In the first part of the present study (Fig. 1), we evaluated the potential genotoxicity of GO sheets after single (30 µg) or repeated (3x1 µg or 3x10 µg) pulmonary exposure. The aim was to compare outcomes from standard exposure protocol (i.e., single exposure triggering acute response) to outcomes from chronic exposure that may better represent exposure at the workplace [26]. This would help to estimate whether the more practical and faster single exposure protocol can predict with enough accuracy the genotoxicity of GO or is missing out on subtle effects only appearing with chronicity. To address the role of time, we investigated the DNA damages at 1, 7 and 28 days after single exposure and 1, 7, 28 and 84 days after repeated exposure. Building on our previous works [26, 27, 39], we also interrogated whether lateral dimensions of the GO sheets could be a key factor of their lung genotoxicity. Hence we compared micrometric LGO sheets (1–25 µm) with nanometric USGO sheets (10–300 nm) that had both similar thickness (1–2 nm) and the same physicochemical properties (Figure S1, Table S1) [40]. Both LGO and USGO suspensions were made of thin, endotoxin-free and metal-free GO sheets that have been fully characterized previously [26]. Multi-walled CNTs (MWCNTs, Mitsui-7) were used in both exposure scenarios as a positive control for the intended biological endpoints, based on the extensive number of reports in the literature demonstrating their genotoxicity [16, 41, 42]. The second part of the study aimed to reveal whether DNA repair mechanisms were triggered alongside the DNA damages. To explain the mechanisms behind the DNA damages or their recovery, we correlated the present results with our already published data on inflammation and oxidative stress caused by repeated exposure to high doses of GO sheets [26] (Fig. 1).
Lung DNA damages induced by pulmonary exposure to GO depend on size, dose, time, and exposure regimen
First, the potential DNA damages caused by GO after either single or repeated pulmonary exposure were investigated in lung sections using γ-H2A.X (S139 phosphorylated H2AX) immunostaining. We considered in our analysis either the total intensity of γ-H2A.X per field of view (FOV), which represents the overall/global DNA damage irrespective of cell type (i.e., it includes immune cell infiltrates, granulomatous structures and remaining lung parenchyma), or the number of γ-H2A.X positive nuclei in the non-inflammatory areas of the lung parenchyma (i.e., it excludes inflammatory areas). To get spatial insights and a better understanding of the cell types that may be affected by the material-induced DNA damages, we performed co-immunostaining for γ-H2A.X (DNA damage marker) and CD45 (immune cell marker) or E-Cad (epithelial cell marker). This differential analysis was particularly important because, genetic damages in epithelial cells can induce loss of proliferative control, and potentially leading to neoplastic lesions [3].
Location and cell type dependent DNA damages after single exposure
When performing global analysis of γ-H2A.X immuno-reactivity (i.e., total fluorescence intensity for γ-H2A.X; Figs. 2A and 2E), a single exposure to GO was found to cause significant DNA damages in lungs, but only at day 1 and for LGO. In contrast, no significant increase in global DNA damages could be measured in USGO exposed animals at any time point, highlighting that DNA damages were GO size dependent. When looking at the spatial location of the DNA damages, it is worth noting that a substantial amount of damages induced by LGO were concentrated in areas with immune cell infiltrates (Figs. 1 and 2B), visible in H&E stained lung sections (Figure S2), and represented by a high density of CD45+ cells (Fig. 1). Up to 28.5% of the global fluorescence intensity for LGO induced γ-H2A.X positive signal could be ascribed to these immune cell clusters at day 1 (i.e., 71.5% of the remaining LGO induce DNA damages were in non-inflammatory areas), increasing to 33.5% at day 7, but disappearing by day 28 (Fig. 2B). Although we did not observe any significant increase in DNA damage for the USGO, it is worth mentioning that most of the DNA strand breaks events recorded were in the parenchyma (Fig. 2B). On the other hand, DNA damages induced by MWCNTs that were significant at both days 1 and 7 (Figs. 2A and 2E) were concentrated primarily in inflammatory areas at early time points (58.3% at day 1 and 61.7% at day 7; Fig. 2B) and in non-inflammatory areas at the latest time points (93.6% at day 28; Fig. 2B).
Focusing on γ-H2A.X immuno-reactivity in non-inflammatory areas of the lung parenchyma, we found a statistically significant increase in the number of γ-H2A.X positive cells only at 1 day and for both USGO and LGO (Fig. 2C). Importantly, a majority of these DNA damages were found in lung epithelial cells, corresponding to 75.3% and 79.2% for USGO and LGO respectively (Fig. 2D), as demonstrated using co-immunostaining (E-cad+; Figure S3A and CD45+; S3B). However, at day 7 and 28, neither LGO nor USGO were causing significant DNA damages in non-inflammatory areas, suggesting that DNA repair may have happened between day 1 and day 7, or after clearance of dead cells. For MWCNTs, there was a statistically significant increase in the number of γ-H2A.X positive cells in non-inflammatory areas at both day 1 and day 28 in comparison to the negative control (Fig. 2C). At day 1, 82.8% of these DNA damages were found in lung epithelial cells; decreasing to 76.2% at day 7 and 70.5% at day 28 (Figs. 2D and S3A), suggesting persistent damages to the epithelium or at least longer lasting damages compared to GO. Taken together, these data suggested that an efficient lung recovery, leading to a rapid eradication of DNA damages, coupled with a fast resolution of the acute inflammation (evidenced by H&E staining, Figure S2), took place early on in the lungs after exposure to GO. However, this was not the case after exposure to MWCNTs.
Location and cell type dependent DNA damages after repeated exposure
After repeated exposure to GO, we found that only the high dose of LGO induced a statistically significant increase in DNA double strand breaks in the lungs, and only 1 day after the last exposure (Figs. 3A, 3B, and 4 for high dose; 3C and 3D for low dose). This suggests that GO genotoxicity was both dose and time dependent. Importantly, irrespective of the time point, neither LGO nor USGO induced significant DNA damages at the low dose (1 µg repeated 3 times), which we consider a possible occupational exposure level in a worst case scenario (Figs. 3C and 3D). When looking at the location of the LGO induced damages at the high dose, 45.8% of them were located in inflammatory areas (Fig. 3B). These results suggest that GO-induced immune cell infiltrates, which size increase with the dose applied or the dimension of the material used [26], may be hotspots for DNA damages. This agrees with previous studies reporting DNA damages in immune cells collected from BALF but not in lungs after pulmonary exposure to GO [28]. This may be due to the ability of these immune cells to rapidly phagocyte nanomaterials that may have genotoxic effects, hence preventing damages in lung epithelial cells. This is also in line with previous work showing lung genotoxicity in overload condition, when clearance by phagocytes is impaired due to an excess of nanomaterial in the lungs, but not in a non-overload condition [43].
Interestingly, the DNA damages found in the infiltrates were alleviated over time, dropping to 29.8% at day 7, then 18.3% at day 28, and disappearing entirely by day 84 (Fig. 3B). This suggests some recovery (i.e., DNA repair) and/or clearance of the damaged (immune) cells from the lungs. Immune cells called to resolve an inflammatory event in lungs are known to reverse migrate to other locations after inflammation has vanished [44]. The decrease in LGO induced DNA damages over time (Fig. 3B), which is aligned with the resolution of the inflammation over time [26], might hence be explained by the relocation of the damaged cells to extra-pulmonary locations. Future research should aim at investigating this possibility in expected locations, such as lymph nodes or bone marrow.
Finally and as expected, high dose of MWCNTs induced significant DNA double strand breaks at any of the tested time points (Figs. 3A and 4), affecting primarily immune cell infiltrates (Fig. 3B). These results are consistent with the persistence of immune cell infiltrates, for up to 84 days after the last exposure to MWCNTs [26], where the materials are accumulated. In contrast, low dose MWCNTs induced statistically significant DNA damages only at day 28 and primarily in the lung parenchyma (Fig. 3C and 3D), which could be explained by the lower accumulation of the materials in immune infiltrates at the low dose.
Focusing on γ-H2A.X immuno-reactivity in non-inflammatory areas of the lung parenchyma, we found using co-immunostaining that a high dose of USGO induced significant DNA damages at day 1 (Fig. 3E) and mostly in alveolar epithelial cells (E-Cad+; 63.1% ; Figs. 3F and S4A). There was no other statistically significant result for USGO at any other time point or in the low dose groups (Figs. 3G, 3H and S4), suggesting that the USGO induced DNA damages in these cells were both time and dose dependent. For LGO, we found significant DNA damages only in the high dose group and at 84 days after the last exposure (Fig. 3E), with 76.4% of the damages found in alveolar epithelial cells (Figs. 3F and S4A). Nevertheless, when evaluating DNA damages only in the epithelial cells (E-Cad+), there was also significant DNA damages at day 1 (Figure S4). Importantly, there was no significant DNA damages at the low dose (Figs. 3G, 3H and S4B), suggesting that LGO induced DNA damages in epithelial cells are mostly dose dependent. The fact that a high dose of LGO can induce DNA damages in lung epithelial cells, not only at an early time point after exposure but also at a late stage of the washout period (here 3 months after last exposure; Figure S4A), raises some concerns about the long(er) term impact of these DNA damages, and warrant further research to address these questions.
On the other hand, and surprisingly for MWCNTs, there was no statistically significant DNA damages at the high dose in non-inflammatory areas (Fig. 3E and 3F); significance was found only at 7 days when isolating the results obtained for lung epithelial cells (E-Cad+; Figure S4A). However, in agreement with the results reported above (using global analysis that includes immune cell infiltrates, granulomatous structures and lung parenchyma; Fig. 3C), we found significant DNA damages at day 28 for the low dose (Fig. 3G) and most of them were located in alveolar epithelial cells (75%; Figs. 3H and S4B). This could be explained by a lower agglomeration of these materials at low dose, allowing them to diffuse more easily throughout the parenchyma, and not being entrapped in immune infiltrates.
Potential genotoxicity of carbon nanomaterials in lungs
Overall, the above results suggest that lungs were able to recover rapidly (within 7 days) from exposure to GO sheets with nanometric dimensions even after multiple exposures, whereas exposure to GO sheets with micrometric dimensions may cause long-term genotoxic effect. Noticeably, the latter result was only perceivable in the repeated exposure at the latest time point after exposure, namely 84 days, highlighting the value of chronic exposure over acute exposure to reveal subtle long–term changes. Beyond size, these results further demonstrate that dose (‘high’ worse than ‘low’) and exposure regimen (‘repeated’ worse than single’) are critical factors to consider when assessing the potential genotoxic impact of GO in lungs, especially for alveolar epithelial cells (Figures S3A and S4). In contrast to these findings, Bengtson et al. have previously shown using comet assay on cell isolates from whole tissue that a single dose of GO (18 µg) delivered to mice by intratracheal instillation could not induce significant DNA damages in lung or liver [28]. Exploring the transcriptomic differences in whole lung and liver tissues after pulmonary exposure to either GO or reduced GO (from 18 µg to 162 µg/ mouse), they confirmed that there was no significant activation of genotoxicity pathways in lung tissue [29]. Interestingly and in agreement with some of our present results, they also demonstrated that despite the lack of DNA damages in whole lungs, cells collected from lung BAL displayed GO-induced genotoxic effects [28]. However, further comparison with the present study are limited because of the differences in methods used to evaluate genotoxicity or the physicochemical features of the tested GO materials. In the same study, Bengtson et al. reviewed the lung genotoxicity profile of other carbon nanomaterials and reported that reduced GO was also not inducing significant DNA damages to lungs [28].
Conversely, carbon black (18–162 µg) was shown to induce significant DNA double strand breaks in BAL cells and lungs for up to 28 days after single intratracheal instillation [45]. The authors emphasized the contribution of a persistent inflammation in lungs, especially with the increased expression of SAA3 at all time-points (i.e., 1, 3 and 28 days after exposure). They further correlated the measured DNA double strand breaks with oxidative DNA damages and inflammation levels, and concluded that the lung genotoxicity of carbon black was both oxidative stress and inflammation dependent [45]. Similarly, Kato et al. demonstrated in mice that MWCNTs (50–200 µg, Mitsui-7, same MWCNTs as in the present study) induced significant DNA damages in lungs at 3 h after instillation, and significant oxidative DNA damages in lungs at any tested time point (up to 7 days) [46]. The authors also highlighted that these MWCNTs elicited mutagenicity in lungs of transgenic mice (gpt delta, used for rodent gene mutation assays, TG488 [35]), but only after multiple instillation to the highest dosage (4 x 200 µg). Going further, Kasai et al. demonstrated that chronic exposure to MWCNTs (Mitsui-7) aerosols led to lung carcinogenicity in rats [15].
In the present study, a majority of the DNA double strand breaks induced by GO after either single or repeated exposure was in alveolar epithelial cells (Figs. 2 and 3). While there is a relative paucity of in vivo studies on the genotoxicity of GO, the number of predictive in vitro studies based on lung epithelial cell models is more substantial. Moreover, despite the limitations and differences in terms of dose applied or model used when comparing in vivo and in vitro data [47, 48], in vitro models may provide more detailed insights to the mechanisms leading to genotoxicity. In murine pulmonary epithelial cell line FE1, GO (5–200 µg/mL) did not induce significant DNA damages after 24 h of exposure, although strong ROS generation was reported [49]. In both alveolar epithelial A549 and bronchial epithelial BEAS-2B models, GO (10–100 µg/mL) induced significant micronuclei formation after 6 h of exposure in a dose-dependent manner, and significant ROS production at the highest dose tested [50]. Alongside direct DNA damages due to the interaction of nanomaterials with the genetic materials, it is well-known that DNA damages may result from bystander effects due to nanomaterials (i.e., inflammation mediated ROS level). This type of DNA damages, called secondary genotoxicity [51], has been recently reported for graphene nanomaterials in human-transformed type-I (TT1) alveolar epithelial cell [52]. In this in vitro study, both inflammation and oxidative stress were the two factors associated with either transient or persistent DNA damages. In another study using the BEAS-2B model, both single- and few- layer GO sheets (50 µg/mL) induced significant DNA damages after 24 h of exposure, reduction in LIG4 (DNA ligase 4) expression, but no variation in RAD51 (DNA repair protein RAD51 homolog 1) expression; while a significant increase in OGG1 (8-oxoguanine DNA glycosylase) expression was noticed for single-layer GO only [53].
In summary, these different in vitro data confirm that GO and other graphene based materials can cause DNA damages in lung epithelial cells and that these effects are often associated with oxidative stress and inflammation. In fact, both in vivo and in vitro existing data for GO and other carbon nanomaterials suggest that genotoxicity is primarily due to the unbalance between a high ROS level typically found in an inflammatory environment, and the anti-inflammatory, anti-oxidant and DNA repair defense mechanisms trying to counteract the effects of inflammation and ROS. This led us to seek whether the spatially-resolved DNA damages found here with GO could be correlated to the induction of DNA repair mechanisms and to previously published in vivo data on inflammation and oxidative stress after repeated exposure to GO.
Lung DNA damages induced by repeated pulmonary exposure to GO are correlated to inflammation, oxidative stress, and DNA repair events
To evaluate the extent of ongoing DNA repair in the lungs of mice repeatedly exposed to a high dose of either USGO or LGO, we assessed the mRNA expression level of three essential DNA repair proteins, namely RAD51, LIG4, and OGG1 (Figure S5). This first data set was then cross-correlated in a correlation matrix with a second data set based on our previously published data on lung inflammation and oxidative stress induced by GO [26] (Figure S6 and S7) in order to identify the possible cause of GO induced DNA damages (Fig. 5). In both cases, only the data related to repeated exposure to a high dose of USGO or LGO, and MWCNTs used as positive control, were considered. A similar strategy has been previously applied for MWCNTs in order to reveal which nanomaterial physicochemical features could predict pulmonary inflammation and genotoxicity [9, 54]. To confirm the spatial distribution of DNA damages with respect to inflammatory areas and the rest of the lung parenchyma, we then performed correlative imaging. For that, we correlated H&E staining (to reveal immune structures) and γ-H2A.X related DNA double strand breaks immunostaining at day 1 and 84 after repeated exposure to the different materials (Figure S8).
Correlation analysis after repeated exposure to a high dose of USGO
Correlation with inflammation At day 1 after exposure to USGO, we previously demonstrated that there was a significant secretion of pro-inflammatory cytokines and evidence of acute inflammation (i.e., significant increase in SAA3 expression level, Figure S6; [26]). However, despite a significant increase in expression of RAD51 mRNA level (Figure S5), this was not correlated to any inflammation event (Fig. 5). Nevertheless, there was a positive correlation (Fig. 5) between the mRNA expression level of OGG1 (decrease in expression compared to the control, although non-significant; Figures S5) and the mRNA expression levels of both IL1β and IL1α (non-significant increase for both; Figures S6). At day 7, there was neither a significant increase in immune cell infiltrates, nor significant secretion in pro-inflammatory cytokines. Despite a significant increase in expression of LIG4 mRNA level (Figure S5), this was not correlated to any inflammation event (Fig. 5). Increased expression levels of RAD51 at day 1 and LIG4 at day 7, which are both proteins able to repair DNA double strand breaks [55], suggest that DNA defense mechanisms were counteracting the negative effects on DNA found for high dose USGO in the non-inflammatory lung parenchyma areas (Fig. 3). At days 28 and 84, there was neither significant increase in inflammatory markers (including both recruited immune cells and secreted cytokines; group (a) in Fig. 5), nor significant increase in the expression of DNA repair proteins (Figure S5 and group (c) in Fig. 5), suggesting a successful resolution from both the initial mild inflammation and DNA damages observed in the lungs at day 1 after USGO treatment. However, we identified at day 84 a positive correlation (Fig. 5) between the mRNA expression level of LIG4 (non-significant decrease; Figures S5) and the mRNA expression levels of both TNFα and IL6 (non-significant increase for both; Figures S5), suggesting that lungs were still in a recovery phase.
Correlation with oxidative stress As mentioned above, there was a significant increase in the mRNA expression level of RAD51 at day 1 (Figure S5) that correlated positively with the expression level of LIG4 (non-significant decrease; Figures S5 and 5). More interestingly, both of these proteins were found to have a positive correlation with the expression level of SOD2 (non-significant increase; Figures S7 and 5). Noticeably for this time point, there was also a positive correlation (Fig. 5) between the mRNA expression level of LIG4 (not significant decrease; Figures S5) and the expression levels of GSH (significant decrease) and HO1 (non-significant decrease) (Figures S5). Although significant lung DNA damages were no longer detected at day 7 (Fig. 3), the significant increase in LIG4 mRNA expression level (Figure S5) was positively correlated with SOD1 and SOD2 expression levels (non-significant increase in both cases; Figure S7), highlighting the contribution of antioxidant enzymes in the DNA repair (Fig. 5). At day 28 and 84, there was no positive correlation between the expression levels of DNA repair proteins and oxidative stress markers (Fig. 5).
Correlation analysis after repeated exposure to a high dose of LGO
Correlation with inflammation At day 1, LGO induced a significant inflammation and the formation of multinucleated macrophages [26] (Figures S6 and S7). However here, only the non-significant increase in mRNA expression of IL1β (Figure S5) correlated positively with the non-significant decrease in mRNA expression of LIG4 (Figures S5 and 5). At day 7, the LGO-induced inflammation, which included the presence of multinucleated macrophages in the lungs, was still above the level found for the negative control, despite evidence of resolution (Figure S6; [26]). Interestingly, there was a negative correlation (Fig. 5) between the mRNA expression level of RAD51 (undisturbed in comparison to the control; Figure S5) and the presence of multinucleated macrophages in BALF (significant increase; Figure S6). Although negative correlation was observed in this case, it is known that BALF cells might be sensitive to DNA double strand breaks [28, 51]. This was demonstrated in our above mentioned findings in which immune cell infiltrates were identified as hotspots of GO-induced DNA damages (Fig. 3B). This negative correlation can be explained by either poor ongoing DNA repair or limitations in the whole lung analysis (used here for determining the mRNA expression level), which is not efficient and sensitive enough to detect discrete DNA repairs happening locally at the site of DNA damages. On the other hand, a positive correlation was observed (Fig. 5) between the mRNA expression level of RAD51 (undisturbed in comparison to the control; Figure S5) and the expression level of SAA3 (undisturbed in comparison to the control; Figure S6). More importantly, there was a strong negative correlation (Fig. 5) between the mRNA expression of LIG4 (non-significant increase; Figure S5) and the presence of neutrophils in BALF (undisturbed in comparison to the control; Figure S6). This suggested no association between the ongoing inflammation due to LGO and this DNA repair enzyme (Fig. 5). At day 28, the presence of multinucleated macrophages and inflammatory mediators, such as IL1 α, in the lungs (statistically significant for both) was suggesting that the inflammation was still ongoing (Figure S6; [26]). Interestingly, we found a positive correlation (Fig. 5) between the mRNA expression level of LIG4 (non-significant increase; Figure S5) and the presence of macrophages as well as the increase in mRNA expression level of IL1α (Figure S6). At day 84, we only found a positive correlation (Fig. 5) between the mRNA expression level of LIG4 (non-significant increase; Figure S5) and the mRNA expression level of SAA3 (undisturbed in comparison to the control; Figure S6).
Correlation with oxidative stress OGG1 is a DNA repair enzyme associated to both inflammation and oxidative stress [56]. Its main role is the excision of oxidized guanine nucleotides, which can turn into DNA mutation hotspots if they are not eliminated. At day 1, we found a positive correlation (Fig. 5) between the mRNA expression level of OGG1 (not significant decrease; Figure S5) and the mRNA expression level GSH (non-significant decrease; Figure S7). This positive correlation between OGG1 and GSH was also present for day 28 and 84 (Figs. 5, S5 and S7). Interestingly at day 84, we also found a positive correlation (Fig. 5) between the mRNA expression level of LIG4 (non-significant decrease; Figure S5) and the expression levels of SOD2 and GSH (non-significant decrease; Figure S7). These decreases in DNA repair proteins and correlation with antioxidant enzymes at day 84 may explain the significant DNA damages found in mouse lungs at 84 days after exposure to high dose LGO (Fig. 3).
Correlation analysis after repeated exposure to a high dose of MWCNTs
Correlation with inflammation At day 1, there was a significant influx of immune cells in the BALF, with presence of multi-nucleated macrophages and pro-inflammatory cytokines, all revealing a strong inflammation (Figures S6, S7 and S8). From day 1 to 84, lung inflammation (Figures S6 and S7; [26]) and DNA damages (Figs. 3A and S8) after repeated exposure to MWCNTs were still significantly above the negative control levels. However, none of the DNA repair proteins were upregulated at any time points; in fact the mRNA expression level of OGG1 was decreased in comparison to the negative control (statistically significant decrease at day 1, 7 and 28, but not at day 84; Figure S5). Despite DNA damages (Figs. 3 and S8) and strong immune response involving both innate and adaptive immune cells (Fig. 6; [26]), no correlation were found at day 1 and day 7 (Fig. 5). At day 28, there was a positive correlation (Fig. 5) between the mRNA expression level of RAD51 (not significant decrease; Figure S5) and the influx of macrophages (significant increase compared to control; Figure S6). Interestingly both RAD51 and LIG4 mRNA expression levels (Figure S5) were negatively correlated with the mRNA expression level of IL6 (non-significant increase; Figure S6). At day 84, RAD 51 expression level (Figure S5) was also negatively correlated (Fig. 5) with the presence of multinucleated macrophages in the BALF (Figure S6). The same negative correlation between RAD51 and BAL multinucleated macrophages was found for LGO at day 7 (Fig. 5), demonstrating that LGO in contrast to USGO may present some similarities to MWCNTs in terms of impact (i.e., inflammation and DNA damages).
Correlation with oxidative stress After exposure to MWCNTs, along the inflammation, a rapid response to oxidative stress occurred in the form of an upregulation of mRNA expression for HSP70 and HO1 (significant at days 1 and 7, and then going back to baseline by day 28; Figure S7; [26]), whereas SOD1 and GSH expression levels were downregulated compared to the negative control (not significant for SOD1, but significant for GSH, at days 1 and 7; Figure S7). At day 1, HSP70 expression levels were positively correlated with inflammatory markers (SAA3 and IL6; Fig. 5), as was the expression levels of SOD2 with SAA3, or the expression levels of GSH with IL1β, confirming the strong link between oxidative stress and inflammation in the impact of MWCNTs, as previously described [57]. In addition, a positive correlation between the mRNA expression of RAD51 with some oxidative stress markers (such as HO-1 at day 1, 28 and 84; or SOD2 at day 1) was found (Fig. 5). There was also a positive correlation between LIG4 and SOD1 at day 84 (Fig. 5). Taken together these results support the idea that MWCNT-induced DNA damages and their repair were related to inflammation-related oxidative stress and the regulation of ROS levels. On the other hand, there was a negative correlation between the mRNA expression level of LIG4 and HO1 at day 7 and 84 (Fig. 5), as well as between expression level of OGG1 and HO1 at day 7 (Fig. 5). Interestingly, the mRNA expression levels of the DNA repair protein OGG1 were downregulated at all tested time points (significant at day 1, 7, 28 but not 84; Figure S5). However, there was no correlation with any of the endpoints tested here (Fig. 5). Since OGG1 acts on the excision of oxidized nucleotides due to ROS in the DNA repair process, we expected to observe an increase of this marker to counteract the ROS-induced DNA damages after repeated exposure to MWCNTs (Fig. 3A). However, the only significant correlation found between this DNA repair marker and any marker associated to the oxidative stress response was at day 28 and for HSP70. Therefore, the decrease in OGG1 expression are likely explained by other feedback loop regulations not investigated here.
Predicting DNA damages in function of GO aspect ratio
Owing to the correlation analysis performed here, subtle variations in oxidative stress and inflammation could be associated to ongoing DNA repair mechanisms, although some markers were not expressed significantly. We found correlations between DNA repair mechanisms and oxidative stress markers, as well as with secretion of pro-inflammatory mediators, or the presence of neutrophils and multinucleated macrophages in BALF. Nevertheless, an important difference was noted between the USGO and the LGO. For USGO, correlations between DNA repair and oxidative stress were identified only at the earliest time point and for up to 7 days, although correlations with pro-inflammatory cytokine secretion were still measured at day 84. For LGO, we found correlations at all tested time points (up to 84 days) for both oxidative stress and inflammation events. Interestingly, MWCNT results showed absence of correlation between DNA repair and inflammatory events before day 28, but strong correlation with oxidative stress from day 1 till day 84. Taken together, these results underline the importance of prolonged oxidative stress on DNA damage and repair mechanisms and could explain the presence of DNA damages at day 84 for the LGO, but not for the USGO.
The differences between USGO and LGO in pace of recovery and repair mechanisms can explain the increased impact of LGO on DNA. On the one hand, USGO sheets due to their nanometric dimensions were promptly internalized by immune cells and cleared from the lungs [26]. Hence they did not elicit a strong and long-lasting inflammatory response or oxidative stress, but led only to short-term repairable genotoxic effects (Figs. 3 and S8). On the other hand, LGO sheets, due to their micrometric dimensions, were entrapped in granulomatous formations inside multinucleated macrophages [26, 58] and could not be eliminated rapidly from the lungs. This was evidenced by the presence of positive Raman signals in the lung sections at 84 days [26] and the persistence of multinucleated macrophages in the BALF at the same time point (Figure S6). This could explain the long-lasting inflammation [26] and long-term bystander genotoxic effects found in the LGO high dose group (Figs. 3E, 3F and S4A, S8). Persistence of multinucleated macrophages in lung granulomas has been reported previously for nanomaterials [59] and is often attributed to the dimensions of the considered nanomaterials. In particular, the phagocytosis capacity of macrophages is reduced in presence of high aspect ratio materials, hence requiring the fusion of these macrophages to allow appropriate internalization [26]. Therefore, it can be inferred that the long-lasting LGO-induced DNA damages could be ascribed to both the persistence of materials in the lungs and the persistent cell-mediated inflammation and oxidative stress resulting from their presence. Similar conclusions were reached previously for MWCNTs [60] and single-walled CNTs [10]. Future work should aim at determining whether these long-term DNA damages after LGO exposure can be repaired at later time points (e.g., > 3 months) or lead to mutation and/or cancer. This is particularly important considering that these DNA damages were associated here with an acute neutrophil response (Figure S6) that has been shown previously to increase the long-term risk of carcinogenesis [38].