An in-depth multi-omics analysis in RLE-6TN rat alveolar epithelial cells allows for nanomaterial categorization

Abstract

BackgroundNanomaterials (NMs) can be fine-tuned in their properties resulting in a high number of variants, each requiring a thorough safety assessment. Grouping and categorization approaches that would reduce the amount of testing are in principle existing for NMs but are still mostly conceptual. One drawback is the limited mechanistic understanding of NM toxicity. Thus, we conducted a multi-omics in vitro study in RLE-6TN rat alveolar epithelial cells involving 12 NMs covering different materials and including a systematic variation of particle size, surface charge and hydrophobicity for SiO2 NMs. Cellular responses were analyzed by global proteomics, targeted metabolomics and SH2 profiling. Results were integrated using Weighted Gene Correlation Network Analysis (WGCNA).ResultsCluster analyses involving all data sets separated Graphene Oxide, TiO2_NM105, SiO2_40 and Phthalocyanine Blue from the other NMs as their cellular responses showed a high degree of similarities, although apical in vivo results may differ. SiO2_7 behaved differently but still induced significant changes. In contrast, the remaining NMs were more similar to untreated controls. WGCNA revealed correlations of specific physico-chemical properties such as agglomerate size and redox potential to cellular responses. A key driver analysis could identify biomolecules being highly correlated to the observed effects, which might be representative biomarker candidates. Key drivers in our study were mainly related to oxidative stress responses and apoptosis.ConclusionsOur multi-omics approach involving proteomics, metabolomics and SH2 profiling proved useful to obtain insights into NMs Mode of Actions. Integrating results allowed for a more robust NM categorization. Moreover, key physico-chemical properties strongly correlating with NM toxicity were identified. Finally, we suggest several key drivers of toxicity that bear the potential to improve future testing and assessment approaches.

Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

“…This is in accordance with the literature, where it has been shown that SiO2_15_Unmod is "active" in vitro [38] and in vivo [25,73]. Importantly, SiO2_15_Amino was classi ed to be "passive" based on all conducted studies, which has been described before as well [11,25,38]. This illustrates that the used surface modi cation can in uence the effects, since the only difference between SiO2_15_Unmod and SiO2_15_Amino are amino groups on the surface of latter, which is in accordance to the literature [73].…”

“…SiO2_15_Amino. Overall, the NM treatment of type II cells led to increased abundances of proteins and metabolites that are part of the GSH/GSSG signaling, thus indicating an increased production of GSH, especially in case of SiO2_40, SiO2_7, and TiO2_NM105, con rming that not only SiO2_40 and SiO2_7 are "active" NMs but also TiO2_NM105, which is consistent with our previous study [11].…”

“…In contrast, almost no signi cantly altered proteins were observable in type II cells, while dose-dependent increases of the portion of signi cantly altered proteins were noticeable for SiO2_40 and SiO2_7 after treatment of macrophages. Thus, in accordance to our previous studies, SiO2_40, SiO2_7, and TiO2_NM105 were considered "active" NMs [11,12], while SiO2_15_Unmod and SiO2_15_Amino were classi ed as "passive".…”

“…Since the level of GSH is highly connected to the formation of oxidative stress, proteins and metabolites that are linked to GSH/GSSG signaling [41,42,43,44,45,46,47] were examined rst ( Figure 1b). In type II cells (Figure 1b, left), SiO2_40, SiO2_7 and TiO2_NM105 were the only NMs with signi cant changes after treatment with 10 µg/cm 2 , which is the dose we used in previous screening experiments with twelve NMs in type II cells as well [11]. Interestingly, SiO2_15_Unmod and SiO2_15_Amino showed the same trends, with SiO2_15_Unmod leading to higher elevations (expressed as fold changes, FCs) than…”

“…Cell culture of alveolar macrophages (cell line NR8383, ATCC, CRL-2192, USA) and alveolar type II cells (cell line RLE-6TN, ATCC, CRL-2300, USA) was conducted as described before [11,12].…”