A549 transcriptomic response to nanomaterials, intact and degraded Mn3O4-functionalized graphene, Mn3O4 nanoparticles

Background, Motivation and Objective Nanotechnology has revolutionized many fields of knowledge or application1, due to their differential properties at the scale of 1 to 100 nm when compared with the original macroscopic materials2. The speed in the development of new materials poses potential risks to health, either from a societal perspective or an occupational hazard one. Studies evidencing nanoparticle cell-internalization3 can easily be extrapolated to a manufacturing scenario where interactions are sustained in time. Among different toxicological effects of nanoparticles during cell interaction, internalization can pose a potential release of ions that can impair cell metal homeostasis4 and exert negative consequences for viability and normal functioning5. Manganese, for instance, is essential for manganese superoxide dismutase (Mn-SOD)6, but in high concentrations can react with hydrogen peroxide and release ROS7,8, subsequently leading to malignant transformation of cells9 or triggering apoptosis10. Side by side with classic cellular assays, high-resolution omics approaches such as transcriptomics can provide a closer evaluation of the effects of xenobiotics. Nanotoxicology appeared to provide answers in terms of safety11. We were motivated to dive into a molecular level, in order to decipher the underlying mechanisms of toxicity caused upon exposure of A549 cell line to three different nanomaterials. These mechanisms are unknown given that toxicity from these materials has not been previously characterized. The first of the nanoparticles is a novel engineered graphene decorated with manganese oxide (GNA15), while the second is the degraded form of this material (GNA15d). Lastly, we also tested the engineered manganese oxide that was used to produce GNA15. Our study has as an overall objective the assessment of exposure hazards from an occupational risk perspective, specifically inhalation risk by using a human pulmonary cell line. The specific objectives of our study were: - Dissection of underlying molecular mechanisms triggered upon nanomaterial exposure - Strengthening of an incipient research line to assist manufacturers in Safe-by-Design strategies Statement of Contribution/Methods We carried out classical cellular toxicological assays where cells were exposed to each of the nanomaterials. The assays comprised a cell survival evaluation by MTT viability assays (24 h post-exposure), and further evaluation of the oxidative stress by testing of Reactive Oxygen Species (ROS) (0, 30 and 60 min post-exposure). For transcriptomics assays cells were exposed cells for 24 h, and then harvested for RNA isolation. Normalized reads obtained through Illumina sequencing were used for statistical analysis of gene expression. Results/Discussion Results show at first a different trend between the cellular and the transcriptomic assays. In overall, the cellular assays indicate that both forms of functionalized graphene are more harmful than Mn3O4 towards A549 cell line. Interestingly, the transcriptomic evaluation showed a different trend. Expression analyses revealed that both intact and degraded forms of the decorated graphene trigger a dramatically lower number of genes than the manganese oxide. According to our results and other reports in literature, we believe that transcriptomics can inform of early onsets on future negative outcomes, by revealing mechanistic deregulations that are not evident at the cellular assays. Even to the point of showing apparently opposed patterns. These results are not only important for the field of toxicology, but can be translated as well to other fields such as drug screening. Funding information This project was funded by Junta de Castilla y León-FEDER under grant N° BU058P20 (NANOCOMP). References 1. De Marchi, L. et al. An overview of graphene materials: Properties, applications and toxicity on aquatic environments. Science of The Total Environment 631–632, 1440–1456 (2018). 2. Choi, J. Y. et al. In vitro cytotoxicity screening of water-dispersible metal oxide nanoparticles in human cell lines. Bioprocess and Biosystems Engineering 33, 21–30 (2010). 3. Frick, R. et al. Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells. Toxicology Letters 205, 163–172 (2011). 4. Ermini, M. L. & Voliani, V. Antimicrobial Nano-Agents: The Copper Age. ACS Nano (2021) doi:10.1021/acsnano.0c10756. 5. Martinez-Finley, E. J., Gavin, C. 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