 40]. The valuable spatial information. For instance, using immu- aim was to compare outcomes from standard exposure nostaining and γ-H2A.X (S139 phosphorylated H2AX) as protocol (i.e., single exposure triggering acute response) marker for DNA double strand breaks (DSB) [37, 38] may to outcomes from chronic exposure that may better rep- provide better spatial insights to evaluate the genotoxic- resent exposure at the workplace [26]. This would help ity of GO in lungs after pulmonary exposure. In particu- to estimate whether the more practical and faster single lar, immunostaining used in a multiplex format (i.e., to exposure protocol can predict with enough accuracy the reveal other protein biomarkers) or in combination with genotoxicity of GO or is missing out on subtle effects only correlative imaging may help to determine the spatial dis- appearing with chronicity. To address the role of time, we tribution of DNA damages within the lungs while simul- investigated the DNA damages at 1, 7 and 28 days after taneously identify the cells affected by these damages. single exposure and 1, 7, 28 and 84 days after repeated With this in mind, the primary aim of the present study exposure. was to reveal whether GO could induce long-lasting Building on our previous works [26, 27, 41], we also DNA damages in the lungs of mice, and whether these interrogated whether lateral dimensions of the GO sheets effects align with previous evidence of inflammation and could be a key factor of their lung genotoxicity. Hence oxidative stress [26]. To get a better mechanistic under- we compared micrometric LGO sheets (1–25 µm) with standing of GO effects, we designed the study to evaluate nanometric USGO sheets (10–300 nm) that had both not only the role of dose and time but also lateral dimen- similar thickness (1–2 nm) and the same physicochemi- sion and exposure regimen. We compared the impact cal properties (Additional file 1: Fig. S1, Table S1) [42]. of single exposure to a high-dose (30 µg) of GO, to the Both LGO and USGO suspensions were made of thin, impact of repeated exposure to two doses (cumulated low endotoxin-free and metal-free GO sheets that have been dose 3 µg or cumulated high dose 30 µg) of GO, using fully characterized previously [26]. Importantly, a pre- micrometric GO (LGO) and nanometric GO (USGO) in vious study has determined that 0.1 µm thick graphene both cases. Impact of single oro-pharyngeal aspiration based materials of 25 µm lateral size would have an were assessed for up to 28 days after exposure, whereas aerodynamic diameter of 2.97 µm, well below the 5 µm the impact of repeated exposure was assessed for up to threshold for reaching the alveolar region [43], suggest- 84 days. All results were compared with the correspond- ing that both LGO and USGO used here may reach the ing impact of long and rigid MWCNTs (Mitsui-7) at the distal lung. In fact, we demonstrated in our previous same time points and doses. To get spatial information work that about 85% of alveolar macrophages present in about DNA damages and avoid the dilution effect inher- the broncho-alveolar lavage had engulfed GO materials ent to whole tissue analysis, we used immunostaining of 1 day after pharyngeal aspiration and that it represented lung sections and quantified the level of genetic mate- about 60% of the macrophages’ internalization capacity rial alterations under the different conditions. Moreo- at day 1 for both USGO and LGO exposures [26]. This ver, we used co-immunostaining to reveal the identity result underlines that despite clear differences in size, of cells presenting DNA damages in the lung sections similar amounts of either USGO or LGO could reach the de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 4 of 21 Fig. 1 Experimental design of the study. Single (high-dose) and repeated (low and high-dose) exposure to nanometric (USGO) or micrometric (LGO) graphene oxide sheets or MWCNTs were delivered to mouse lungs by oro-pharyngeal aspiration. Quantification of DNA damages in formalin-fixed paraffin embedded lung sections was performed using recombinant rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139, combined with anti-mouse CD45 Alexa Fluor 594 and anti-mouse E-Cadherin Alexa Fluor 488 to phenotype the damaged cells (n = 3). A correlation matrix was performed for the repeated exposure (high-dose) study using inflammation parameters obtained from BALF, whole lung ELISA, and RT-qPCR obtained from the same animals (n = 6). Figure created with BioRender.com de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 5 of 21 alveolar space [26]. This similarity allowed in return an for LGO. In contrast, no significant increase in global accurate evaluation of the impact of GO lateral dimen- DNA damages could be measured in USGO exposed sion on lung genotoxicity. In parallel, multi-walled CNTs animals at any time point, highlighting that DNA dam- (MWCNTs, Mitsui-7) were used in both exposure sce- ages were GO size dependent. When looking at the spa- narios as a positive control for the intended biological tial location of the DNA damages, it is worth noting that endpoints, based on the extensive number of reports in a substantial amount of damages induced by LGO were the literature demonstrating their genotoxicity [16, 44, concentrated in areas with immune cell infiltrates (Figs. 1 45]. and 2B), visible in H&E stained lung sections (Addi- The second part of the study aimed to reveal whether tional file 1: Fig. S2), and represented by a high density DNA repair mechanisms were triggered alongside the of ­CD45+ cells (Fig. 1). Up to 28.5% of the global fluores- DNA damages. To explain the mechanisms behind the cence intensity for LGO induced γ-H2A.X positive signal DNA damages or their recovery, we correlated the pre- could be ascribed to these immune cell clusters at day 1 sent results with our already published data on inflam- (i.e., 71.5% of the remaining LGO induce DNA damages mation and oxidative stress caused by repeated exposure were in non-inflammatory areas), increasing to 33.5% at to high doses of GO sheets [26] (Fig. 1). day 7, but disappearing by day 28 (Fig. 2B). Although we did not observe any significant increase in DNA damage Lung DNA damages induced by pulmonary exposure to GO for USGO, it is worth mentioning that most of the DNA depend on size, dose, time, and exposure regimen strand breaks events recorded were in the parenchyma First, potential DNA damages caused by GO after either (Fig. 2B). On the other hand, DNA damages induced single or repeated pulmonary exposure were investigated by MWCNTs that were significant at both days 1 and 7 in lung sections using γ-H2A.X (S139 phosphorylated (Fig. 2A, E) were concentrated primarily in inflammatory H2AX) immunostaining. We considered in our analy- areas at early time points (58.3% at day 1 and 61.7% at day sis either the total intensity of γ-H2A.X per field of view 7; Fig. 2B) and in non-inflammatory areas at the latest (FOV), which represents the overall/global DNA dam- time points (93.6% at day 28; Fig. 2B). age irrespective of cell type (i.e., it includes immune cell Focusing on γ-H2A.X immuno-reactivity in non- infiltrates, granulomatous structures and remaining lung inflammatory areas of the lung parenchyma, we found parenchyma), or the number of γ-H2A.X positive nuclei a statistically significant increase in the number of in the non-inflammatory areas of the lung parenchyma γ-H2A.X positive cells only at 1 day and for both USGO (i.e., it excludes inflammatory areas). To get spatial and LGO (Fig. 2C). Importantly, a majority of these DNA insights and a better understanding of the cell types that damages were found in lung epithelial cells, correspond- may be affected by material-induced DNA damages, we ing to 75.3% and 79.2% for USGO and LGO respectively performed co-immunostaining for γ-H2A.X (DNA dam- (Fig. 2D), as demonstrated using co-immunostaining age marker) and CD45 (immune cell marker) or E-Cad (E-cad+; Additional file 1: Fig. S3A and C ­ D45+; S3B). (epithelial cell marker). This differential analysis was par- However, at day 7 and 28, neither LGO nor USGO were ticularly important because genetic damages in epithelial causing significant DNA damages in non-inflammatory cells can induce loss of proliferative control, and poten- areas, suggesting that DNA repair may have happened tially lead to neoplastic lesions [3]. between day 1 and day 7, or there was clearance of DNA damaged cells. For MWCNTs, there was a statistically Location and cell type dependent DNA damages after single significant increase in the number of γ-H2A.X positive exposure cells in non-inflammatory areas at both day 1 and day 28 When performing global analysis of γ-H2A.X immuno- in comparison to the negative control (Fig. 2C). At day reactivity (i.e., total fluorescence intensity for γ-H2A.X; 1, 82.8% of these DNA damages were found in lung epi- Fig. 2A, E), a single exposure to GO was found to cause thelial cells; decreasing to 76.2% at day 7 and 70.5% at significant DNA damages in lungs, but only at day 1 and day 28 (Figs. 2D and Additional file 1: S3A), suggesting (See figure on next page.) Fig. 2 Evaluation of DNA damages induced by GO sheets after single exposure. A Quantification of lung DNA damages using rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139 immunostaining after single exposure to 30 µg of GO sheets (USGO or LGO) and MWCNTs (positive control/reference materials) expressed as fold change in fluorescence intensity (negative control = water for injection). B Percentage of γ-H2Ax imunoreactivity (fluorescence intensity) located in lung immune cell infiltrates or in lung parenchyma at day 1, 7 and 28 after exposure. C Total number of γ-H2Ax positive cells in lung parenchyma (outside inflammatory infiltrates) expressed as fold change in fluorescence intensity. D Percentage of E-cadherin+ γ-H2Ax+ cells and ­CD45+ γ-H2Ax+ cells in lung parenchyma. E Representative images of lung section after DAPI staining and immunostaining (primary: rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139; secondary: donkey anti-rabbit Alexa Fluor 647) for DNA damages. Mice were exposed by single oro-pharyngeal aspiration to a high dose GO sheets (USGO or LGO), MWCNTs, or water for injection. Scale bar = 50 µm. Significance level *p < 0.05 **p < 0.01, ***p < 0.001 (One-Way ANOVA; n = 3) de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 6 of 21 Fig. 2 (See legend on previous page.) de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 7 of 21 persistent damages to the epithelium or at least longer recovery (i.e., DNA repair) and/or clearance of the dam- lasting damages compared to GO. Taken together, these aged (immune) cells from the lungs. Immune cells called data suggested that an efficient lung recovery, leading to to resolve an inflammatory event in lungs are known to a rapid eradication of DNA damages, coupled with a fast reverse migrate to other locations after inflammation has resolution of the acute inflammation (evidenced by H&E vanished [47]. The decrease in LGO induced DNA dam- staining, Additional file 1: Fig. S2), took place early on in ages over time (Fig. 3B), which is aligned with the resolu- the lungs after exposure to GO. However, this was not tion of the inflammation over time [26], might hence be the case after exposure to MWCNTs. explained by the relocation of damaged cells to extra-pul- monary locations. Future research should aim at inves- Location and cell type dependent DNA damages tigating this possibility in expected locations, such as after repeated exposure lymph nodes or bone marrow. After repeated exposure to GO, we found that only the Finally and as expected, high dose of MWCNTs high dose of LGO induced a statistically significant induced significant DNA double strand breaks at any of increase in DNA double strand breaks in the lungs, and the tested time points (Figs. 3A and 4), affecting primar- only 1 day after the last exposure (Figs. 3A, B and 4 for ily immune cell infiltrates (Fig. 3B). These results are con- high dose; 3C and 3D for low dose). This suggests that sistent with the persistence of immune cell infiltrates, for GO genotoxicity was both dose and time dependent. up to 84 days after the last exposure to MWCNTs [26], Importantly, irrespective of the time point, neither LGO where the materials are accumulated. In contrast, low nor USGO induced significant DNA damages at the low dose MWCNTs induced statistically significant DNA dose (1 µg repeated 3 times) (Fig. 3C, D), which repre- damages only at day 28 and primarily in the lung paren- sents a realistic dose relevant to human exposure at the chyma (Fig. 3C, D), which could be explained by the workplace [39, 40]. When looking at the location of LGO lower accumulation of materials in immune infiltrates at induced damages at the high dose, 45.8% of them were the low dose. located in inflammatory areas (Fig. 3B). These results Focusing on γ-H2A.X immuno-reactivity in non- suggest that GO-induced immune cell infiltrates, which inflammatory areas of the lung parenchyma, we found size increased with the dose applied or the dimension of using co-immunostaining that a high dose of USGO the material used [26], may be areas with higher DNA induced significant DNA damages at day 1 (Fig. 3E) and damage prevalence. This agrees with previous studies mostly in alveolar epithelial cells (E-Cad+; 63.1%; Figs. 3F reporting DNA damages in immune cells collected from and Additional file 1: S4A). There was no other statisti- BALF but not in lungs after pulmonary exposure to GO cally significant result for USGO at any other time point [28]. This may be due to the ability of these immune cells or in the low dose group (Fig. 3G, H and Additional to rapidly phagocyte nanomaterials that may have geno- file 1: Fig. S4), suggesting that USGO induced DNA dam- toxic effects, hence preventing damage to lung epithelial ages in these cells were both time and dose dependent. cells. This is also in line with previous work showing gen- For LGO, we found significant DNA damages only in the otoxicity in lung overload condition, when clearance by high dose group and at 84 days after the last exposure phagocytes is impaired due to an excess of nanomaterial (Fig. 3E), with 76.4% of the damages found in alveolar in the lungs, but no genotoxicity in a non-overload condi- epithelial cells (Figs. 3F and Additional file 1: Fig. S4A). tion [46]. Nevertheless, when evaluating DNA damages only in the Interestingly, the DNA damages found in the immune epithelial cells (E-Cad +), there was also some signifi- cell infiltrates were alleviated over time, dropping to cant DNA damages at day 1 (Additional file 1: Fig. S4). 29.8% at day 7, then 18.3% at day 28, and disappear- Importantly, there was no significant DNA damages at ing entirely by day 84 (Fig. 3B). This suggests some the low dose (Fig. 3G, H and Additional file 1: Fig. S4B), (See figure on next page.) Fig. 3 Evaluation of DNA damages induced by GO sheets after repeated exposure. A Quantification of lung DNA damages using rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139 immunostaining after repeated exposure to 3 × 10 µg (high dose) of GO sheets (USGO or LGO) and MWCNTs (positive control/reference materials), expressed as fold change in fluorescence intensity (negative control = water for injection). B Percentage of γ-H2Ax imunoreactivity (fluorescence intensity) located in lung immune cell infiltrates or in lung parenchyma at day 1, 7, 28 and 84 after repeated exposure to 3 × 10 µg. C Quantification of lung DNA damages after repeated exposure to 3 × 1 µg (low dose) of materials. D Percentage of γ-H2Ax imunoreactivity (fluorescence intensity) located in lung immune cell infiltrates or in lung parenchyma at day 1, 7 and 28 after repeated exposure to 3 × 1 µg. E Total number of γ-H2Ax positive cells in lung parenchyma (outside inflammatory infiltrates) expressed as fold change in fluorescence intensity, after repeated exposure to 3 × 10 µg. F Percentage of E-cadherin+ γ-H2Ax+ cells and C ­ D45+ γ-H2Ax+ cells in lung parenchyma at day 1, 7, 28 and 84 after exposure. G Total number of γ-H2Ax positive cells in lung parenchyma (outside inflammatory infiltrates) expressed as fold change in fluorescence intensity, after repeated exposure to 3 × 1 µg. H Percentage of E-cadherin+ γ-H2Ax+ cells and C ­ D45+ γ-H2Ax+ cells in lung parenchyma at day 1, 7 and 28 after exposure. Significance level *p < 0.05 **p < 0.01, ***p < 0.001 (One-Way ANOVA; n = 3) de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 8 of 21 Fig. 3 (See legend on previous page.) de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 9 of 21 Fig. 4 Representative images of immunostaining for DNA damages in lung sections after repeated exposure to a high dose of materials. DNA damages was performed using rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139, and donkey anti-rabbit Alexa Fluor 647. Mice were exposed by single oro-pharyngeal aspiration to a high dose GO sheets (USGO or LGO), MWCNTs, or water for injection. All sections were counterstained for DAPI. Scale bar = 50 µm. Inset boxes highlight positive nuclei with higher magnification (100×) suggesting that LGO induced DNA damages in epithelial the high dose in non-inflammatory areas (Fig. 3E, F); cells are mostly dose dependent. The fact that a high dose significance was found only at 7 days when isolating of LGO can induce DNA damages in lung epithelial cells, the results obtained for lung epithelial cells (E-Cad + ; not only at an early time point after exposure but also at Additional file 1: Fig. S4A). However, in agreement a late stage of the washout period (here 3 months after with the results reported above (using global analysis last exposure; Additional file 1: Fig. S4A), raises some that includes immune cell infiltrates, granulomatous concerns about the long(er) term impact of these DNA structures and lung parenchyma; Fig. 3C), we found damages, and warrant further research to address these significant DNA damages at day 28 for the low dose questions. (Fig. 3G) and most of them were located in alveolar On the other hand, and surprisingly for MWCNTs, epithelial cells (75%; Figs. 3H and Additional file 1: Fig. there was no statistically significant DNA damages at S4B). This could be explained by a lower agglomeration of these materials at low dose, allowing them to diffuse de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 10 of 21 more easily throughout the parenchyma, and not being after instillation, and significant oxidative DNA dam- entrapped in immune cell infiltrates. ages in lungs at any tested time point (up to 7 days) [49]. The authors also highlighted that these MWCNTs elic- Potential genotoxicity of carbon nanomaterials in lungs ited mutagenicity in lungs of transgenic mice (gpt delta, Overall, the above results suggest that lungs were able used for rodent gene mutation assays, TG488 [35]), but to recover rapidly (within 7 days) from exposure to GO only after multiple instillation to the highest dosage sheets with nanometric dimensions even after multiple (4 × 200 µg). Going further, Kasai et al. demonstrated exposures, whereas exposure to GO sheets with micro- that chronic exposure to MWCNT (Mitsui-7) aerosols metric dimensions may cause long-term genotoxic effect. led to lung carcinogenicity in rats [15]. Noticeably, the latter result was only perceivable in the In the present study, a majority of the DNA dou- repeated exposure at the latest time point after expo- ble strand breaks induced by GO after either single or sure, namely 84 days, highlighting the value of chronic repeated exposure was in alveolar epithelial cells (Figs. 2 exposure over acute exposure to reveal subtle long–term and 3). While there is a relative paucity of in vivo stud- changes. Beyond size, these results further demonstrate ies on the genotoxicity of GO, the number of predic- that dose (‘high’ worse than ‘low’) and exposure regi- tive in vitro studies based on lung epithelial cell models men (‘repeated’ worse than ‘single’) are critical factors to is more substantial. Moreover, despite the limitations consider when assessing the potential genotoxic impact and differences in terms of dose applied or model used of GO in lungs, especially for alveolar epithelial cells when comparing in vivo and in vitro data [50, 51], (Additional file 1: Figs. S3A, S4). In contrast to these find- in vitro models may provide more detailed insights to ings, Bengtson et al. have previously shown using comet the mechanisms leading to genotoxicity. In murine pul- assay on cell isolates from whole tissue that a single dose monary epithelial cell line FE1, GO (5–200 µg/mL) did of GO (18 µg) delivered to mice by intratracheal instilla- not induce significant DNA damages after 24 h of expo- tion could not induce significant DNA damages in lung sure, although strong ROS generation was reported [52]. or liver [28]. Exploring the transcriptomic differences in In both alveolar epithelial A549 and bronchial epithe- whole lung and liver tissues after pulmonary exposure lial BEAS-2B models, GO (10–100 µg/mL) induced sig- to either GO or reduced GO (from 18 to 162 µg/mouse), nificant micronuclei formation after 6 h of exposure in a they confirmed that there was no significant activation of dose-dependent manner, and significant ROS production genotoxicity pathways in lung tissue [29]. Interestingly at the highest dose tested [53]. Alongside direct DNA and in agreement with some of our present results, they damages due to the interaction of nanomaterials with also demonstrated that despite the lack of DNA damages the genetic materials, it is well-known that DNA dam- in whole lungs, (immune) cells collected from lung BAL ages may result from bystander effects due to nanoma- displayed GO-induced genotoxic effects [28]. However, terials (i.e., inflammation mediated ROS level). This type further comparison with the present study are limited of DNA damages, called secondary genotoxicity [54], has because of the differences in methods used to evaluate been recently reported for graphene nanomaterials in genotoxicity or the physicochemical features of the tested human-transformed type-I (TT1) alveolar epithelial cell GO materials. In the same study, Bengtson et al. reviewed [55]. In this in vitro study, both inflammation and oxi- the lung genotoxicity profile of other carbon nanomateri- dative stress were the two factors associated with either als and reported that reduced GO was also not inducing transient or persistent DNA damages. In another study significant DNA damages to lungs [28]. using the BEAS-2B model, both single- and few- layer Conversely, carbon black (18–162 µg) was shown to GO sheets (50 µg/mL) induced significant DNA damages induce significant DNA double strand breaks in BAL after 24 h of exposure, reduction in LIG4 (DNA ligase cells and lungs for up to 28 days after single intratracheal 4) expression, but no variation in RAD51 (DNA repair instillation [48]. The authors emphasized the contribu- protein RAD51 homolog 1) expression; while a signifi- tion of a persistent inflammation in lungs, especially with cant increase in OGG1 (8-oxoguanine DNA glycosylase) the increased expression of SAA3 at all time-points (i.e., expression was noticed for single-layer GO only [56]. 1, 3 and 28 days after exposure). They further correlated In summary, these different in vitro data confirm that the measured DNA double strand breaks with oxidative GO and other graphene based materials can cause DNA DNA damages and inflammation levels, and concluded damages in lung epithelial cells and that these effects are that the lung genotoxicity of carbon black was both oxi- often associated with oxidative stress and inflammation. dative stress and inflammation dependent [48]. Simi- In fact, both in vivo and in vitro data existing for either larly, Kato et al. demonstrated in mice that MWCNTs GO or other carbon nanomaterials suggest that geno- (50–200 µg, Mitsui-7, same MWCNTs as in the present toxicity is often primarily due to the unbalance between study) induced significant DNA damages in lungs at 3 h a high ROS level typically found in an inflammatory de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 11 of 21 environment, and the anti-inflammatory, anti-oxidant in SAA3 expression level, Additional file 1: Fig. S6; [26]). and DNA repair defense mechanisms trying to counter- However, despite a significant increase in expression of act the effects of inflammation and ROS. This led us to RAD51 mRNA level (Additional file 1: Fig. S5), this was seek whether the spatially-resolved DNA damages found not correlated to any inflammation event (Fig. 5). Never- here with GO could be correlated to the induction of theless, there was a positive correlation (Fig. 5) between DNA repair mechanisms and to previously published the mRNA expression level of OGG1 (decrease in expres- in vivo data on inflammation and oxidative stress after sion compared to the control, although non-significant; repeated exposure to GO. Additional file 1: Fig. S5) and the mRNA expression levels of both IL1β and IL1α (non-significant increase for both; Lung DNA damages induced by repeated pulmonary Additional file 1: Fig. S6). At day 7, there was neither a exposure to GO are correlated to inflammation, oxidative significant increase in immune cell infiltrates, nor signifi- stress, and DNA repair events cant secretion in pro-inflammatory cytokines. Despite a To evaluate the extent of ongoing DNA repair in the significant increase in expression of LIG4 mRNA level lungs of mice repeatedly exposed to a high dose of either (Additional file 1: Fig. S5), this was not correlated to any USGO or LGO, we assessed the mRNA expression level inflammation event (Fig. 5). Increased expression levels of three essential DNA repair proteins, namely RAD51, of RAD51 at day 1 and LIG4 at day 7, which are both pro- LIG4, and OGG1 (Additional file 1: Fig. S5). This first teins able to repair DNA double strand breaks [58], sug- data set was then cross-correlated in a correlation matrix gest that DNA defense mechanisms were counteracting with a second data set based on our previously published the negative effects on DNA found for high dose USGO data on lung inflammation and oxidative stress induced in the non-inflammatory lung parenchyma areas (Fig. 3). by GO [26] (Additional file 1: Figs. S6, S7) in order to At days 28 and 84, there was neither significant increase identify the possible cause of GO induced DNA damages in inflammatory markers (including both recruited (Fig. 5). In both cases, only the data related to repeated immune cells and secreted cytokines; group (a) in Fig. 5), exposure to a high dose of USGO or LGO, and MWC- nor significant increase in the expression of DNA repair NTs used as positive control, were considered. A simi- proteins (Additional file 1: Fig. S5 and group (c) in lar strategy has been previously applied for MWCNTs Fig. 5), suggesting a successful resolution from both the in order to reveal which nanomaterial physicochemi- initial mild inflammation and DNA damages observed cal features could predict pulmonary inflammation and in the lungs at day 1 after USGO treatment. However, genotoxicity [9, 57]. To confirm the spatial distribution we identified at day 84 a positive correlation (Fig. 5) of DNA damages with respect to inflammatory areas and between the mRNA expression level of LIG4 (non-signif- the rest of the lung parenchyma, we then performed cor- icant decrease; Additional file 1: Fig. S5) and the mRNA relative imaging. For that, we correlated H&E staining (to expression levels of both TNFα and IL6 (non-significant reveal immune structures) and γ-H2A.X related DNA increase for both; Additional file 1: Fig. S5), suggesting double strand breaks immunostaining at day 1 and 84 that lungs were still in a recovery phase. after repeated exposure to the different materials (Addi- Correlation with oxidative stress As mentioned above, tional file 1: Fig. S8). there was a significant increase in the mRNA expression level of RAD51 at day 1 (Additional file 1: Fig. S5) that Correlation analysis after repeated exposure to a high dose correlated positively with the expression level of LIG4 of USGO (non-significant decrease; Additional file 1: Fig. S5 and Correlation with inflammation At day 1 after exposure Fig. 5). More interestingly, both of these proteins were to USGO, we previously demonstrated that there was a found to have a positive correlation with the expres- significant secretion of pro-inflammatory cytokines and sion level of SOD2 (non-significant increase; Additional evidence of acute inflammation (i.e., significant increase file 1: Fig. S7 and Fig. 5). Noticeably for this time point, (See figure on next page.) Fig. 5 Correlation matrix of inflammation, oxidative stress and DNA repair in lungs of mice repeatedly exposed by oro-pharyngeal aspiration to a high dose of GOs or MWCNTs. Data of multiple biomarkers were gathered and then transformed in log2 of fold change compared to negative control (water for injection) before the Pearson correlation analysis was performed. Group (a) represents the inflammation markers: with cellular markers determined from BALF analysis [26] (A = Total Mac (number of macrophages); B = Multi Mac (number of multinucleated macrophages); C = Neutrophils (number of Neutrophils)) and with pro-inflammatory cytokines detected by ELISA in lung tissue (D = Saa3; E = TNF-α; F = IL-1β; G = IL-1α; H = IL-6). Group (b) represents oxidative stress markers (I = HSP70, J = HO-1, K = SOD1, L = SOD2 and M = GSH) assessed by RT-qPCR [26]. Group (c) represents DNA repair proteins (N = RAD51, O = LIG4 and P = OGG1) assessed by RT-qPCR in whole lung tissue. Increased cell counts, secretion and upregulated expression are highlighted by upward arrows adjacent to the matrix, and decreased or downregulated are highlighted by downward arrows. Pearson positive correlation is highlighted by red squared boxes and negative correlation by blue squared boxes. Significance level of the correlation *p < 0.05. The Pearson values and their respective p values of significance are reported in tables in the supplementary content de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 12 of 21 Fig. 5 (See legend on previous page.) de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 13 of 21 there was also a positive correlation (Fig. 5) between (undisturbed in comparison to the control; Additional the mRNA expression level of LIG4 (not significant file 1: Fig. S6). This suggested no association between the decrease; Additional file 1: Fig. S5) and the expression ongoing inflammation due to LGO and this DNA repair levels of GSH (significant decrease) and HO1 (non-sig- enzyme (Fig. 5). At day 28, the presence of multinucle- nificant decrease) (Additional file 1: Fig. S5). Although ated macrophages and inflammatory mediators, such as significant lung DNA damages were no longer detected IL1 α, in the lungs (statistically significant for both) was at day 7 (Fig. 3), the significant increase in LIG4 mRNA suggesting that the inflammation was still ongoing (Addi- expression level (Additional file 1: Fig. S5) was positively tional file 1: Fig. S6; [26]). Interestingly, we found a posi- correlated with SOD1 and SOD2 expression levels (non- tive correlation (Fig. 5) between the mRNA expression significant increase in both cases; Additional file 1: Fig. level of LIG4 (non-significant increase; Additional file 1: S7), highlighting the contribution of antioxidant enzymes Fig. S5) and the presence of macrophages as well as the in the DNA repair (Fig. 5). At day 28 and 84, there was increase in mRNA expression level of IL1α (Additional no positive correlation between the expression levels of file 1: Fig. S6). At day 84, we only found a positive cor- DNA repair proteins and oxidative stress markers (Fig. 5). relation (Fig. 5) between the mRNA expression level of LIG4 (non-significant increase; Additional file 1: Fig. S5) Correlation analysis after repeated exposure to a high dose and the mRNA expression level of SAA3 (undisturbed in of LGO comparison to the control; Additional file 1: Fig. S6). Correlation with inflammation At day 1, LGO induced Correlation with oxidative stress OGG1 is a DNA a significant inflammation and the formation of multi- repair enzyme associated to both inflammation and oxi- nucleated macrophages [26] (Additional file 1: Figs. S6, dative stress [59]. Its main role is the excision of oxidized S7). However here, only the non-significant increase in guanine nucleotides, which can turn into DNA mutation mRNA expression of IL1β (Additional file 1: Fig. S5) cor- hotspots if they are not eliminated. At day 1, we found a related positively with the non-significant decrease in positive correlation (Fig. 5) between the mRNA expres- mRNA expression of LIG4 (Additional file 1: Fig. S5 and sion level of OGG1 (not significant decrease; Additional Fig. 5). At day 7, the LGO-induced inflammation, which file 1: Fig. S5) and the mRNA expression level GSH included the presence of multinucleated macrophages (non-significant decrease; Additional file 1: Fig. S7). This in the lungs, was still above the level found for the nega- positive correlation between OGG1 and GSH was also tive control, despite evidence of resolution (Additional present for day 28 and 84 (Fig. 5, Additional file 1: Figs. file 1: Fig. S6; [26]). Interestingly, there was a negative S5, S7). Interestingly at day 84, we also found a positive correlation (Fig. 5) between the mRNA expression level correlation (Fig. 5) between the mRNA expression level of RAD51 (undisturbed in comparison to the control; of LIG4 (non-significant decrease; Additional file 1: Fig. Additional file 1: Fig. S5) and the presence of multi- S5) and the expression levels of SOD2 and GSH (non- nucleated macrophages in BALF (significant increase; significant decrease; Additional file 1: Fig. S7). These Additional file 1: Fig. S6). Although negative correlation decreases in DNA repair proteins and correlation with was observed in this case, it is known that BALF cells antioxidant enzymes at day 84 may explain the signifi- might be sensitive to DNA double strand breaks [28, 54]. cant DNA damages found in mouse lungs at 84 days after This was demonstrated in our above mentioned find- exposure to high dose LGO (Fig. 3). ings in which immune cell infiltrates were identified as hotspots of GO-induced DNA damages (Fig. 3B). This Correlation analysis after repeated exposure to a high dose negative correlation can be explained by either poor of MWCNTs ongoing DNA repair or limitations in the whole lung Correlation with inflammation At day 1, there was a analysis (used here for determining the mRNA expres- significant influx of immune cells in the BALF, with sion level), which is not efficient and sensitive enough to presence of multi-nucleated macrophages and pro- detect discrete DNA repairs happening locally at the site inflammatory cytokines, all revealing a strong inflamma- of DNA damages. On the other hand, a positive correla- tion (Additional file 1: Figs. S6, S7 and S8). From day 1 tion was observed (Fig. 5) between the mRNA expres- to 84, lung inflammation (Additional file 1: Figs. S6, S7; sion level of RAD51 (undisturbed in comparison to the [26]) and DNA damages (Fig. 3A and Additional file 1: control; Additional file 1: Fig. S5) and the expression Fig. S8) after repeated exposure to MWCNTs were still level of SAA3 (undisturbed in comparison to the control; significantly above the negative control levels. However, Additional file 1: Fig. S6). More importantly, there was a none of the DNA repair proteins were upregulated at any strong negative correlation (Fig. 5) between the mRNA time points; in fact the mRNA expression level of OGG1 expression of LIG4 (non-significant increase; Additional was decreased in comparison to the negative control (sta- file 1: Fig. S5) and the presence of neutrophils in BALF tistically significant decrease at day 1, 7 and 28, but not at de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 14 of 21 day 84; Additional file 1: Fig. S5). Despite DNA damages excision of oxidized nucleotides due to ROS in the DNA (Fig. 3 and Additional file 1: Fig. S8) and strong immune repair process, we expected to observe an increase of this response involving both innate and adaptive immune marker to counteract the ROS-induced DNA damages cells (Fig. 5; [26]), no correlation were found at day 1 and after repeated exposure to MWCNTs (Fig. 3A). However, day 7 (Fig. 5). At day 28, there was a positive correlation the only significant correlation found between this DNA (Fig. 5) between the mRNA expression level of RAD51 repair marker and any marker associated to the oxidative (not significant decrease; Additional file 1: Fig. S5) and stress response was at day 28 and for HSP70. Therefore, the influx of macrophages (significant increase compared the decrease in OGG1 expression are likely explained by to control; Additional file 1: Fig. S6). Interestingly both other feedback loop regulations not investigated here. RAD51 and LIG4 mRNA expression levels (Additional file 1: Fig. S5) were negatively correlated with the mRNA expression level of IL6 (non-significant increase; Addi- Predicting DNA damages in function of GO aspect ratio tional file 1: Fig. S6). At day 84, RAD 51 expression level Owing to the correlation analysis performed here, subtle (Additional file 1: Fig. S5) was also negatively correlated variations in oxidative stress and inflammation could be (Fig. 5) with the presence of multinucleated macrophages associated to ongoing DNA repair mechanisms (i.e., indi- in the BALF (Additional file 1: Fig. S6). The same nega- rect indicators of DNA damages), although some markers tive correlation between RAD51 and BAL multinucleated were not expressed significantly. We found clear correla- macrophages was found for LGO at day 7 (Fig. 5), dem- tions between DNA repair mechanisms and oxidative onstrating that LGO in contrast to USGO may present stress markers, as well as with secretion of pro-inflamma- some similarities to MWCNTs in terms of impact (i.e., tory mediators, or the presence of neutrophils and multi- inflammation and DNA damages). nucleated macrophages in BALF. Taken together, the Correlation with oxidative stress After exposure to correlation analysis results were therefore suggesting the MWCNTs, along the inflammation, a rapid response to key role of inflammation and oxidative stress in driving oxidative stress occurred in the form of an upregulation the in vivo genotoxicity of GO, as reported before in vitro of mRNA expression for HSP70 and HO1 (significant at and discussed above. Despite the lack of comparative days 1 and 7, and then going back to baseline by day 28; in vivo studies demonstrating a link between inflamma- Additional file 1: Fig. S7; [26]), whereas SOD1 and GSH tion and genotoxicity, several inhalation studies have expression levels were downregulated compared to the confirmed that graphene based materials (GBMs) can negative control (not significant for SOD1, but significant induce inflammation. For instance, Ma-Hock et al. have for GSH, at days 1 and 7; Additional file 1: Fig. S7). At shown a dose-dependent increase in the number of BALF day 1, HSP70 expression levels were positively correlated cells (especially neutrophils and lymphocytes) and LDH with inflammatory markers (SAA3 and IL6; Fig. 5), as was release, as well as an increase in the BALF concentrations the expression levels of SOD2 with SAA3, or the expres- of pro-inflammatory mediators at both 7 and 28 days sion levels of GSH with IL1β, confirming the strong link after acute nose-only inhalation (6 h/day for 5 days) between oxidative stress and inflammation in the impact of graphene nanoplatelets (up to 10 µm; mean median of MWCNTs, as previously described [60]. In addition, aerodynamic diameter < 400 nm) in mice [61]. A series a positive correlation between the mRNA expression of studies using the pharyngeal aspiration or tracheal of RAD51 with some oxidative stress markers (such as instillation routes have also reported various patterns of HO-1 at day 1, 28 and 84; or SOD2 at day 1) was found inflammatory response to GO [62, 63] or graphite nano- (Fig. 5). There was also a positive correlation between plates [64]. On the other hand, no evident lung inflam- LIG4 and SOD1 at day 84 (Fig. 5). Taken together these mation tested for up to 21 days after exposure to GO results support the idea that MWCNT-induced DNA (0.5–5 µm lateral dimension; mass median aerodynamic damages and their repair were related to inflammation- diameter (MMAD) of 134 nm) was found after a short- related oxidative stress and the regulation of ROS lev- term nose-only inhalation study (6 h/day for 5 days) in els. On the other hand, there was a negative correlation rats [65]. Similarly, after chronic nose-only inhalation between the mRNA expression level of LIG4 and HO1 (6 h/day, 5 days per week, for 28 days) of graphene nano- at day 7 and 84 (Fig. 5), as well as between expression platelets (2 µm lateral dimension; MMAD of 123 nm) in level of OGG1 and HO1 at day 7 (Fig. 5). Interestingly, rats, no dose dependent effects on body weight, distinct the mRNA expression levels of the DNA repair protein lung pathology or immune cell infiltration were identi- OGG1 were downregulated at all tested time points (sig- fied at any time points (up to 90 days after exposure) [66]. nificant at day 1, 7, 28 but not 84; Additional file 1: Fig. In this study, the lack of inflammation was interestingly S5). However, there was no correlation with any of the matched with a lack of genotoxicity, with an absence of endpoints tested here (Fig. 5). Since OGG1 acts on the DNA damage measured by comet assay [66], supporting de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 15 of 21 the idea that inflammation and genotoxicity may work ascribed to both the persistence of materials in the lungs hand in hand. and the persistent cell-mediated inflammation and oxida- In addition to a correlation between inflammation, tive stress resulting from their presence. And it appears oxidative stress and genotoxicity, our findings were also that the geometric dimensions of LGO in comparison to underlining an important difference between USGO and USGO were the primary drivers of the material persis- LGO. For USGO, correlations between DNA repair and tence, enhanced inflammation and oxidative stress, and oxidative stress were identified only at the earliest time ultimately DNA damages. point and for up to 7 days, although correlations with Overall, the results of the present study and our previ- pro-inflammatory cytokine secretion were still measured ous works [26, 27, 41] are all pointing at the importance at day 84. For LGO, we found correlations at all tested of geometric dimensions in relation to the severity of time points (up to 84 days) for both oxidative stress and the lung response, with GO materials of larger geomet- inflammation events. Interestingly, MWCNT results ric dimensions causing the most adverse effects. Inter- showed absence of correlation between DNA repair and estingly, similar conclusions were reached previously inflammatory events before day 28, but strong correla- for MWCNTs [69] and single-walled CNTs [10], two tion with oxidative stress from day 1 till day 84. Taken high aspect ratio materials, well-known for their ability together, these results highlight the importance of pro- to agglomerate and their bio persistence in lung paren- longed oxidative stress (and likely ROS production) on chyma. The present findings are also in agreement with DNA damage and repair mechanisms, which may explain several in vivo pulmonary studies in which GBMs of the presence of DNA damages at day 84 for LGO, but varying lateral dimensions were compared to assess the not for USGO. Taking into account the size difference importance of GBM dimension in respect to their effect between the two GOs, these results were also suggesting on lungs. Using either small GO (100–200 nm) or large that lateral dimensions may be an essential driver of the GO (800–1100 nm) sheets, Ma et al. reported a more initial response to GO materials leading to different pat- pronounced inflammatory response with large GO than terns of toxicological profile. with small GO in lungs or BAL fluids, in particular for With respect to mechanisms of toxicity, the differences cytokine expression levels, 72 h after intra-tracheal instil- between USGO and LGO in terms of repair mechanisms lation [62]. Similarly, Roberts et al. demonstrated that a and pace of tissue recovery could explain the increased high dose of either 20 µm or 5 µm graphite nanoplates impact of LGO on DNA. On the one hand, USGO sheets (8–25 nm thick) was more potent than < 2 µm graphite due to their nanometric dimensions were promptly nanoplates to induce lung inflammation after pharyn- internalized by immune cells and cleared from the lungs geal aspiration [64]. In addition, none of these graphite [26]. Hence they did not elicit a strong and long-lasting nanoplates, regardless of their geometric dimension, inflammatory response or oxidative stress, but led only to were able to induce fibrosis at high dose, or able to trig- short-term repairable genotoxic effects (Fig. 3 and Addi- ger any adverse response at low dose [64]. A dimension- tional file 1: Fig. S8). On the other hand, LGO sheets, dependent response was also identified when comparing due to their micrometric dimensions, were entrapped in small GO (179 nm) with large GO (1676 nm) sheets in granulomatous formations inside multinucleated mac- mice after oro-pharyngeal aspiration [63]. In this case, rophages [26, 67] and could not be eliminated rapidly small GO induced a higher IL1 β secretion than large GO from the lungs. This was evidenced by the presence of at day 2, but the difference between the two GOs and the positive Raman signals in the lung sections at 84 days negative control was negligible by day 6 and up to day [26] and the persistence of multinucleated macrophages 21 after administration. Importantly, both GO materials in the BALF at the same time point (Additional file 1: were inducing TGF β secretion at day 21 after exposure, Fig. S6). This could explain the long-lasting inflammation with large GO inducing a more robust response than [26] and long-term bystander genotoxic effects found small GO, and this translated into collagen deposition in the LGO high dose group (Fig. 3E, F and Additional for both GO types, with the lungs of large GO exposed file 1: Figs. S4A, S8). Persistence of multinucleated mac- mice showing clear evidence of pulmonary fibrosis [63]. rophages in lung granulomas has been reported previ- Of note, the large GO tested in this study was induc- ously for nanomaterials [68] and is often attributed to the ing a greater extend of pulmonary fibrosis than quartz, dimensions of the considered nanomaterials. In particu- used as positive control and a well-known lung carcino- lar, the phagocytosis capacity of macrophages is reduced gen [63]. Even though none of these in vivo studies have in presence of high aspect ratio materials, hence requir- directly investigated the possible impact of the GBM ing the fusion of these macrophages to allow appropri- induced inflammation on DNA integrity, it is probable ate internalization [26]. Therefore, it can be inferred that that the largest materials had the ability to induce geno- the long-lasting LGO-induced DNA damages could be toxicity, especially for those materials inducing chronic de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 16 of 21 inflammation. In respect of the present study, future Denmark) was heated in oven (150–180 °C) overnight work should aim at determining whether the long-term to remove potential microorganisms. Aqueous suspen- DNA damages found after LGO exposure can be repaired sion of MWCNTs was achieved by re-suspended the at later time points (e.g., > 3 months) or lead to mutation MWCNT powder in sterile water for injection contain- and/or cancer. This is particularly important considering ing 0.5% bovine serum albumin (Gibco, Thermo Fisher that these DNA damages were associated with an acute Scientific) and using bath sonication (VWR essential) neutrophil response (Additional file 1: Fig. S6) that has for 5–7 min at nominal 80 W. MWCNT suspensions been shown to increase the risk of carcinogenesis [38]. were prepared the day before administration to animals and then stored at 4 °C until further use. Nanomaterials Conclusions contamination by endotoxins was evaluated according DNA damages induced by GO sheets in mouse lung tis- to Mukherjee et al. [70] and all the nanomaterials tested sues after pulmonary exposure appear dependent on the negative (data not shown). material lateral dimensions, dose and exposure regimen. The GO sheets with nanometric dimensions (USGO) did Single and repeated exposure of animals to nanomaterials not induce persistent DNA damages, irrespective of dose Female mice C57BL/6 J, 6–8 weeks old, were purchased or chronicity. On the contrary, GO sheets with micro- from Envigo, UK. The experiments were carried out in metric dimensions (LGO) triggered long-term DNA the University of Manchester’s Biological Services Facil- double strand breaks after repeated exposure to a high ity where animals had access to water and food ad libi- dose. Interestingly, a quick recovery was measured after tum. Mice were housed in ventilated cages (4 per cage) single exposure to the same high dose of these larger GO in a room with appropriate light cycle, temperature and sheets, highlighting the key role of exposure chronicity humidity. Prior to the exposure, animals were anaesthe- in the development of durable DNA damages. Combin- tized in an inhalation chamber using a mixture of iso- ing multiparametric data analysis and spatial location of flurane (4%) and oxygen (96%) at 2 L ­min−1 flowing rate the DNA damages, we were able to reveal that inflamma- for 5 min. Animals were then held in supine position in tion and oxidative stress were two mechanisms involved order to deliver 30 µL of the material suspension by oro- in either recovery from or persistence of lung DNA dam- pharyngeal aspiration. After anaesthesia recovery, mice ages. For nanometric GO, the recovery from DNA dam- were put in randomized cages. The administered dose ages was strongly associated with the recovery from for the single exposure study was 30 µg for each nano- acute inflammation. On the other hand, the continuous material, then animals were housed for 1, 7 or 28 days presence of multinucleated macrophages that are linked (n = 3). For the repeated exposure study, the adminis- to the persistence of micrometric GO in the lungs, was tered dose was either 1 or 10 µg of nanomaterials per ani- associated to DNA damages after 3 months. In summary, mal, performed three times with a 14 day washout period the present work provides some foundations towards between administrations. After the last exposure, animals a better understanding of the key elements (causes and (n = 6) were housed for 1, 7, 28 or 84 days. In both stud- mechanisms) guiding the development and/or resolu- ies, water for injection (30 µL) was used as negative con- tion of DNA damages in mouse lungs after exposure to trol while exposure to MWCNTs was the positive control GO sheets. In particular, it emphasizes the importance of for the endpoints considered in the present work. Proce- their lateral dimensions, which will be a crucial consid- dures were performed after ethical approval from the UK eration for the risk classification of these materials and Home Office, under the Project License no. P089E2E0A, towards designing safer two dimensional materials for and in accordance with ARRIVE 2.0 guidelines for animal future commercial applications. research reporting [71]. Experimental section Lung tissue collection and processing Preparation of carbon nanomaterial suspensions Mice were culled by overdose with intraperitoneal injec- Aqueous suspensions of ultrasmall (USGO) and large tion of Pentobarbitone. The right lungs were cut in pieces (LGO) GO sheet were produced in house, as previously and then stored in microtubes containing either 1 mL of described [42]. GO stock suspensions prepared in ster- RNAlater Storage Solution (Merck Sigma) or 0.5 mL of ile water for injection, with neutralised pH, were stored RIPA Buffer (Merck Sigma), for RNA or protein extrac- at 4 °C until further use. Detailed information about tion respectively. The left lungs were collected, washed in the characterization of these materials can be found in PBS (Gibco, ThermoFisherScientific), inflated with neu- Loret et al. [26]. Multi-walled carbon nanotube powder tral buffered formalin solution (10%; i.e., 4% (v/v) formal- (MWCNTs, Mitsui-7, kind gift from Prof Ulla B. Vogel, dehyde solution), and then stored in tubes filled with 10% National Research Centre for the Working Environment, neutral buffered formalin solution. After 24 h, these lungs de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 17 of 21 were transferred to 70% alcohol solution for histological fluorescence threshold before image acquisition in each processing. They were then embedded in paraffin and channel. Seven images per animal from different parts of 5.0 µm thick sections were obtained using a microtome the lungs were acquired in a Zeiss AxioImager.D2 upright (Leica RM2255). fluorescence microscope (Zeiss) using a 40×/0.5 EC Plan-neofluar objective and captured using a Coolsnap Evaluation of cell phenotype and spatial location of DNA HQ2 camera (Photometrics), piloted by the μManager damages in lungs Software v1.4.23 (GitHub open source). Specific band DNA double-strand breaks were evaluated by immu- pass filter sets for DAPI, FITC, Cy3 and Cy5 were used nostaining of formalin-fixed, paraffin-embedded (FFPE) to prevent spectral bleed-through (crossover) between lung sections with rabbit anti-mouse γ-H2A.X (phos- channels. pho S139) recombinant antibodies (BLR053F, ab243906; For IHC-DAB, following the incubation with the pri- Abcam). We performed either multiplex fluorescence mary antibodies (rabbit anti-mouse γ-H2A.X (phospho immunostaining (IHC-IF) to correlate DNA damages S139) BLR053F, ab243906; Abcam) as described above, with specific cell phenotype or peroxidase immunostain- we applied horseradish peroxidase (HRP) labeled anti- ing (IHC-DAB) combined with hematoxylin and eosin rabbit IgG secondary antibodies (ABC detection IHC Kit, (H&E) to locate the DNA damages in respect to lung ab64261; Abcam), for 1 h at room temperature. Follow- immune response and material accumulation. ing instructions from the supplier, the DAB peroxidase For the IHC-IF, Alexa Fluor 647 labeled donkey anti- substrate was then applied. In parallel to the immu- rabbit IgG (H + L) highly cross-adsorbed secondary nostaining, H&E staining using an automatic stainer (XL antibodies (Invitrogen, ThermoFisherScientific) were autostainer, Leica Biosystems) was performed. Bright used to enhance the signal of the primary antibodies. field images of H&E and IHC-DAB stained sections were In order to differentiate between DNA damages in lung generated with a slide scanner (Pannoramic 250 Flash, alveolar epithelial cells and those in immune cells, we 3DHistech Ltd) and visualized with CaseViewer (software added either the Alexa Fluor 488 labeled rat anti-mouse/ version 2.4.0.11902, 3DHISTECH Ltd) in order to reveal human CD324 (E-Cadherin) monoclonal antibody (clone the spatial location of inflammation hot-spots, material DECMA-1, eBioscience; ThermoFisherScientific) [72] to accumulation, and DNA damages. identify epithelial cells or the Alexa Fluor 594 labeled rat anti-mouse CD45 monoclonal antibody (clone 30-F11, Biolegend) [73] to identify immune cells. Prior to immu- Global and cell segmented analysis of DNA damages nostaining, FFPE lung sections were deparaffinized Captured images were processed and analyzed using with Histo-clear (HistoChoice clearing agent; Merck ImageJ software (National Institutes of Health, USA). For Sigma), followed by re-hydration in series of ethanol the cell segmentation analysis in the lung parenchyma, and ultrapure water. Antigen retrieval was performed we used both DAPI (nucleus) and Cy5 (γ-H2A.X) masks in sodium citrate buffer (10 mM sodium citrate, 0.05% to co-localize and count the number of nuclei positive tween 20; pH = 6) and microwave heating for 10 min. for γ-H2A.X. In order to distinguish between cell types, After cooling down at room temperature, the sections channels for lung epithelial cells (­CD324+, FTIC) and were permeated with Triton X-100 at 0.1% in PBS (Merck immune cells (­ CD45+, Cy3) were overlaid with DAPI and Sigma) for 10 min. Un-specific antigen blocking was Cy5 channels. In the inflammatory areas, performing cell performed using 10% normal goat serum (Invitrogen, segmentation was not possible due to a highly compact ThermoFisherScientific), 1% BSA (Gibco; ThermoFish- number of DAPI positive nuclei. For this reason and to erScientific) in PBS for 2 h. Next, the sections were gen- allow later comparison, a total fluorescence intensity for tly drained with a pipette to remove the blocking agent. γ-H2A.X (Cy5) was taken per (field of view) FOV and The primary antibodies suspended in PBS with 1% BSA for each animal (i.e., global DNA damage analysis). After were then applied. The sections were incubated overnight analysis, global DNA damage data was plotted as total at 4 °C. The next day, the sections were gently washed fluorescence intensity for γ-H2A.X (mean ± SD) per ani- with 0.1% Triton X-100 in PBS and then incubated for mal, and specific DNA damages in the lung parenchyma 1 h with the secondary antibodies suspended in PBS as the number of γ-H2A.X positive nuclei (mean ± SD) with 1% BSA, at room temperature and protected from per animal. DNA damages in immune infiltrates were light. Sections were finally mounted using mounting therefore reported as percentage of the total γ-H2A.X medium (ProLong Gold Antifade Mountant with DAPI, fluorescence intensity, whereas the distribution of DNA Invitrogen, ThermoFisherScientific) and glass coverslips. damages between epithelial and immune cells in the Non-stained sections (blank) were similarly prepared parenchyma was reported as percentage of total γ-H2A.X in order to identify auto-fluorescence and determine positive cells. de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 18 of 21 Evaluation of whole lung DNA repair by multiplex RT‑qPCR Supporting information available The gene expression for key DNA repair enzymes (i.e., Supplementary Information can be found online at: RAD51, OGG1 and Ligase IV) was assessed in lung https://​parti​clean​dfibr​etoxi​cology.​biome​dcent​ral.​com/ homogenates at the various tested time points. The description of the procedures for these samples, includ- Abbreviations ing tissue lysis, DNA extraction, RNA isolation, synthe- BALF: Broncho alveolar lavage fluid; DSB: Double strand breaks; E-Cad: E-cad- sis of cDNA, and quantification of mRNA by Multiplex herin; FOV: Field of view; GSH: Gluthatione S-transferase; HO-1: Heme oxyge- RT-qPCR can be found in Loret et al. [26]. The primers nase-1; HSP70: Heat shock protein 70; IL: Interleukin; LGO: Large GO; MWCNT: Multi-walled carbon nanotubes; Multi Mac: Multinucleated macrophages; sequences are detailed in Additional file 1: Table S2. Gene SAA3: Serum amyloid A 3; SOD: Superoxide dismutase; SWCNT: Single-walled expression values were normalized against GAPDH and carbon nanotubes; TNF-α: Tumor necrosis factor-α; USGO: Ultra small GO. were presented as fold change compared to the negative control. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12989-​022-​00502-w. Multiparametric data correlation analysis Additional file 1. Figure S1. Structural and morphological characteriza- To identify in a time-dependent manner key biomarkers tion of USGO and LGO. A) and B) Height AFM images, C) and D) scanning associated to the observed DNA damages in lungs repeat- electron micrographs. Figure S2. Lung inflammation after single exposure edly exposed to high dose of GO, we built a correlation to GOs. A) Immune cells infiltration and granulomatous formation in mice lungs at 1, 7 and 28 days after exposure to a single dose of carbon matrix for multiple comparisons. For this purpose, we nanomaterials (USGO, LGO or MWCNT) compared to water for injection included the new data on DNA repair markers (RAD51, (= control). Lung infiltrates (Red arrow), granulomas formation (green OGG1 and Ligase IV, expressed in log twofold change to arrows). Figure S3. DNA damages in lung cells after single exposure. (A) Quantification of γ-H2Ax positive nuclei of epithelial cells (E-Cadherin+) negative control; n = 6; Additional file 1: Fig. S4), and our in lung parenchyma (outside inflammatory infiltrates and excluding previously obtained data [26] on inflammatory markers CD45+ cells per FOV, n = 7) with immunostaining using Alexa Fluor® 594 CD45+ cells). (B) Quantification of immune cells in mice lungs (number of (n = 6) including total number of macrophages, multinu- labelled rat anti-mouse CD45 monoclonal antibody. A single exposure to cleated macrophages and neutrophils measured in bron- high dose of carbon nanomaterials (USGO, LGO or MWCNTs) compared to cho-alveolar lavages, SAA3 expression level (obtained by water for injection (= control). Statistical significance: *P<0.05, **P<0.01, multiplex RT-qPCR), a panel of secreted cytokines (TNF- ***P<0.001. (n = 3). Figure S4. DNA damages in lung epithelial cells after repeated exposure. Quantification of γ-H2Ax positive nuclei of α, IL-1α, IL-1β and IL-6; obtained by multiplex ELISA; epithelial cells (E-Cadherin+) in lung parenchyma (outside inflamma- Additional file 1: Fig. S5) and oxidative stress mark- tory infiltrates and excluding CD45+ cells) was performed after repeated ers (OH-1, HSP70, SOD1, SOD2 and GSH; obtained by exposure (x3) to high dose (A) or low dose (B) of carbon nanomaterials (USGO, LGO or MWCNTs), compared to water for injection (= control). multiplex RT-qPCR; Additional file 1: Fig. S6). The cor- Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 3). Figure relation is presented as a categorical heat-map for each S5. DNA repair activity in whole lung tissue after repeated exposure to considered time-point, and includes the Pearson corre- a high dose. (A - C) mRNA expression level measured by RT-qPCR for three genes coding for DNA repair proteins, expressed as fold change, lation values between − 1 (negative correlation; the two in whole lung tissue lysate after exposure to carbon materials (USGO, variables move in opposite directions) and + 1 (positive LGO or MWCNTs) or negative control (water for injection). Statistical correlation; the two variables move in the same direc- significance: *P<0.05, **P<0.01, ***P<0.001. (n = 6). Figure S6. Published data on inflammation markers after repeated exposure to a high dose. tion), with statistically significant Pearson correlation val- (A - C) BALF cell counts measured after repeated exposure of mice to ues highlighted with * (p < 0.05). GraphPad Prism v. 8.4.3 carbon materials (USGO, LGO or MWCNTs) or negative control (water was used for this analysis. The raw Pearson R values can for injection). (D) mRNA expression level measured by RT-qPCR for SAA3, expressed as fold change, in whole lung tissue lysate after exposure to be found in Additional file 1: Table S3–S26. carbon materials (USGO, LGO or MWCNTs) or negative control (water for injection). (E-H) Pro-inflammatory cytokines measured by ELISA multiplex. Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 6). From [ref 26; Loret et al, Advanced Science, 2022;9:2104559] Figure S7. Published Statistical analysis data on oxidative stress markers after repeated exposure to a high dose. Raw data were imported in Graphpad Prism 8.0 (Graph- (A - E) mRNA expression levels measured by RT-qPCR for different genes Pad Software Inc.) for producing graphs and performing involved in the regulation of oxidative stress, expressed as fold change, in whole lung tissue lysate after exposure to carbon materials (USGO, LGO or statistical analyses. Depending on data normality (Sha- MWCNTs) or negative control (water for injection). Statistical significance: piro–Wilk test), either one-way ANOVA followed by *P<0.05, **P<0.01, ***P<0.001. (n = 6). From [ref 26; Loret et al, Advanced Dunnett’s post-hoc test was used to evaluate statistical Science, 2022;9:2104559]. Figure S8. Evaluation of DNA damages after repeated exposure to a high dose of GO sheets or MWCNTs. Lung differences compared to the negative control (*p < 0.05, parenchyma, infiltrates, granulomatous areas and nanomaterial deposition **p < 0.01, ***p < 0.001), or Kruskall-Wallis followed by (brown colour) are highlighted in the H&E staining. The corresponding Dunn’s post-hoc test, was used to evaluate significant dif- DNA damages after immunostaining (IHC) with γ-H2AX (DAB+ nuclei) and counterstaining with haematoxylin are highlighted. The framed ferences compared to the negative control at each time- inset pictures represent the same area of IHC without incubation with point (*p < 0.05, **p < 0.01, ***p < 0.001). the primary antibody. The scale bar is equivalent to 20 µm. Table S1. de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 19 of 21 Funding Summary of the physicochemical characterization of USGO and LGO The present study was supported by the UK Research and Innovation sheets. Table S2. List of PCR primers. Table S3. Pearson R values (n = 6) for Engineering and Physical Sciences Research Council (UKRI-EPSRC) Centre for USGO, day 1. Table S4. P values (n = 6) for USGO, day 1. Table S5. Pearson Doctoral Training programme Graphene NOWNANO (EP/L01548X/1), and the R values (n = 6) for USGO, day 7. Table S6. P values (n = 6) for USGO, day European Union (EU) ­8th Framework Programme for Research and Technologi- 7. Table S7. Pearson R values (n = 6) for USGO, day 28. Table S8. P values cal Development, H2020 Future and Emerging Technologies, GrapheneFlag- (n = 6) for USGO, day 28. Table S9. Pearson R values (n = 6) for USGO, ship project (H2020-FET- GrapheneCore2 - #785219 and H2020-FET- Graphe- day 84. Table S10. P values (n = 6) for USGO, day 84. Table S11. Pearson neCore3 - #881603). R values (n = 6) for LGO, day 1. Table S12. P values (n = 6) for LGO, day 1. Table S13. Pearson R values (n = 6) for LGO, day 7. Table S14. P values Availability of data and materials (n = 6) for LGO, day 7. Table S15. Pearson R values (n = 6) for LGO, day All data associated with the study are included in this article and the Supple- 28. Table S16. P values (n = 6) for LGO, day 28. Table S17. Pearson R mentary Information. Raw data are available from the University of Manches- values (n = 6) for LGO, day 84. Table S18. P values (n = 6) for LGO, day 84. ter research repository https://​figsh​are.​manch​ester.​ac.​uk/ (project code # Table S19. Pearson R values (n = 6) for MWCNT, day 1. Table S20. P values xxxxxx). GO materials are available from KK and NL upon reasonable request. (n = 6) for MWCNT, day 1. Table S21. Pearson R values (n = 6) for MWCNT, day 7. Table S22. P values (n = 6) for MWCNT, day 7. Table S23. Pearson R values (n = 6) for MWCNT, day 28. Table S24. P values (n = 6) for MWCNT, Declarations day 28. Table S25. Pearson R values (n = 6) for MWCNT, day 84. Table S26. P values (n = 6) for MWCNT, day 84. Ethics approval and consent to participate Animal research was performed under Project License no. P089E2E0A from the UK Home Office and approved by the University of Manchester animal Acknowledgements welfare and ethical review boy (AWERB). The study was carried out in accord- AF and AA would like to acknowledge studentships co-funded by the Lloyds ance with the ARRIVE 2.0 guidelines and following procedures regulated by Register Foundation funded International Consortium on Nanotechnology the Animals (Scientific Procedures) Act 1986 (ASPA) and Amendment Regula- (LRF-ICON) and the UK Research and Innovation Engineering and Physical Sci- tions 2012 (“the Act”). ences Research Council (UKRI-EPSRC) Centre for Doctoral Training programme Graphene NOWNANO (EP/L01548X/1). This work was also supported by the Consent for publication European Union (EU) 8th Framework Programme for Research and Techno- The authors agree for the manuscript to be submitted for publication. logical Development, GrapheneFlagship project (H2020-FET- GrapheneCore2 - #785219 and H2020-FET- GrapheneCore3 - #881603). We also thank Dr Adrian Competing interests Esteban Arranz, Dr Miguel Arellano, Ms Irene Rebollido, Ms Angeliki Karakasidi The authors declare no competing interests. and Dr Nerea Murillo for their contributions to the synthesis and characterisa- tion of the GO materials used in the present study. In addition, we would like Author details to acknowledge the following groups at ICN2 (Catalan Institute of Nanosci- 1 Nanomedicine Lab 2.0, Faculty of Biology, Medicine and Health, Manchester ence and Nanotechnology, Barcelona, Spain): Advanced Electronic Materials Academic Health Science Centre, The University of Manchester, Manches- and Devices led by Prof. Jose Garrido for the AFM and Raman instrumentation ter M13 9PT, UK. 2 National Graphene Institute, The University of Manchester, and assistance, and Supramolecular NanoChemistry and Materials led by Prof. Manchester M13 9PL, UK. 3 Lydia Becker Institute of Immunology and Inflam- Daniel Maspoch for the UV-Vis equipment and assistance. The ICN2 is funded mation, Faculty of Biology, Medicine and Health, Manchester Academic Health by the CERCA programme, Generalitat de Catalunya, and is supported by the Science Centre, The University of Manchester, Manchester M13 9PT, UK. Severo Ochoa Centres of Excellence programme by the Spanish Research 4 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Agency (AEI, grant no. SEV-2017-0706). At the Faculty of Biology, Medicine Campus UAB, Bellaterra, 08193 Barcelona, Spain. and Health (FBMH Platform Sciences, Enabling Technologies & Infrastructure, FBMH Research & Innovation), The University of Manchester, UK, we would like Received: 30 May 2022 Accepted: 6 September 2022 to thank the following staff for their advices and their provision of technical support services: Peter Walker and Grace Bako (Histology Facility), Dr Gareth Howell (Flow Cytometry Facility), Claire Morrison (Genomic Facility), Dr Peter March (Bioimaging Facility), and Dr. N. Hodson (Bio-AFM Facility). The Bioimag- ing Facility microscopes used in the present study were purchased with grants References from the UKRI Biotechnology and Biological Sciences Research Council (UKRI- 1. Donaldson K, Mills N, MacNee W, Robinson S, Newby D. Role of inflam- BBSRC), the Wellcome Trust, and the University of Manchester Strategic Fund. mation in cardiopulmonary health effects of PM. Toxicol Appl Pharmacol. 2005;207:483–8. Author contributions 2. Arden Pope C, Dockery DW. Epidemiology of particle effects. Air Pollut LAVdeL and TL contributed equally to this work. LAVdeL: Conceptualiza- Heal. 1999; 673-705. https://​doi.​org/​10.​1016/​B978-​01235​2335-8/​50106-X. tion (lead); investigation (lead for biological analyses); formal analysis (lead); 3. Knaapen AM, Borm PJA, Albrecht C, Schins RPF. Inhaled particles and lung methodology (lead); visualization (lead); writing – original draft (lead); writing cancer. Part A Mechanisms Int J Cancer. 2004;109:799–809. – review and editing (equal). TL: Conceptualization (lead); investigation (lead 4. Pope CA, Burnett RT, Thun MJ, Calle EE, Krewski D, Ito K, et al. Lung cancer, for biological analyses); formal analysis (lead); methodology (lead); visualiza- cardiopulmonary mortality, and long-term exposure to fine particulate air tion (lead); writing – original draft (supporting); writing – review and editing pollution. J Am Med Assoc. 2002;287:1132–41. (equal). AF: Investigation (supporting); visualization (supporting); writing – 5. Jeggo PA, Pearl LH, Carr AM. DNA repair, genome stability and cancer: a review and editing (equal). AA: Investigation (supporting). MD: Investigation historical perspective. Nat Rev Cancer. 2016;16:35–42. (supporting). AD: Investigation (supporting). NL: Investigation (lead for materi- 6. Oberdörster G. Pulmonary effects of inhaled ultrafine particles. Int Arch als); resources (lead for GO materials); formal analysis (lead for GO materials); Occup Environ Health. 2000;74:1–8. visualization (lead for materials); supervision (supporting); writing – review and 7. Fadeel B, Bussy C, Merino S, Vázquez E, Flahaut E, Mouchet F, et al. Safety editing (equal). KK: Conceptualization (lead); resources (lead for GO materials); assessment of graphene-based materials: focus on human health and funding acquisition (lead); supervision (lead); writing – review and editing the environment. ACS Nano. 2018;12:10582–620. https://​doi.​org/​10.​1021/​ (equal). CB: Conceptualization (lead); investigation (supporting); resources acsna​no.​8b047​58. (lead for MWCNT materials); funding acquisition (lead); supervision (lead); 8. Godwin H, Nameth C, Avery D, Bergeson LL, Bernard D, Beryt E, et al. visualization (supporting); writing – review and editing (equal). All authors Nanomaterial categorization for assessing risk potential to facilitate read and approved the final manuscript. regulatory decision-making. ACS Nano. 2015;9:3409–17. 9. Knudsen KB, Berthing T, Jackson P, Poulsen SS, Mortensen A, Jacob- sen NR, et al. Physicochemical predictors of multi-walled carbon de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 20 of 21 nanotube–induced pulmonary histopathology and toxicity one year after after pulmonary exposure to graphene and graphene oxide. Toxicol Appl pulmonary deposition of 11 different multi-walled carbon nanotubes in Pharmacol. 2021;410:115343. mice. Basic Clin Pharmacol Toxicol. 2019;124:211–27. 30. Chortarea S, Kuru OC, Netkueakul W, Pelin M, Keshavan S, Song Z, et al. 10. Shvedova AA, Kisin E, Murray AR, Johnson VJ, Gorelik O, Arepalli S, et al. Hazard assessment of abraded thermoplastic composites reinforced with Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 reduced graphene oxide. J Hazard Mater. 2022;435:129053. Available mice: inflammation, fibrosis, oxidative stress, and mutagenesis. Am J from: https://​linki​nghub.​elsev​ier.​com/​retri​eve/​pii/​S0304​38942​20084​21 Physiol Lung Cell Mol Physiol. 2008;295:L552–65. 31. Yang S, Huang Y, Zhao Q. Epigenetic alterations and inflammation as 11. Poland CA, Duffin R, Kinloch IA, Maynard A, Wallace WAH, Seaton A, emerging use for the advancement of treatment in non-small cell lung et al. Carbon nanotubes introduced into the abdominal cavity of mice cancer. Front Immunol. 2022;4:5. https://​doi.​org/​10.​3389/​fimmu.​2022.​ show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 878740. 2008;3:423–8. 32. Van Rijt SH, Bein T, Meiners S. Medical nanoparticles for next generation 12. Donaldson K, Poland CA, Murphy FA, MacFarlane M, Chernova T, Schin- drug delivery to the lungs. Eur Respir J Eur Respir Soc. 2014;44:765–74. wald A. Pulmonary toxicity of carbon nanotubes and asbestos - Similari- 33. Williams RO, Carvalho TC, Peters JI. Influence of particle size on ties and differences. Adv Drug Deliv Rev. 2013;65:2078–86. regional lung deposition - what evidence is there? Int J Pharm Elsevier. 13. Ryman-Rasmussen JP, Cesta MF, Brody AR, Shipley-Phillips JK, Everitt JI, 2011;406:1–10. Tewksbury EW, et al. Inhaled carbon nanotubes reach the subpleural 34. Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis tissue in mice. Nat Nanotechnol. 2009;4:747–51. and silicosis. Am J Respir Crit Care Med. 1998;157:1666–80. 14. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, 35. Elespuru R, Pfuhler S, Aardema MJ, Chen T, Doak SH, Doherty A, et al. et al. Unusual inflammatory and fibrogenic pulmonary responses to Genotoxicity assessment of nanomaterials: Recommendations on best single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol practices, assays, and methods. Toxicol Sci. 2018;164:391. Physiol. 2005;289:L698. 36. Azqueta A, Dusinska M. The use of the comet assay for the evaluation of 15. Kasai T, Umeda Y, Ohnishi M, Mine T, Kondo H, Takeuchi T, et al. Lung carci- the genotoxicity of nanomaterials. Front Genet. 2015;6:239. nogenicity of inhaled multi-walled carbon nanotube in rats. Part Fibre 37. Li M, Gu MM, Tian X, Xiao BB, Lu S, Zhu W, et al. Hydroxylated-graphene Toxicol. 2015;13:53 https://​doi.​org/​10.​1186/​s12989-​016-​0164-​24. quantum dots induce DNA damage and disrupt microtubule structure in 16. Hojo M, Maeno A, Sakamoto Y, Ohnuki A, Tada Y, Yamamoto Y, et al. human esophageal epithelial cells. Toxicol Sci. 2018;164:339–52. Two-year intermittent exposure of a multiwalled carbon nanotube by 38. Wculek SK, Bridgeman VL, Peakman F, Malanchi I. Early neutrophil intratracheal instillation induces lung tumors and pleural mesothelio- responses to chemical carcinogenesis shape long-term lung cancer mas in F344 rats. Part Fibre Toxicol. 2022;19:38. https://​doi.​org/​10.​1186/​ susceptibility. iScience. 2020;23:101277. s12989-​022-​00478-7. 39. Boccuni F, Ferrante R, Tombolini F, Natale C, Gordiani A, Sabella S, et al. 17. Bussy C, Ali-Boucetta H, Kostarelos K. Safety considerations for graphene: Occupational exposure to graphene and silica nanoparticles. Part I: work- lessons learnt from carbon nanotubes. Acc Chem Res. 2013;46:692–701. place measurements and samplings. Nanotoxicology. 2020;14:1280–300. 18. Moon JJ, Xu C, Hong H, Lee Y, Park KS, Sun M, et al. Efficient lymph node- 40. Lovén K, Franzén SM, Isaxon C, Messing ME, Martinsson J, Gudmundsson targeted delivery of personalized cancer vaccines with reactive oxygen A, et al. Emissions and exposures of graphene nanomaterials, titanium species-inducing reduced graphene oxide nanosheets. ACS Nano. dioxide nanofibers, and nanoparticles during down-stream industrial 2020;14:13268–78. handling. J Expo Sci Environ Epidemiol. 2021;31:736. 19. De Lázaro I, Vranic S, Marson D, Rodrigues AF, Buggio M, Esteban-Arranz 41. Vranic S, Rodrigues AF, Buggio M, Newman L, White MRH, Spiller DG, et al. A, et al. Graphene oxide as a 2D platform for complexation and intracel- Live imaging of label-free graphene oxide reveals critical factors causing lular delivery of siRNA. Nanoscale. 2019;11:13863–77. oxidative-stress-mediated cellular responses. ACS Nano. 2018;12:1373–89. 20. De Sousa M, Visani De Luna LA, Fonseca LC, Giorgio S, Alves OL. 42. Rodrigues AF, Newman L, Lozano N, Mukherjee SP, Fadeel B, Bussy C, et al. Folic-acid-functionalized graphene oxide nanocarrier synthetic A blueprint for the synthesis and characterisation of thin graphene oxide approaches, characterization, drug delivery study, and antitumor screen- with controlled lateral dimensions for biomedicine. 2D Mater. IOP Pub- ing. ACS Appl Nano Mater. 2018;1:922–32. lishing; 2018;5:035020. Available from: http://​stacks.​iop.​org/​2053-​1583/5/​ 21. Yang K, Zhang S, Zhang G, Sun X, Lee ST, Liu Z. Graphene in mice: Ultra- i=3/​a=​035020?​key=​cross​ref.​1d398​b3c03​16dee​ca39e​85128​a3426​dc high in vivo tumor uptake and efficient photothermal therapy. Nano Lett 43. Schinwald A, Murphy FA, Jones A, MacNee W, Donaldson K. Gra- Am Chem Soc. 2010;10:3318–23. phene-based nanoplatelets: a new risk to the respiratory system as 22. Di Cristo L, Grimaldi B, Catelani T, Vázquez E, Pompa PP, Sabella S. a consequence of their unusual aerodynamic properties. ACS Nano. Repeated exposure to aerosolized graphene oxide mediates autophagy 2012;6:736–46. inhibition and inflammation in a three-dimensional human airway 44. Grosse Y, Loomis D, Guyton KZ, Lauby-Secretan B, El Ghissassi F, Bouvard model. Mater Today Bio. 2020;6:100050. V, et al. Carcinogenicity of fluoro-edenite, silicon carbide fibres and whisk- 23. Drasler B, Kucki M, Delhaes F, Buerki-Thurnherr T, Vanhecke D, Korejwo ers, and carbon nanotubes. Lancet Oncol. 2014;15:1427–8. D, et al. Single exposure to aerosolized graphene oxide and graphene 45. Toyokuni S. Genotoxicity and carcinogenicity risk of carbon nanotubes. nanoplatelets did not initiate an acute biological response in a 3D human Adv Drug Deliv Rev. 2013;65:2098–110. Available from: https://​www.​scien​ lung model. Carbon. 2018;137:125–35. cedir​ect.​com/​scien​ce/​artic​le/​pii/​S0169​409X1​30014​9X?​via%​3Dihub 24. Spinazzè A, Cattaneo A, Campagnolo D, Bollati V, Bertazzi PA, Cavallo 46. Relier C, Dubreuil M, Garcìa OL, Cordelli E, Mejia J, Eleuteri P, et al. Study of DM. Engineered nanomaterials exposure in the production of graphene. ­TiO2 P25nanoparticles genotoxicity on lung, blood, and liver cells in lung Aerosol Sci Technol. 2016;50:812–21. overload andnon-overload conditionsafter repeated respiratory exposure 25. Su WC, Ku BK, Kulkarni P, Cheng YS. Deposition of graphene nanomaterial in rats. Toxicol Sci. 2017;156:527–37. aerosols in human upper airways. J Occup Environ Hyg. 2016;13:48–59. 47. Nourshargh S, Renshaw SA, Imhof BA. Reverse migration of neutrophils: 26. Loret T, de Luna LAV, Fordham A, Arshad A, Barr K, Lozano N, et al. Innate where, when, how, and why? Trends Immunol. 2016;37:273–86. but not adaptive immunity regulates lung recovery from chronic expo- 48. Bourdon JA, Saber AT, Jacobsen NR, Jensen KA, Madsen AM, Lamson JS, sure to graphene oxide nanosheets. Adv Sci. 2022;9:2104559. et al. Carbon black nanoparticle instillation induces sustained inflamma- 27. Rodrigues AF, Newman L, Jasim D, Mukherjee SP, Wang J, Vacchi IA, et al. tion and genotoxicity in mouse lung and liver. Part Fibre Toxicol BioMed Size-dependent pulmonary impact of thin graphene oxide sheets in Central. 2012;9:5. mice: toward safe-by-design. Adv Sci. 2020;7:1903200. 49. Kato T, Totsuka Y, Ishino K, Matsumoto Y, Tada Y, Nakae D, et al. Genotoxic- 28. Bengtson S, Knudsen KB, Kyjovska ZO, Berthing T, Skaug V, Levin M, ity of multi-walled carbon nanotubes in both in vitro and in vivo assay et al. Differences in inflammation and acute phase response but similar systems. Nanotoxicology. 2013;7:452–61. genotoxicity in mice following pulmonary exposure to graphene oxide 50. Oberdörster G. Safety assessment for nanotechnology and nanomedi- and reduced graphene oxide. PLoS One. 2017;12:e0178355. https://​doi.​ cine: concepts of nanotoxicology. J Intern Med. 2010;267:89–105. org/​10.​1371/​journ​al.​pone.​01783​55. 51. Landsiedel R, Sauer UG, Ma-Hock L, Schnekenburger J, Wiemann M. 29. Poulsen SS, Bengtson S, Williams A, Jacobsen NR, Troelsen JT, Halappana- Pulmonary toxicity of nanomaterials: a critical comparison of published var S, et al. A transcriptomic overview of lung and liver changes one day de Luna et al. Particle and Fibre Toxicology (2022) 19:62 Page 21 of 21 in vitro assays and in vivo inhalation or instillation studies. Nanomedicine. 72. Lovisa S, LeBleu VS, Tampe B, Sugimoto H, Vadnagara K, Carstens JL, et al. 2014;9:2557–85. Epithelial-to-mesenchymal transition induces cell cycle arrest and paren- 52. Bengtson S, Kling K, Madsen AM, Noergaard AW, Jacobsen NR, Clausen chymal damage in renal fibrosis. Nat Med. 2015;21:998–1009. PA, et al. No cytotoxicity or genotoxicity of graphene and graphene 73. Cornet A, Savidge TC, Cabarrocas J, Deng WL, Colombel JF, Lassmann oxide in murine lung epithelial FE1 cells in vitro. Environ Mol Mutagen. H, et al. Enterocolitis induced by autoimmune targeting of enteric glial 2016;57:469–82. https://​doi.​org/​10.​1002/​em.​22017. cells: A possible mechanism in Crohn’s disease? Proc Natl Acad Sci USA. 53. Mittal S, Kumar V, Dhiman N, Chauhan LKS, Pasricha R, Pandey AK. 2001;98:13306–11. Physico-chemical properties based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through oxidative stress. Sci Rep. 2016;6:39548. Available from: http://​www.​nature.​ Publisher’s Note com/​artic​les/​srep3​9548 Springer Nature remains neutral with regard to jurisdictional claims in pub- 54. Schins RPF, Knaapen AM. Genotoxicity of poorly soluble particles. Inhal lished maps and institutional affiliations. Toxicol. 2007;19:189–98. 55. Burgum MJ, Clift MJD, Evans SJ, Hondow N, Tarat A, Jenkins GJ, et al. Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro. J Nanobiotechnol. 2021;19:1–15. 56. Chatterjee N, Yang J, Choi J. Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells. Mutat Res Toxicol Environ Mutagen. 2016;798–799:1–10. Available from: https://​www.​scien​cedir​ect.​com/​scien​ce/​artic​le/​pii/​S1383​ 57181​63002​62?​via%​3Dihub 57. Poulsen SS, Jackson P, Kling K, Knudsen KB, Skaug V, Kyjovska ZO, et al. Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity. Nanotoxicology. 2016;10:1263–75. 58. Postel-Vinay S, Vanhecke E, Olaussen KA, Lord CJ, Ashworth A, Soria JC. The potential of exploiting DNA-repair defects for optimizing lung cancer treatment. Nat Rev Clin Oncol. 2012;9:144–55. 59. Visnes T, Cázares-Körner A, Hao W, Wallner O, Masuyer G, Loseva O, et al. Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation. Science. 2018;362:834–9. 60. Dong J, Ma Q. Suppression of basal and carbon nanotube-induced oxida- tive stress, inflammation and fibrosis in mouse lungs by Nrf2. Nanotoxi- cology. 2016;10:699–709. 61. Ma-Hock L, Strauss V, Treumann S, Küttler K, Wohlleben W, Hofmann T, et al. Comparative inhalation toxicity of multi-wall carbon nanotubes, graphene, graphite nanoplatelets and low surface carbon black. Part Fibre Toxicol. 2013;10:23. 62. Ma J, Liu R, Wang X, Liu Q, Chen Y, Valle RP, et al. Crucial role of lateral size for graphene oxide in activating macrophages and stimulat- ing pro-inflammatory responses in cells and animals. ACS Nano. 2015;9:10498–515. 63. Wang X, Duch MC, Mansukhani N, Ji Z, Liao Y-P, Wang M, et al. Use of a pro-fibrogenic mechanism-based predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials. ACS Nano. 2015;9:3032–43. https://​doi.​org/​10.​1021/​nn507​243w. 64. Roberts JR, Mercer RR, Stefaniak AB, Seehra MS, Geddam UK, Chaudhuri IS, et al. Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: a member of the graphene-based nanomaterial family. Part Fibre Toxicol. 2016;13:34. 65. Kim YH, Jo MS, Kim JK, Shin JH, Baek JE, Park HS, et al. Short-term inhalation study of graphene oxide nanoplates. Nanotoxicology. 2018;12:224–38. 66. Kim JK, Shin JH, Lee JS, Hwang JH, Lee JH, Baek JE, et al. 28-Day inhalation toxicity of graphene nanoplatelets in Sprague-Dawley rats. Nanotoxicol- ogy. 2016;10:891–901. 67. Pagán AJ, Ramakrishnan L. The formation and function of granulomas. Ready to submit your research ? Choose BMC and benefit from: Annu Rev Immunol. 2018;36:639–65. 68. Trout KL, Holian A. Macrophage fusion caused by particle instillation. Curr • fast, convenient online submission Res Toxicol. 2020;1:42–7. 69. Muller J, Huaux F, Moreau N, Misson P, Heilier JF, Delos M, et al. Respira- • thorough peer review by experienced researchers in your field tory toxicity of multi-wall carbon nanotubes. Toxicol Appl Pharmacol. • rapid publication on acceptance 2005;207:221–31. • support for research data, including large and complex data types 70. Mukherjee SP, Lozano N, Kucki M, Del R-C, Newman L, Vázquez E, et al. Detection of endotoxin contamination of graphene based materials • gold Open Access which fosters wider collaboration and increased citations using the TNF-α expression test and guidelines for endotoxin-free gra- • maximum visibility for your research: over 100M website views per year phene oxide production. PLoS ONE. 2016;11:1–17. 71. du Sert NP, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The arrive At BMC, research is always in progress. guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol. 2020;18:e3000410. Learn more biomedcentral.com/submissions