To get insights into NMs MoAs, the effects of four silica NMs (SiO2_15_Unmod, SiO2_15_Amino, SiO2_40, SiO2_7) and TiO2_NM105 were investigated in vitro and in vivo. The physico-chemical properties of these NMs have been described before [11, 12, 25, 38, 39, 40], and a summary of their key physico-chemical properties is presented in Table 1.
3.1 In vitro investigations
First, the effects of these five NMs were investigated in vitro in alveolar type II cells and alveolar macrophages at three different doses after an exposure time of 24 h. Changes of protein (Additional file 2: Figure S1 – Figure S4) and metabolite (Additional file 2: Figure S5 – Figure S8) abundances were considered relative to untreated controls and the summary of the percentages of significantly (p.adj ≤ 0.05) altered proteins and metabolites (Figure 1a) shows that significant changes appeared for metabolites only at the highest investigated dose in type II cells. Interestingly, the pyrogenic SiO2_40 and SiO2_7 were the only NMs, which significantly affected metabolites also at a dose of 10 µg/cm2 in type II cells and macrophages, where none of the other tested NMs resulted in significant alterations. TiO2_NM105 showed significantly changed metabolites at a dose of 10 µg/cm2 in type II cells but not in macrophages. In contrast, almost no significantly altered proteins were observable in type II cells, while dose-dependent increases of the portion of significantly 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 classified as “passive”.
Additionally, exposure times of 6 h and 48 h were investigated for type II cells. After 6 h of NM treatment, a maximum of 72 significantly altered proteins was observable for 10 µg/cm2 SiO2_7, compared to 220 and 462 proteins under the same conditions but after 24 h and 48 h incubation, respectively (Table E3). Thus, the 6 h time point was excluded from further analyses.
An enrichment analysis was applied to the significantly (p.adj ≤ 0.05) altered proteins and metabolites, which showed mainly enrichment of pathways that are related to oxidative stress, e.g. mitochondrial dysfunction, Nrf2-mediated oxidative stress response and oxidative phosphorylation (Additional file 2: Figure S9). Thus, we focused on the investigation of the levels of oxidative stress that were reached under the tested conditions. For this purpose, we used only results after 24 h treatment because only minor time-dependent changes were observable between 24 h and 48 h (Additional file 2: Figure S9 and Figure S10).
3.1.1 GSH/GSSG signaling
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 first (Figure 1b). In type II cells (Figure 1b, left), SiO2_40, SiO2_7 and TiO2_NM105 were the only NMs with significant changes after treatment with 10 µg/cm2, 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 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, confirming that not only SiO2_40 and SiO2_7 are “active” NMs but also TiO2_NM105, which is consistent with our previous study [11].
Interestingly, the opposite effects were observed for macrophages (Figure 1b, right) compared to type II cells (Figure 1b, left). Most significant changes were obtained for SiO2_7 and SiO2_40. SiO2_15_Unmod showed the same trends, even though with less significance. The decreased abundances of proteins and metabolites suggest a decreased GSH production for these three NMs in macrophages. This in turn may have led to an insufficient neutralization of appearing ROS, thus inducing further oxidative stress reactions. The opposite changes in type II cells and macrophages occurred especially in the treatment with 10 µg/cm2 SiO2_7 (Figure 1c).
3.1.2 Comparison of oxidative stress levels
To clarify whether the observed effects on the GSH/GSSG signaling for the two cell lines led to a different assignment to the three tiers of oxidative stress, the changes of proteins and metabolites that are connected to these tiers were investigated. Interestingly, all the proteins that are shown in Figure 1b and Figure 1c are regulated by the transcription factor Nrf2 [48, 49, 50, 51], a hallmark of tier 1, suggesting that the NMs led to tier 1 in the used type II cells at the highest applied dose. In macrophages, decreased abundances of analytes that are connected to GSH/GSSG signaling were observed for SiO2_7, SiO2_40, and SiO2_15_Unmod, suggesting that these three NMs led to a decreased GSH/GSSG ratio, while SiO2_15_Amino and TiO2_NM105 did not.
Besides the proteins that are part of the GSH/GSSG signaling, Gstm1, Nqo1, Hmox1, Txn, Txnrd1, Cat, Sod1, Sod2 and Lamp2 have been described to be Nrf2 targets [48, 49, 52] and thus appear relevant for tier 1 (Figure 2a).
Tier 2 is induced by activation of Nfκb and Ap-1 target genes. Importantly, several of the already described Nrf2 target genes have also a binding position for Nfκb or Ap-1. Examples are Gclc, Idh, Pgd, Phgdh, Hmox1, Nqo1, Cat, Sod2, and Lamp2 [53, 54]. Furthermore, Icam1 [55, 56], Il18 [57, 58], B2m [59], and Tnfaip8 [60] have been described to be target genes of Nfκb or Ap-1 and related to either inflammation or apoptosis. Ccr1 has been described to be expressed mitogen-activated protein kinase (Mapk)-dependently [61]. Bax has been described to be an Nfκb target protein [62, 63], and it is a pro-apoptotic protein since it is involved in the formation of mitochondrial pores that allow for the release of pro-apoptotic molecules from the mitochondria into the cytosol [64]. Another protein that is involved in the formation of mitochondrial pores and thus in the induction of apoptosis is Vdac1 [65], which consequently should be assigned to tier 3. Furthermore, it has been shown that an increased citric acid cycle (TCA) leads to the increased formation of ROS, followed by apoptosis [66]. Besides the already mentioned proteins, Idh, Glud1, and Fh are involved in the TCA [67, 68]. In addition, sphingomyelins (SMs), which belong to the class of sphingolipids and are mainly found in plasma membranes and lipoproteins, are relevant for the formation of ROS. SMs have been shown to be hydrolyzed in response to oxidative stress, thus resulting in the formation of ceramides, which act as second messengers that are involved in the induction of apoptosis. Importantly, the concrete mechanisms are not fully understood, yet [69, 70]. A summary of all mentioned analytes and their assignment to the different tiers of oxidative stress can be found in Tables S1 and S2.
In type II cells, almost no significant changes were noticed for all these candidates up to the highest tested dose (Figure 2a), while treatment of macrophages with 10 µg/cm2 SiO2_40 and SiO2_7 led to significant alterations. Importantly, SiO2_15_Unmod again showed the same trends as SiO2_40 and SiO2_7 but effects were less pronounced. Furthermore, major differences were recognizable between treated type II cells and macrophages once again, especially after treatment with 10 µg/cm2 SiO2_7 (Figure 2b). For type II cells significant changes appeared only in proteins that are Nrf2 target genes, thus confirming that in type II cells only tier 1 of oxidative stress was affected under the tested conditions. The significantly affected proteins were Sod1, Txn, Gstm1, and Lamp2. In contrast, the used macrophages led to significant changes over all tiers after treatment with 10 µg/cm2 SiO2_7. Interestingly, there are several cases in which opposite changes in protein abundance were visible in type II cells and macrophages. Examples are Sod1 and Sod2 with opposite changes among themselves and additionally, opposite changes in the two investigated cell lines. The fact that Sod1 and Sod2 resulted in different directions of alteration can be explained by their localization within the cell. While Sod1 can be found in the cytoplasm, Sod2 is responsible for neutralizing ROS in the mitochondria. Furthermore, both proteins have not only an Nrf2 binding site but also an Nfκb binding site in their promoter region. While the Nfκb binding site in Sod1 has been described to be rather insensitive, the binding site in Sod2 seems to be highly sensitive [71], thus suggesting the Sod1 expression to be predominantly induced by Nrf2, while the Sod2 expression may be induced by Nfκb. This is an additional evidence that type II cells led only to tier 1, whereas in macrophages all three tiers were initiated.
3.2 Comparison of in vivo and in vitro results
Next, we aimed to investigate the in vivo effects of SiO2_7 and TiO2_NM105 using STIS and of SiO2_15_Amino, SiO2_15_Unmod, SiO2_7, and SiO2_40 applying instillations. For both methods, changes in lung proteome and metabolome were investigated at several doses. Exposure groups (E) were sacrificed directly after treatment for 5 d or 3 d in STIS and instillations, respectively. In contrast, recovery groups (R) had a recovery time of 21 d after the treatment period. The percentages of significantly altered proteins (Additional file 2: Figure S11 – Figure S14) and metabolites (Additional file 2: Figure S15 – Figure S18) indicated dose-dependent increases of significantly altered analytes mainly for STIS exposure groups (Figure 3a). The assignment to the three tiers of oxidative stress in vivo was not as clear (Figure 3b) as for the in vitro results, even though significant changes were observable for all three tiers, especially in case of SiO2_7, SiO2_40, and TiO2_NM105, indicating those to be “active” in vivo. Furthermore, analytes with dose-dependent changes were detectable, e.g. Ornithine, Sod1, Sod2, and Txn.
The different results of the assignment to the three tiers in vitro and in vivo may be explained by the cellular composition of rat lungs (Figure 4a), with only 14.2% alveolar type II epithelial cells and 3% alveolar type II epithelial cells [72], and the circumstance that the two cell types investigated within the present study showed opposite effects. The comparison of analytes that are connected to the three tiers of oxidative stress after treatment with SiO2_7 in vitro and in vivo (Figure 4b) exemplifies this. For GSH/GSSG signaling, opposite effects were obtained for type II cells and macrophages, but no clear trends were observed in STIS and instillations. Analytes connected to the three tiers revealed no clear trends either, but the distributions (Figure 4b) indicated higher changes in macrophages and STIS, suggesting these model systems to be more sensitive.
Since the GSH/GSSG signaling did not reflect many of the significant changes in vivo (Figure 3a), Ingenuity Pathway Analysis (IPA, Qiagen) was used to identify NM effects in vivo. This analysis confirmed that oxidative stress pathways like glutathione redox reactions I, Nrf2-mediated oxidative stress response, and mitochondrial dysfunction were not among the most significantly enriched pathways (Additional file 2: Figure S19). Furthermore, chemokine signaling was found to be enriched mainly in case of the instillation studies with SiO2_7 and SiO2_40 (Figure 4c). Examples for pathways with higher enrichment were endocytosis signaling, phagosome maturation, leukocyte extravasion signaling, remodeling of epithelial adherens junctions, integrin signaling, and actin cytoskeleton signaling. Apoptosis signaling was again mainly enriched for SiO2_7 and SiO2_40 in the instillation studies, thus indicating that these two NMs had the strongest effects. Interestingly, most pathways showed more significant enrichment in the recovery groups than in the exposure groups. The complete results from the enrichment analysis can be found in the Additional file 1 (Table E25 – E30).
In summary, these results indicate the same classification of NMs in vitro and in vivo, although biological effects differed. SiO2_7, SiO2_40, and TiO2_NM105 were classified to be “active”, while SiO2_15_Amino led to almost no changes, thus suggesting it to be “passive”. SiO2_15_Unmod induced the same trends as SiO2_7 and SiO2_40, thus indicating that it might have adverse effects at higher doses.