The differentially expressed gene lists were uploaded to Ingenuity Pathway Analysis (IPA) to delineate the signaling pathways affected. IPA advises that more than 200 DEGs are needed to get a reasonable picture of the pathway alteration, therefore, the lists from X5 at 30 µg/ml, Q at 100 µg/ml and Y6 at 3 and 100 µg/ml were not used in this analysis since they had less than 200 genes altered. The top 12 altered signaling pathways of each CeO2 treatment are listed in Table 4. The most affected signaling pathway category was stress induced response (highlighted yellow in Table 4); a total 45 pathways out of possible 132 (12 x11) pathways were in this category. This was followed by cell cycle-related pathway, a total 18 pathways out of 132.
3.4.1. Stress induced response
a. Sirtuin signaling pathway
The sirtuin signaling pathway was altered by many of the treatments in this study; the only one treatment where the sirtuin signaling pathway was not affected is the X5 at 3 µg/ml (Tables 5 and 6). Sirtuin signaling pathway ranked very high in most of the treatments, usually among top 3, but for X5 (0.3 µg/ml) and Y6, they ranked lower than 15. Sirtuins are a family of proteins that possess either ADP-ribosyltransferase or deacetylase activity, including deacetylase, desuccinylase, depalmitoylase, demyristoylase and demalonylase activity [30, 31]. The redox sensitive sirtuin family of 7 proteins have been shown to play a key roles in many cellular functions like protein acylation and deacetylation, histone deacetylation [32], as well as several antioxidant and oxidative stress related processes, such as DNA damage response, mitochondrial biogenesis and function, glucose and fatty acid metabolism, cell cycle regulation, inflammation and more [37]. The sirtuin signaling pathway also crosstalks with NRF-2 mediated oxidative response pathway [33, 34]. Sirtuin proteins use NAD+ as a cofactor. NAD+ is a key redox signaling molecule in the cells. The NAD+/NADH ratio is a key component of the redox state of a cell and an indication of the status of metabolism and health of cells [35].
Table 5
Ranking of some relevant signaling pathways from IPA analysis.
CeO2 particle name | W4 | X5 | Y6 | Z7 | Q |
Concentration (µg/ml) | 3 | 30 | 100 | 0.3 | 3 | 30 | 0.3 | 3 | 30 | 30 | 300 |
# of DEGs | 5,164 | 6,553 | 5,937 | 2,063 | 1,064 | 2,096 | 8,598 | 4,976 | 3,768 | 2,029 | 3,118 |
# of altered pathways | 216 | 257 | 271 | 235 | 93 | 94 | 278 | 200 | 201 | 155 | 128 |
| Protein Ubiquitination Pathway | Sirtuin Signaling Pathway | NRF2-mediated Oxidative Stress Response | Acute Phase Response Signaling | Cleavage and Polyadenylation of Pre-mRNA | Kinetochore Metaphase Signaling Pathway | Protein Ubiquitination Pathway | Sirtuin Signaling Pathway | Sirtuin Signaling Pathway | NER Pathway | Sirtuin Signaling Pathway |
| Sirtuin Signaling Pathway | Protein Ubiquitination Pathway | Sirtuin Signaling Pathway | ERK/MAPK Signaling | Intrinsic Prothrombin Activation Pathway | Mitotic Roles of Polo-Like Kinase | Sirtuin Signaling Pathway | Protein Ubiquitination Pathway | HER-2 Signaling in Breast Cancer | Colanic Acid Building Blocks Biosynthesis | Kinetochore Metaphase Signaling Pathway |
| NRF2-mediated Oxidative Stress Response | NRF2-mediated Oxidative Stress Response | Molecular Mechanisms of Cancer | Telomerase Signaling | IGF-1 Signaling | Cell Cycle: G2/M DNA Damage Checkpoint Regulation | Mitochondrial Dysfunction | Cell Cycle Control of Chromosomal Replication | NER Pathway | Sirtuin Signaling Pathway | Protein Ubiquitination Pathway |
| Germ Cell-Sertoli Cell Junction Signaling | Senescence Pathway | Cell Cycle Control of Chromosomal Replication | SAPK/JNK Signaling | Role of Tissue Factor in Cancer | NER Pathway | NRF2-mediated Oxidative Stress Response | PI3K/AKT Signaling | PEDF Signaling | Protein Ubiquitination Pathway | Mitochondrial Dysfunction |
| Kinetochore Metaphase Signaling Pathway | NER Pathway | Mitochondrial Dysfunction | Non-Small Cell Lung Cancer Signaling | Colanic Acid Building Blocks Biosynthesis | Inhibition of ARE-Mediated mRNA Degradation Pathway | NER Pathway | Molecular Mechanisms of Cancer | IL-15 Signaling | Mitochondrial Dysfunction | Oxidative Phosphorylation |
| Mitochondrial Dysfunction | Mitochondrial Dysfunction | Acute Phase Response Signaling | Chronic Myeloid Leukemia Signaling | Virus Entry via Endocytic Pathways | Role of CHK Proteins in Cell Cycle Checkpoint Control | Acute Phase Response Signaling | Colanic Acid Building Blocks Biosynthesis | Molecular Mechanisms of Cancer | Mitotic Roles of Polo-Like Kinase | ATM Signaling |
| Cell Cycle Control of Chromosomal Replication | Aryl Hydrocarbon Receptor Signaling | PDGF Signaling | Prostate Cancer Signaling | Extrinsic Prothrombin Activation Pathway | Osteoarthritis Pathway | Aryl Hydrocarbon Receptor Signaling | Germ Cell-Sertoli Cell Junction Signaling | Mitochondrial Dysfunction | Kinetochore Metaphase Signaling Pathway | Cell Cycle Control of Chromosomal Replication |
| NER Pathway | Cell Cycle Control of Chromosomal Replication | Germ Cell-Sertoli Cell Junction Signaling | Oncostatin M Signaling | MSP-RON Signaling In Cancer Cells Pathway | ATM Signaling | Molecular Mechanisms of Cancer | FXR/RXR Activation | Role of Tissue Factor in Cancer | Phagosome Maturation | Noradrenaline and Adrenaline Degradation |
| Acute Phase Response Signaling | IGF-1 Signaling | Protein Ubiquitination Pathway | Role of Tissue Factor in Cancer | Angiopoietin Signaling | Mismatch Repair in Eukaryotes | Senescence Pathway | Cyclins and Cell Cycle Regulation | Hypoxia Signaling in the Cardiovascular System | GDP-mannose Biosynthesis | Cell Cycle: G2/M DNA Damage Checkpoint Regulation |
| Molecular Mechanisms of Cancer | Molecular Mechanisms of Cancer | 14-3-3-mediated Signaling | Regulation of eIF4 and p70S6K Signaling | ERK/MAPK Signaling | BEX2 Signaling Pathway | Cell Cycle: G1/S Checkpoint Regulation | Cell Cycle: G2/M DNA Damage Checkpoint Regulation | 14-3-3-mediated Signaling | Role of PKR in Interferon Induction and Antiviral Response | Cyclins and Cell Cycle Regulation |
| Oxidative Phosphorylation | Colanic Acid Building Blocks Biosynthesis | HER-2 Signaling in Breast Cancer | PI3K/AKT Signaling | Galactose Degradation I (Leloir Pathway) | Androgen Signaling | Hypoxia Signaling in the Cardiovascular System | ERK/MAPK Signaling | Pancreatic Adenocarcinoma Signaling | Acute Phase Response Signaling | Aryl Hydrocarbon Receptor Signaling |
| TWEAK Signaling | Hypoxia Signaling in the Cardiovascular System | PTEN Signaling | IL-6 Signaling | Acute Phase Response Signaling | Nucleotide Excision Repair Pathway | Cell Cycle Control of Chromosomal Replication | 14-3-3-mediated Signaling | Oxidative Phosphorylation | NRF2-mediated Oxidative Stress Response | Hypoxia Signaling in the Cardiovascular System |
Key: | | stress response | | |
| | cancer related pathways | |
| | biochemical/metabolism pathways | |
| | cell cycle regulation | |
| | receptor mediated response | |
| | protein synthesis related | |
| | inflammation related | |
| | cell proliferation related | |
| | aging related | | |
| | sirtuin signaling pathway | |
Table 6
Gene expression fold changes (treated compared to untreated controls) in key signaling pathways in HepG2 cells following CeO2 treatment.
| Particle name | W4 | X5 | Y6 | Z7 | Q |
| Concentration (µg/ml) | 3 | 30 | 100 | 0.3 | 3 | 30 | 0.3 | 3 | 30 | 30 | 300 |
| # of altered pathways | 216 | 257 | 271 | 235 | 93 | 94 | 236 | 200 | 201 | 155 | 128 |
Stress response | Sirtuin signaling pathway | 2 | 1 | 2 | 26 | | 19 | 2 | 1 | 1 | 3 | 1 |
NRF2-mediated oxidative stress response | 3 | 3 | 1 | 104 | | | 4 | 38 | 15 | 12 | 24 |
Mitochondrial Dysfunction | 6 | 6 | 5 | | | | 3 | 18 | 7 | 5 | 4 |
Protein Ubiquitination Pathway | 1 | 2 | 9 | 27 | | | 1 | 2 | 30 | 4 | 3 |
Acute Phase Response Signaling | 9 | 13 | 6 | 1 | 12 | 45 | 6 | 37 | 113 | 11 | 50 |
p53 | 139 | 69 | 55 | 149 | | | 14 | 55 | 81 | | |
NER pathway | 8 | 5 | 19 | | | 4 | 5 | 13 | 3 | 1 | 22 |
Glutathione redox reaction I | 116 | 119 | | | | | 146 | 137 | 194 | 54 | 46 |
Fatty acid metabolism | TR/RXR Activation | 128 | 173 | | 130 | 61 | | 201 | | | | |
PPARα/RXRα Activation | | 196 | | 82 | 68 | | 243 | | | | |
LXR/RXR activation | 154 | | 186 | 198 | | 22 | 120 | | | | 96 |
FXR/RXR activation | 43 | 90 | 65 | | | 50 | 74 | 8 | 22 | 21 | 41 |
PPAR signaling pathway | 102 | 46 | 86 | 23 | 49 | 64 | 44 | 129 | 90 | 20 | |
fatty acid beta oxidation | | | 171 | 195 | | | 173 | | | | |
Cell cycle regulations | Cell Cycle Control of Chromosomal Replication | 7 | 8 | 4 | | | 82 | 12 | 3 | 28 | 34 | 7 |
Cell Cycle: G2/M DNA Damage Checkpoint Regulation | 30 | 24 | 21 | 19 | | 3 | 18 | 10 | 62 | 62 | 9 |
Cell Cycle: G1/S Checkpoint Regulation | 42 | 48 | 75 | 66 | | | 10 | 43 | 107 | | 37 |
Kinetochore Metaphase Signaling Pathway | 5 | 43 | 124 | 28 | | 1 | 69 | 50 | 35 | 7 | 2 |
Metabolism related | Glycolysis I | 143 | 159 | 168 | | 84 | | | | | 76 | |
TCA cycle | | 229 | | | | | 73 | | | | 21 |
Fatty acid beta oxidation I | | | 171 | 195 | | | | | | | |
Oxidative phosphorylation | 11 | 27 | 87 | | | | 16 | 45 | 12 | 16 | 6 |
Proliferation | IGF-1 signaling | 13 | 9 | 30 | 16 | 3 | | 34 | 26 | 42 | 19 | |
mTOR signaling | 54 | 32 | 68 | 105 | | | 77 | 57 | 49 | | |
HGF signaling | 68 | 38 | 14 | 71 | 80 | | 94 | 92 | 43 | | |
PI3/AKT signaling | 36 | 29 | 15 | 11 | 57 | 28 | 53 | 4 | 31 | 129 | |
ERK/MAPK signaling | 40 | 19 | 32 | 2 | 10 | | 86 | 11 | 33 | | |
Inflammation | NF-κB Activation by Viruses | 46 | 101 | 39 | 106 | 19 | | 55 | 73 | 77 | 94 | |
IL-6 signaling | 101 | 75 | 34 | 12 | 18 | | 81 | 150 | 166 | 118 | |
Interferon signaling | na | 86 | 41 | | | | 27 | | | | |
IL-3 signaling | 90 | 96 | 50 | 32 | 51 | | 215 | 169 | 65 | 106 | |
IL-8 signaling | 106 | 149 | 74 | 213 | | | 72 | | | | |
Cancer-related pathways | Molecular Mechanisms of Cancer | 10 | 10 | 3 | 64 | 64 | 65 | 8 | 5 | 6 | 23 | 55 |
Non-Small Cell Lung Cancer Signaling | 191 | 62 | 69 | 5 | 65 | | 50 | 82 | 156 | 126 | 100 |
Chronic Myeloid Leukemia Signaling | 25 | 28 | 79 | 6 | 45 | | 29 | 41 | 58 | | 79 |
Prostate Cancer Signaling | 64 | 22 | 56 | 7 | 47 | | 20 | 86 | 50 | 144 | 89 |
Role of Tissue Factor in Cancer | 120 | 33 | 36 | 9 | 4 | | 65 | 81 | 8 | 32 | 76 |
HER-2 Signaling in Breast Cancer | 15 | 16 | 11 | 52 | 31 | | 13 | 40 | 2 | 18 | 94 |
Glioma Invasiveness Signaling | 16 | 105 | 89 | 127 | | | 102 | 126 | | | |
Pancreatic Adenocarcinoma Signaling | 100 | 120 | 71 | 63 | 54 | | 22 | 87 | 11 | 46 | 47 |
Empty cell: not altered in IPA analysis. | | | | | | | | | | |
| Particle name | W4 | X5 | Y6 | Z7 | Q |
| Concentration (µg/ml) | 3 | 30 | 100 | 0.3 | 3 | 30 | 3 | 30 | 0.3 | 3 | 30 | 3 | 30 | 300 |
Sirtuin | SIRT7 | | | 1.14 | | | | | | | | | | | |
SIRT5 | | -1.14 | -1.14 | | | | | -1.15 | -1.15 | -1.16 | -1.18 | | | |
SIRT6 | | | | | | | | 1.19 | 1.13 | | | | | |
SIRT2 | | | | | | | | 1.14 | | | | | | |
NFR2-mediated oxidative stress response | NRF2 | | | | | | | | | | | | | | -1.3 |
KEAP1 | -1.38 | | | | | | | | -1.34 | | | | -1.29 | |
JUN | | | 1.26 | | | | | | | | | | | |
JUNB | | | 1.27 | | | | | | | | | | | |
Mitochondrial dysfunction | SOD2 | 1.67 | 1.47 | | 1.50 | | 1.27 | | | 1.33 | 1.48 | 1.37 | | 1.27 | 1.34 |
GPX1 | | -1.93 | | | | | | | | | -1.55 | | -1.65 | -1.94 |
GPX2 | | -1.29 | | | | | | | | | -1.28 | | | |
MAOB | | | -1.41 | | | | | | | | | | | -1.30 |
CYB5R3 | | | -1.42 | | | | | | | | | | | |
CASP 3 | | | 1.42 | | | | | | | | 1.32 | | | 1.21 |
Cytochrome C | | | 1.38 | | | | | | | | 1.27 | | | 1.25 |
CAT | | | | | | | | | | | | | | |
TRAK1 | | | | | | | | | | | | | | |
ACO2 | | | | | | | | 1.25 | | | | | | |
mtSOD | | | 1.35 | | | | | | | | 1.37 | | | 1.34 |
Fatty acid synthesis | FASN | | | -1.54 | | | | | | -1.65 | | | | | |
SCD1 | | | | | | | | -1.23 | | | | | | |
Fatty acid uptake | CD36 | | | | | | | | -1.17 | | | | | | |
SLC27A1 (FATP1) | | -1.36 | | | | | | | | | -1.33 | | | |
SLC27A2 | | -1.23 | | | | | | -1.28 | | | -1.18 | | | -1.16 |
SLC27A3 | | -1.21 | | | | | | | | | | | | |
SLC27A5 (FATP5) | | -1.41 | | | | | | | | | | | | |
Fatty acid export | APO A1 | | -1.34 | -1.41 | | | | | | | -1.34 | | | | -1.28 |
APO A5 | | | | | | | | 1.26 | | | | | | |
APOB100 | | | -1.3 | | | | | | | -1.51 | -1.45 | | | -1.37 |
APO CIII | | -1.61 | -1.73 | | | | | | | -1.49 | | | -1.38 | 1.59 |
ABCG8 | | -1.21 | -1.19 | | | | | | | -1.22 | -1.18 | | -1.17 | -1.21 |
Fatty acid activation | ACSL3 | | | | | | | | -1.26 | | | | | | |
ACSL4 | | | | | | | | | | | -1.57 | | | -1.67 |
ACSL5 | | 1.56 | | | | | | | | | 1.59 | | | 1.72 |
SLC27A1 (FATP1) | | -1.36 | | | | | | | | | -1.33 | | | |
SLC27A2 | | -1.23 | | | | | | -1.28 | | | -1.18 | | | -1.16 |
SLC27A3 | | -1.21 | | | | | | | | | | | | |
SLC27A5 | | -1.41 | | | | | | | | | | | | |
CPT2 | -1.25 | | -1.23 | | | | | | | | | | | |
Dehydrogen-ation | ACAD11 (Acyl CoA dehydrogenase) | | 1.46 | | | | | | -1.26 | | | | | | |
IVD(ACAD2) | -1.86 | -1.52 | -1.60 | | | | | | | -1.42 | -1.53 | | | -1.42 |
ACAD10 | | | -1.24 | | | | | | | | -1.18 | | -1.43 | |
ACAD9 | | -1.59 | -1.63 | -1.40 | -1.53 | | | -1.27 | | | -1.58 | | -1.38 | -1.34 |
ACADM | 1.50 | 1.73 | 1.65 | | | | | | | 1.35 | | | | |
ACADS | | -1.37 | -1.31 | | | | | | -1.41 | | -1.31 | -1.22 | -1.25 | -1.23 |
ACADSB | | 1.71 | 1.52 | | | | | | | 1.29 | 1.21 | | 1.22 | |
Hydration | ACADVL | | -1.30 | -1.35 | | | | | | | | -1.42 | | | |
Oxidation | ECHS1 (enoyl CoA hydrolase Short-chain 1) | -1.42 | -1.56 | -1.6 | | | | | | -2.1 | | -1.49 | | | |
HADH | | | -1.27 | | | | | -1.37 | | | | | | |
HADHB | | | | 1.22 | | | | | | | | | | |
HSD17B8 | -2.15 | -2 | -2 | 1.54 | | | | | | -2.05 | -1.77 | | -1.58 | -1.63 |
HSD17B4 | | | | | | | | -1.22 | | | | | | -1.24 |
Thiolysis | HADHA | | | -1.34 | | | | | | | | | | | |
ACAA1 | -1.39 | | | | | | | | | | | | -1.34 | |
ACAA2 | | -1.19 | | | | | | -1.19 | | | | | | |
Alpha- oxidation in peroxisomes | ALDH2 (aldehyde-dehydrogenase) | -1.55 | -1.28 | -1.36 | | | | | | -1.5 | -1.3 | | | 1.26 | -1.22 |
ALDH3B1 | -1.23 | -1.19 | -120 | | -1.14 | | | | -1.21 | -1.22 | -1.22 | | -1.15 | -1.17 |
ALDH4A1 | -1.22 | -1.27 | -1.37 | | | | | | -1.49 | -1.27 | -1.22 | | -1.19 | -1.34 |
ALDH1A1 | -1.27 | -1.2 | | | | | | | | -1.33 | -1.35 | | | -1.42 |
ALDH1B1 | | -1.6 | | | | | | | | | | | | |
ALDH7A1 | | -1.17 | | | | | | | -1.09 | | -1.15 | | | |
ALDH9A1 | 1.32 | 1.33 | | | | | | | | 1.41 | 1.34 | | | |
ALDH3A2 | | | | | | | | -1.24 | | | | | | 1.12 |
Beta oxidation III | ECL1 (enoyl-CoA delta isomerase) | -1.5 | -1.52 | -1.56 | | | | | | -2.08 | | | | -1.32 | |
EHHADH | | | | | | | | | 1.24 | | | | | |
Empty cell: no change detected | | | | | | | | | | | | | |
Table 7. Pattern of HepG2 fatty acid accumulation from metabolomics data compared with fatty acid synthesis, uptake, export and oxidation data from genomic studies. | |
| CeO2 particle name | W4 | X5 | Y6 | Z7 | Q |
Metabolomic data | concentration (µg/ml) | 30 | 30 | 30 | 30 | 100 |
fatty acid accumulation | ↑↑↑ | ↑↑↑ | -- | ↑↑↑ | ↑ |
Genomic data | concentration (µg/ml) | 30 | 30a | 30 | 30 | 300 |
fatty acid synthesis | -- | -- | ↓ | -- | -- |
fatty acid uptake | ↓↓↓ | -- | ↓ | ↓ | ↓ |
fatty acid export | ↓↓↓ | -- | ↑ | ↓ | ↓ |
fatty acid oxidation | ↓↓↓ | -- | ↓ | ↓↓↓ | ↓↓ |
Key: | | | | |
-- | no change detected | |
↑ | small increases | |
↑↑↑ | large increases | |
↓ | small decreases | |
↓↓ | medium decreases | |
↓↓↓ | large decreases | |
a: The number of DEGs for X5 was 2,063 at 0.3, 1,264 at 3.0 and 155 at 30 µg/ml, the |
30 µg/ml genomics data for X5 did not be used in IPA because the number of altered genes was too small. |
The most important and most studied sirtuin genes are SIRT 1 and SIRT3. Even though the expression levels of these 2 genes were not altered in our study, but there are many reports on the sirtuin proteins being post-translationally modified including phosphorylation and ubiquitination [36] and also post-transcriptionally regulated by microRNA [37]. Taken together with the data from metabolomics that NAD+ levels were affected [19], most likely the activities of the sirtuin proteins in these cells were affected and resulted in the altered sirtuin signaling pathway.
We speculate that sirtuin signaling pathway is one of the major contributors to the changes in the metabolism including fatty acid accumulation after treating with the CeO2 particles. Among the 4 CeO2 particles that induced fatty acid accumulation, W4, Z7 and Q all have altered sirtuin signaling pathway, at all concentrations and all ranked very high. Even though X5 also induced fatty acid accumulations at 30 µg/ml, the sirtuin signaling pathway was either not affected or ranked very low. Since there were only 155 genes altered, and did not put into IPA for pathway analysis, whether X5 at 30 µg/ml caused fatty acid accumulation through sirtuin signaling pathway still waits more studies. The direction of the change, either upregulation or downregulation, is not clear at the time of assay which was 72 hours after treatment. The long-term effects of these particles on the sirtuin signaling pathway await longer treatments.
Since sirtuin is an oxidative stress and energy sensor [38], uses NAD+ as a cofactor, and also regulates lipid and glucose metabolism; it is not surprising that glycolysis, fatty acid beta oxidation, tricarboxylic acid cycle and oxidative phosphorylation were also affected in some of the cells treated by these CeO2 particles, e.g., W4, Z7 and Q (Table 5). However, these pathways were not affected in 30 µg/ml Y6 treated cells, indicating a less oxidative stress environment for this treatment.
b. NRF2 mediated oxidative stress response and mitochondrial dysfunction
There have been many reports on oxidative stress induced by nanoparticles [39, 40]. NRF2 is the master regulator of cellular redox homeostasis and is a regulator of cytoprotective genes. The upregulation of NRF2 signaling pathway will lead to upregulation of a group of antioxidant genes, and the protection of the cells from oxidative stress. NRF2 expression also affects mitochondrial function. NRF2 deficiency leads to impaired mitochondrial fatty acid oxidation, while activation of NRF2 supports mitochondrial integrity by conferring resistance to oxidative stress induced membrane permeability changes [41]. These two pathways were not affected in Y6 30 µg/ml and X5 3 µg/ml treated samples and were ranked very low (#104) on the list for X5 0.3 µg/ml treated samples, indicating less oxidative stress than other particles treated samples. This is consistent with the cytotoxicity results showing X5 and Y6 did not induce cytotoxicity at the concentrations we tested.
Mitochondrial dysfunction is ranked high in W4, Q and Z7, and was not altered in Y6. Mitochondria are the powerhouse of the cells. It is where the beta oxidation of fatty acids, tricarboxylic acid cycle (TCA, citric acid or Krebs) cycle and oxidative phosphorylation take place. The pyruvate molecules produced from glycolysis are transported to the mitochondria, converted to acetyl-CoA and enter the TCA cycle in the matrix of mitochondria. Acetyl-CoA could also be derived from catabolism of protein and lipid (beta oxidation). The TCA cycle consumes acetate (in the form of acetyl-CoA) and water, reduces NAD+ to NADH, and releases carbon dioxide. The NADH generated from TCA cycle, then, feeds into oxidative phosphorylation (electron transfer) pathway to generate ATP. This oxidative phosphorylation takes place in the inner membrane of the mitochondrion.
Since beta oxidation, TCA cycle and oxidative phosphorylation all take place in the mitochondria, dysfunction of the mitochondria might cause any of these not working properly. And the lipid accumulation data from prior CeO2 metabolomics correlated well with genomic data. W4, Z7 and Q all induced fatty acid accumulation and also caused alteration in the mitochondrial dysfunction signaling pathway. Conversely, Y6 did not cause fatty acid accumulation and did not cause alteration in this signaling pathway. Mitochondrial dysfunction was not altered in X5, however, X5 did induce accumulation of fatty acid [19]. Therefore, the mechanism(s) for X5 induced fatty acid accumulation might be different from that of W4, Q and Z7 forms of CeO2.
c. Protein ubiquitination pathway
Ubiquitination is a post-translational modification of proteins that involves adding one or more ubiquitin, a small 8.6 KD protein, to the to-be-modified protein. Ubiquitin itself can be ubiquitinated at different positions. The target protein, once ubiquitinated, can change its activity, localization, structure and interaction with its partner proteins [42]. This highly versatile post-translational modification controls virtually all types cellular events [43]. Many of the altered signaling pathways in this study such as stress response, inflammation, endocytic trafficking, DNA repair, cell cycle progression, cancers and many others are regulated by ubiquitination [44].
NRF2 is one of the best studied ubiquitination regulated proteins. When oxidative stress levels are normal, NRF2 protein is bound by KEPA1, its ubiquitin ligase, and cellular NRF2 is constantly ubiquitinated and transported to the proteasome to be degraded. Once the oxidative stress levels are increased, KEAP1 become oxidized at cysteines and this disrupts the KEAP1-NRF2 complex, and NRF2 is released from the KEAP1 inhibition and promotes the stabilization of NRF2 to induce gene expression in this pathway. In addition to NRF2, sirtuin signaling pathway, p53 signaling, Tumor Necrosis Factor (TNF) signaling, and NF-κB signaling are also regulated by ubiquitination. In the sirtuin signaling pathway, SIRT1, SIRT3 and SIRT5 are regulated by ubiquitination. SMURF2, SKP2 and SCF-bound F complex (SCFcyclin−F), are ubiquitin-ligase for SIRT 1, 3 and 5 respectively [45–48]. Many of the DNA damage response pathways, such as the p53 signaling pathway, and many of the cell cycle regulation pathways are also regulated by ubiquitination. In the p53 signaling pathway, MDM2 is the ubiquitin ligase for p53 protein [49], while the anaphase promoting complex/cytochrome (APC/C) and SKP, cullin, F-box containing complex (SCF) are the ubiquitin ligase for many of the cyclins and the cyclin dependent kinase -inhibitors, such as cyclin A, E, p21, p27 [50]. The protein ubiquitination pathway was altered in many of the CeO2 particle treated samples in this study except Y6 30 µg/ml and X5 0.3 µg/ml, indicating the wide range of effects of these CeO2 particles that occur via this signaling pathway. Fatty acid accumulation might be related to the alteration of the ubiquitination pathway [51, 52].