<table><tbody><tr><td>MACF1</td><td>GTPase Binding) 3<br>microtubule-actin crosslinking factor 1</td><td>cytoskeleton<br>stabilization of microtubules</td><td>A_24_P418637</td><td>3.41</td><td>2.10E-04</td></tr><tr><td>PDZD8</td><td>PDZ domain containing 8</td><td>regulation of cell morphology and cytoskeletal organization</td><td>A_23_P321452</td><td>3.38</td><td>5.47E-05</td></tr><tr><td>TJP2</td><td>Tight Junction Protein 2</td><td>tight junctions and adherens junctions</td><td>A_33_P3282963</td><td>2.24</td><td>9.68E-05</td></tr><tr><td>HERC4</td><td>HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 4</td><td>protein trafficking or distribution of cellular structures</td><td>A_33_P3216994</td><td>2.07</td><td>7.18E-04</td></tr><tr><td>BRWD3</td><td>bromodomain and WD repeat domain containing 3</td><td>regulation of cell morphology and cytoskeletal organization</td><td>A_32_P489130</td><td>2.03</td><td>6.45E-04</td></tr><tr><td colspan="6"><strong>Metabolic process</strong></td></tr><tr><td colspan="6"><strong>Glycosylation</strong></td></tr><tr><td>NIPAL2</td><td>NIPA-like domain containing 2</td><td>-</td><td>A_23_P146111</td><td>2.40</td><td>3.40E-04</td></tr><tr><td>CSGALNACT2</td><td>Chondroitin Sulfate N-Acetylgalactosaminyltransferase 2</td><td>addition of the first GalNAc</td><td>A_33_P3266255</td><td>2.43</td><td>1.52E-05</td></tr><tr><td colspan="6"><strong>Regulation of cellular protein metabolic process (GO:0032268)</strong></td></tr><tr><td>EIF4G3</td><td>Eukaryotic Translation Initiation Factor 4 Gamma, 3</td><td>recruitment of mRNA to the ribosome</td><td>A_24_P383850</td><td>2.47</td><td>7.72E-04</td></tr><tr><td>RRBP1</td><td>Ribosome Binding Protein 1</td><td>interaction between the ribosome and the endoplasmic reticulum</td><td>A_33_P3220277</td><td>2.81</td><td>1.11E-04</td></tr><tr><td>PABPC4</td><td>Poly(A) Binding Protein, Cytoplasmic 4</td><td>regulatory processes of mRNA metabolism</td><td>A_33_P3271105</td><td>2.64</td><td>1.07E-04</td></tr><tr><td>LSM14B</td><td>LSM14B, SCD6 Homolog B</td><td>control of mRNA translation</td><td>A_33_P3349774</td><td>2.01</td><td>4.42E-05</td></tr><tr><td colspan="6"><strong>Cellular metabolism</strong></td></tr><tr><td>PRKAA1</td><td>Protein Kinase, AMP-Activated, Alpha 1 Catalytic Subunit</td><td>activates energy-producing pathways</td><td>A_33_P3312212</td><td>3.28</td><td>1.94E-05</td></tr><tr><td>LRRC8C</td><td>Leucine Rich Repeat Containing 8 Family, Member C</td><td>role in adipogenesis</td><td>A_33_P3406030</td><td>2.52</td><td>6.54E-04</td></tr><tr><td>PPP4R1L</td><td>Protein Phosphatase 4, Regulatory Subunit 1-Like</td><td>regulatory subunit of serine/threonine-protein phosphatase 4</td><td>A_19_P00808367</td><td>2.44</td><td>2.06E-04</td></tr><tr><td>MTRR</td><td>Methyltetrahydrofolate-Homocysteine Methyltransferase Reductase</td><td>maintenance of methionine synthase</td><td>A_33_P3304377</td><td>2.43</td><td>5.78E-04</td></tr><tr><td>SLC35A1</td><td>Solute Carrier Family 35 (CMP-Sialic Acid Transporter), Member A1</td><td>transports CMP-sialic acid from the cytosol into Golgi vesicles</td><td>A_33_P3394710</td><td>2.02</td><td>5.91E-04</td></tr><tr><td>TRAPPC2</td><td>Trafficking Protein Particle Complex 2</td><td>vesicular transport from endoplasmic reticulum to Golgi</td><td>A_33_P3407540</td><td>2.04</td><td>5.16E-05</td></tr><tr><td>CCZ1</td><td>CCZ1 Vacuolar Protein Trafficking And Biogenesis Associated Homolog</td><td>protein trafficking and biogenesis</td><td>A_33_P3333863</td><td>2.51</td><td>5.34E-04</td></tr><tr><td colspan="6"><strong>Cell cycle (GO:0007049)</strong></td></tr><tr><td>SESN2</td><td>sestrin 2</td><td>cell growth and survival in response to stress conditions</td><td>A_23_P35082</td><td>3.70</td><td>1.91E-04</td></tr><tr><td>LATS1</td><td>LATS, large tumor suppressor, homolog 1 (Drosophila)</td><td>tumor suppression by restricting proliferation and promoting apoptosis</td><td>A_23_P366230</td><td>2.05</td><td>2.46E-04</td></tr><tr><td>CHTOP</td><td>Chromatin Target Of PRMT1</td><td>cell cycle progression</td><td>A_33_P3243957</td><td>3.75</td><td>2.50E-04</td></tr></tbody></table>
<table><tbody><tr><td>E2F3</td><td>E2F Transcription Factor 3</td><td>control of cell cycle and action of tumor suppressor proteins</td><td>A_33_P3418516</td><td>3.64</td><td>1.47E-04</td></tr><tr><td>SRRT</td><td>Serrate RNA Effector Molecule Homolog (Arabidopsis)</td><td>cell cycle progression at S phase</td><td>A_33_P3299590</td><td>3.51</td><td>6.09E-04</td></tr><tr><td>WDR3</td><td>WD Repeat Domain 3</td><td>cell cycle progression, apoptosis...</td><td>A_33_P3211633</td><td>3.22</td><td>4.65E-04</td></tr><tr><td>PHF8</td><td>PHD Finger Protein 8</td><td>cell cycle progression</td><td>A_33_P3361925</td><td>2.17</td><td>6.45E-05</td></tr><tr><td>WT1</td><td>Wilms tumor 1</td><td>cellular development and cell survival</td><td>A_23_P116280</td><td>2.06</td><td>9.91E-04</td></tr><tr><td colspan="6"><strong>Proliferation</strong></td></tr><tr><td>VEGFA</td><td>vascular endothelial growth factor A</td><td>proliferation, cell migration, inhibits apoptosis</td><td>A_23_P70398</td><td>5.23</td><td>6.39E-04</td></tr><tr><td>PLAU</td><td>Plasminogen Activator, Urokinase</td><td>degradation of extracellular matrix, tumor cell migration, proliferation</td><td>A_33_P3306146</td><td>4.72</td><td>2.05E-04</td></tr><tr><td>PPP1R26</td><td>Protein Phosphatase 1, Regulatory Subunit 26</td><td>positively regulate cell proliferation</td><td>A_33_P3272614</td><td>2.56</td><td>1.82E-04</td></tr><tr><td>MAPK8</td><td>Mitogen-Activated Protein Kinase 8</td><td>proliferation, differentiation, transcription regulation and development</td><td>A_33_P3250289</td><td>2.03</td><td>9.18E-04</td></tr><tr><td colspan="6"><strong>Induction of apoptosis (GO:0006917)</strong></td></tr><tr><td>TRIB3</td><td>tribbles homolog 3 (Drosophila)</td><td>apoptosis, induced by NF-kappaB</td><td>A_23_P210690</td><td>2.34</td><td>1.77E-04</td></tr><tr><td>BBC3</td><td>BCL2 binding component 3</td><td>pro-apoptotic</td><td>A_23_P382775</td><td>3.12</td><td>5.29E-04</td></tr><tr><td>CHAC1</td><td>ChaC, Cation Transport Regulator Homolog 1</td><td>pro-apoptotic</td><td>A_33_P3376971</td><td>5.09</td><td>1.11E-04</td></tr><tr><td>FAM188A</td><td>Family With Sequence Similarity 188, Member A</td><td>pro-apoptotic</td><td>A_33_P3393106</td><td>3.61</td><td>1.42E-05</td></tr><tr><td>MLL</td><td>Myeloid/Lymphoid Or Mixed-Lineage Leukemia</td><td>promotes PPP1R15A-induced apoptosis</td><td>A_33_P3390576</td><td>2.10</td><td>2.85E-04</td></tr><tr><td colspan="6"><strong>Immune response (GO:0006955) and cell activation (GO:0001775)</strong></td></tr><tr><td>NFKBIA</td><td>nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha</td><td>cellular stimulation by immune and proinflammatory responses</td><td>A_23_P106002</td><td>4.27</td><td>6.92E-04</td></tr><tr><td>IRAK2</td><td>Interleukin-1 Receptor-Associated Kinase 2</td><td>participate in the IL1-induced upregulation of NF-kappaB</td><td>A_33_P3352970</td><td>4.05</td><td>1.33E-04</td></tr><tr><td>CCL4</td><td>chemokine (C-C motif) ligand 4</td><td>inflammatory and chemokinetic properties</td><td>A_23_P207564</td><td>15.01</td><td>3.65E-05</td></tr><tr><td>CCL20</td><td>chemokine (C-C motif) ligand 20</td><td>chemotactic factor for lymphocytes and neutrophils</td><td>A_23_P17065</td><td>6.80</td><td>3.19E-04</td></tr><tr><td>CCL3</td><td>chemokine (C-C Motif) Ligand 3</td><td>inflammatory and chemokinetic properties</td><td>A_33_P3316273</td><td>7.29</td><td>2.53E-04</td></tr><tr><td>CCL3L3</td><td>chemokine (C-C Motif) Ligand 3-Like</td><td>chemotactic for lymphocytes and monocytes</td><td>A_33_P3296181</td><td>9.30</td><td>3.08E-04</td></tr><tr><td>IL23A</td><td>interleukin 23, alpha subunit p19</td><td>functions in innate and adaptive immunity</td><td>A_23_P76078</td><td>3.83</td><td>9.11E-05</td></tr><tr><td>FCAR</td><td>Fc fragment of IgA, receptor for</td><td>cytokine production</td><td>A_23_P208768</td><td>3.69</td><td>1.84E-04</td></tr><tr><td>TAPBP</td><td>TAP binding protein (tapasin)</td><td>assembly of MHC class I with peptide</td><td>A_23_P360291</td><td>2.97</td><td>1.16E-04</td></tr><tr><td>LY6G5B</td><td>lymphocyte antigen 6 complex, locus G5B; casein kinase 2, beta polypeptide</td><td>located in the MHC</td><td>A_23_P59130</td><td>2.16</td><td>9.06E-04</td></tr><tr><td>PTGER4</td><td>prostaglandin E receptor 4</td><td>activate T-cell factor signaling</td><td>A_23_P148047</td><td>2.58</td><td>5.58E-04</td></tr></tbody></table>
<table><tbody><tr><td>PTPRC</td><td>(subtype EP4)<br>protein tyrosine phosphatase, receptor type, C</td><td>positive regulator of T-cell coactivation</td><td>A_23_P12392</td><td>2.45</td><td>2.06E-05</td></tr><tr><td>CD84</td><td>CD84 molecule</td><td>signaling lymphocyte activation molecule</td><td>A_24_P362193</td><td>2.83</td><td>6.06E-04</td></tr><tr><td>STAM</td><td>Signal Transducing Adaptor Molecule</td><td>role in T-cell development</td><td>A_33_P3287840</td><td>2.26</td><td>2.10E-04</td></tr><tr><td>INHBA</td><td>inhibin, beta A</td><td>hormone and insulin secretion, germ cell development and maturation...</td><td>A_23_P122924</td><td>6.58</td><td>4.58E-05</td></tr><tr><td>HIVEP1</td><td>Human Immunodeficiency Virus Type I Enhancer Binding Protein 1</td><td>transcriptional regulation of both viral and cellular genes</td><td>A_23_P19619</td><td>5.02</td><td>6.16E-04</td></tr><tr><td>PROS1</td><td>Protein S (Alpha)</td><td>activate protein C in the degradation of coagulation factors</td><td>A_21_P0012453</td><td>2.86</td><td>5.46E-04</td></tr><tr><td>CNTNAP3</td><td>Contactin Associated Protein-Like 3</td><td>NCP family of cell-recognition molecules</td><td>A_21_P0013569</td><td>2.61</td><td>7.29E-04</td></tr><tr><td colspan="6"><strong>Mitochondrion (GO:0005739)</strong></td></tr><tr><td>PPM1K</td><td>protein phosphatase, Mg2+/Mn2+ dependent, 1K</td><td>mitochondrial permeability transition pore, cellular survival and development</td><td>A_24_P214598</td><td>3.22</td><td>1.78E-04</td></tr><tr><td>ALDH1L2</td><td>Aldehyde Dehydrogenase 1 Family, Member L2</td><td>converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2</td><td>A_33_P3282634</td><td>3.45</td><td>9.26E-05</td></tr><tr><td>ZNF746</td><td>Zinc Finger Protein 746</td><td>repress transcription of PGC-1-alpha</td><td>A_33_P3330546</td><td>3.08</td><td>1.50E-04</td></tr><tr><td colspan="6"><strong>Endocytosis</strong></td></tr><tr><td>FAM21C</td><td>Family With Sequence Similarity 21, Member C</td><td>fluid-phase endocytosis</td><td>A_21_P0010870</td><td>3.78</td><td>2.62E-05</td></tr><tr><td>MYOF</td><td>myoferlin</td><td>plasma and nuclear membranes, involved in endocytic recycling</td><td>A_24_P282309</td><td>3.08</td><td>8.66E-05</td></tr><tr><td>ITSN2</td><td>intersectin 2</td><td>regulate formation of clathrin-coated vesicles</td><td>A_23_P434347</td><td>2.23</td><td>1.96E-04</td></tr><tr><td>AAK1</td><td>AP2 Associated Kinase 1</td><td>regulate clathrin mediated endocytosis</td><td>A_33_P3210583</td><td>2.17</td><td>6.45E-04</td></tr><tr><td>FCHO2</td><td>FCH domain only 2</td><td>regulate clathrin mediated endocytosis</td><td>A_23_P420610</td><td>2.02</td><td>9.65E-04</td></tr><tr><td colspan="6"><strong>Oxidative stress</strong></td></tr><tr><td>APP</td><td>Amyloid Beta (A4) Precursor Protein</td><td>Inflammatory response, involved in oxidative stress</td><td>A_33_P3296479</td><td>3.39</td><td>9.49E-04</td></tr><tr><td>MAP2K3</td><td>Mitogen-Activated Protein Kinase Kinase 3</td><td>activated by cytokines and stress, MAP kinase-mediated signaling cascade</td><td>A_33_P3234571</td><td>2.58</td><td>4.80E-04</td></tr><tr><td>GCH1</td><td>GTP Cyclohydrolase 1</td><td>positively regulates nitric oxide synthesis</td><td>A_33_P3311439</td><td>2.23</td><td>3.70E-04</td></tr><tr><td>HIF1A</td><td>Hypoxia Inducible Factor 1, Alpha Subunit</td><td>transcriptional regulator of the adaptive response to hypoxia</td><td>A_33_P3231277</td><td>2.74</td><td>4.90E-04</td></tr><tr><td>E2F7</td><td>E2F Transcription Factor 7</td><td>associates with HIF1A, recognizes VEGFA promoter</td><td>A_33_P3318661</td><td>2.58</td><td>3.13E-04</td></tr><tr><td colspan="6"><strong>Wnt signaling</strong></td></tr><tr><td>DISC1</td><td>Disrupted In Schizophrenia 1</td><td>Wnt-mediated proliferation,</td><td>A_21_P0000047</td><td>2.55</td><td>6.03E-04</td></tr></tbody></table>
<table><tr><td>TLE1</td><td>Transducin-Like Enhancer Of Split 1</td><td>inhibits activation of AKT-mTOR signaling<br/>inhibits the transcriptional activation in Wnt signaling</td><td>A_19_P00804020</td><td>2.18</td><td>7.55E-04</td></tr><tr><td colspan="6"><em>Notch signaling pathway</em></td></tr><tr><td>NOTCH2NL</td><td>ens|notch 2 N-terminal like [ENST00000344859]</td><td>function in the Notch signaling pathway</td><td>A_24_P366777</td><td>3.39</td><td>8.96E-04</td></tr><tr><td>USP12</td><td>Ubiquitin Specific Peptidase 12</td><td>negative regulator of Notch signaling pathway</td><td>A_24_P237613</td><td>3.15</td><td>4.59E-05</td></tr></table>
<table><caption>**Table S4.** Down-regulated genes identified in microarray analysis in response to 4h exposure with 50 µM of GSNO</caption><thead><tr><th>Gene</th><th>Description</th><th>Function</th><th>AGILENT_ID</th><th>FC</th><th>p value</th></tr></thead><tbody><tr><td colspan="6"><strong>Chromosomal part (GO:0044427)</strong></td></tr><tr><td>CENPA</td><td>Centromere Protein A<br>Holliday Junction Recognition</td><td>mitotic progression and<br>chromosome segregation<br>specific chaperone for<br>CENPA</td><td>A_24_P413884</td><td>0.46</td><td>2.43E-04</td></tr><tr><td>HJURP</td><td>Protein</td><td></td><td>A_33_P3807062</td><td>0.45</td><td>9.91E-04</td></tr><tr><td>HIST1H2AE</td><td>Histone Cluster 1, H2ae</td><td></td><td>A_23_P59045</td><td>0.32</td><td>3.06E-04</td></tr><tr><td>HIST1H2BE</td><td>Histone Cluster 1, H2be</td><td></td><td>A_33_P3302632</td><td>0.22</td><td>2.09E-04</td></tr><tr><td>HIST1H2BD</td><td>Histone Cluster 1, H2bd</td><td></td><td>A_24_P146211</td><td>0.46</td><td>7.45E-04</td></tr><tr><td>HIST1H2BI</td><td>Histone Cluster 1, H2bi</td><td></td><td>A_23_P111041</td><td>0.31</td><td>2.57E-05</td></tr><tr><td>HIST2H2BF</td><td>Histone cluster 2, H2bf</td><td></td><td>A_21_P0010654</td><td>0.42</td><td>3.00E-04</td></tr><tr><td>HIST1H2BG</td><td>Histone Cluster 1, H2bg</td><td></td><td>A_23_P167997</td><td>0.32</td><td>9.50E-04</td></tr><tr><td>HIST1H2BJ</td><td>Histone Cluster 1, H2bj</td><td></td><td>A_24_P55148</td><td>0.37</td><td>5.09E-05</td></tr><tr><td>HIST1H2BM</td><td>Histone Cluster 1, H2bm</td><td></td><td>A_24_P3783</td><td>0.31</td><td>6.77E-04</td></tr><tr><td>HIST1H3D</td><td>Histone Cluster 1, H3d</td><td></td><td>A_24_P217834</td><td>0.31</td><td>2.88E-04</td></tr><tr><td>HIST1H3H</td><td>Histone cluster 1, H3h</td><td>transcription regulation,<br>DNA repair and<br>replication, chromosomal<br>stability</td><td>A_33_P3404989</td><td>0.27</td><td>1.36E-05</td></tr><tr><td>HIST2H4B</td><td>Histone Cluster 2, H4b</td><td></td><td>A_23_P436281</td><td>0.38</td><td>1.04E-04</td></tr><tr><td>HIST1H4C</td><td>Histone Cluster 1, H4c<br>Excision Repair Cross-<br>Complementing Rodent Repair<br>Deficiency, Complementation</td><td></td><td></td><td></td><td></td></tr><tr><td>ERCC4</td><td>Group 4<br>PIF1 5'-To-3' DNA Helicase</td><td>DNA repair endonuclease<br>helicase that negatively<br>regulates telomerase<br>regulation of mitotic<br>spindle dynamics</td><td>A_32_P157481</td><td>0.43</td><td>6.18E-05</td></tr><tr><td>PIF1</td><td>Homolog (S. Cerevisiae)</td><td></td><td>A_23_P416468</td><td>0.21</td><td>7.86E-06</td></tr><tr><td>PSRC1</td><td>Proline/Serine-Rich Coiled-Coil 1<br>Putative Uncharacterized Protein</td><td></td><td>A_23_P46539</td><td>0.37</td><td>5.66E-04</td></tr><tr><td>FLJ22184</td><td>FLJ22184</td><td>-<br>mitotic spindle assembly,<br>maintenance of<br>centrosome integrity</td><td>A_24_P147540</td><td>0.44</td><td>6.52E-04</td></tr><tr><td>HAUS3</td><td>HAUS Augmin-Like Complex,<br>Subunit 3</td><td></td><td>A_33_P3385062</td><td>0.40</td><td>6.76E-05</td></tr><tr><td colspan="6"><strong>Microtubule cytoskeleton organization (GO:0000226)</strong></td></tr><tr><td>CKS2</td><td>CDC28 Protein Kinase Regulatory<br>Subunit 2</td><td>essential for biological<br>function of cyclin<br>dependent kinases</td><td>A_23_P71727</td><td>0.36</td><td>8.80E-04</td></tr><tr><td colspan="6"><strong>Integral to membrane (GO:0016021)</strong></td></tr><tr><td>B3GNT8</td><td>UDP-GlcNAc:BetaGal Beta-1,3-N-<br>Acetylglucosaminyltransferase 8<br>Chromosome 17 Open Reading<br>Frame 78</td><td>elongation of specific<br>branch structures of<br>multiantennary N-glycans</td><td>A_23_P101380</td><td>0.44</td><td>1.96E-05</td></tr><tr><td>C17orf78</td><td></td><td>-<br>negative regulator of<br>myoblast differentiation,<br>in part through effects on<br>MTOR signaling</td><td>A_23_P335388</td><td>0.24</td><td>3.15E-06</td></tr><tr><td>PPAPDC3</td><td>Phosphatidic Acid Phosphatase<br>Type 2 Domain Containing 3</td><td></td><td>A_23_P157736</td><td>0.44</td><td>7.95E-05</td></tr></tbody></table>

### Metabolic process

Cellular metabolism
<table><tr><td>EEF1A1</td><td>Eukaryotic Translation Elongation Factor 1 Alpha 1</td><td>promotes GTP-dependent<br>binding of ribosomes<br>during protein<br>biosynthesis</td><td>A_19_P00809682</td><td>0.30</td><td>4.94E-06</td></tr><tr><td colspan="6">Induction of apoptosis (GO:0006917)</td></tr><tr><td>ANGPT2</td><td>Angiopoietin 2</td><td>with VEGF, facilitate cell<br>migration and<br>proliferation</td><td>A_23_P60079</td><td>0.39</td><td>2.11E-04</td></tr><tr><td colspan="6">Notch signaling pathway</td></tr><tr><td>MAML3</td><td>Mastermind-Like 3 (Drosophila)</td><td>coactivator for NOTCH<br>proteins</td><td>A_33_P3335366</td><td>0.50</td><td>6.58E-05</td></tr></table>
Cell structure

Cytoskeletal part (GO:0044430)

<table><tr><td>KAZN</td><td>Kazrin, Periplakin Interacting Protein</td><td>interplay between adherens junctions and desmosomes</td><td>A_24_P331830</td><td>2.59</td><td>9.05E-04</td></tr><tr><td>STARD13</td><td>StAR-Related Lipid Transfer (START) Domain Containing 13</td><td>cytoskeletal reorganization, cell proliferation, tumor suppressor</td><td>A_33_P3382143</td><td>4.30</td><td>2.03E-04</td></tr><tr><td>CDC42EP3</td><td>CDC42 Effector Protein (Rho GTPase Binding) 3</td><td>organization of the actin cytoskeleton</td><td>A_33_P3331307</td><td>5.04</td><td>4.40E-05</td></tr><tr><td>ADD1</td><td>Adducin 1 (Alpha)</td><td>membrane-cytoskeleton-associated protein in anchoring cytoskeleton</td><td>A_24_P100742</td><td>2.03</td><td>1.89E-04</td></tr><tr><td>UTRN</td><td>Utrophin</td><td>to plasma membrane</td><td>A_33_P3234864</td><td>2.95</td><td>9.80E-04</td></tr><tr><td>ARHGAP26</td><td>Rho GTPase Activating Protein 26</td><td>organization of the actin-cytoskeleton</td><td>A_33_P3378680</td><td>3.89</td><td>4.36E-04</td></tr></table>

Metabolic process

Glycosylation

<table><tr><td>CSGALNACT2</td><td>Chondroitin Sulfate N-Acetylgalactosaminyltransferase</td><td>addition of the first GalNAc</td><td>A_33_P3266255</td><td>2.68</td><td>9.45E-06</td></tr></table>

Regulation of cellular protein metabolic process (GO:0032268)

<table><tr><td>PABPC4</td><td>Poly(A) Binding Protein, Cytoplasmic 4</td><td>regulatory processes of mRNA metabolism</td><td>A_33_P3271105</td><td>2.12</td><td>6.24E-05</td></tr><tr><td>RN5-8S1</td><td>RNA, 5.8S Ribosomal 5</td><td>precursor for the 18S, 5.8S and 28S rRNA</td><td>A_33_P3399064</td><td>3.28</td><td>4.18E-04</td></tr><tr><td>RRBP1</td><td>Ribosome Binding Protein 1</td><td>interaction between the ribosome and the endoplasmic reticulum</td><td>A_33_P3220277</td><td>2.31</td><td>2.85E-04</td></tr></table>

Cellular metabolism

<table><tr><td>CCZ1</td><td>CCZ1 Vacuolar Protein Trafficking And Biogenesis</td><td>protein trafficking and biogenesis</td><td>A_33_P3333863</td><td>2.17</td><td>1.96E-04</td></tr><tr><td>PRKAA1</td><td>Associated Homolog Protein Kinase, AMP-Activated, Alpha 1 Catalytic Subunit</td><td>activates energy-producing pathways</td><td>A_33_P3312212</td><td>2.50</td><td>5.13E-04</td></tr><tr><td>ASPH</td><td>Aspartate Beta-Hydroxylase</td><td>role in calcium homeostasis</td><td>A_23_P216094</td><td>2.42</td><td>4.50E-04</td></tr></table>

Catabolic process

Cellular macromolecule catabolic process (GO:0044265~)

<table><tr><td>NAALADL2</td><td>N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 2</td><td>-</td><td>A_21_P0002709</td><td>4.05</td><td>1.92E-04</td></tr></table>

Proteolysis (GO:0006508)

<table><tr><td>HECTD4</td><td>HECT Domain Containing E3 Ubiquitin Protein Ligase 4</td><td>E3 ubiquitin-protein ligase ubiquitination and proteasomal degradation of proteins</td><td>A_23_P309292</td><td>3.00</td><td>5.26E-04</td></tr><tr><td>KLHL15</td><td>Kelch-Like Family Member 15</td><td>hydrolase, remove conjugated ubiquitin from proteins</td><td>A_32_P195647</td><td>2.18</td><td>6.74E-05</td></tr><tr><td>USP10</td><td>Ubiquitin Specific Peptidase 10</td><td>cleaves plasminogen to active form plasmin</td><td>A_32_P26330</td><td>3.08</td><td>4.20E-04</td></tr><tr><td>PLAU</td><td>Urokinase</td><td></td><td>A_33_P3306146</td><td>2.47</td><td>6.07E-04</td></tr></table>
Cell cycle (GO:0007049)

<table><tr><td>SRRT</td><td>Serrathe RNA Effector Molecule Homolog (Arabidopsis)</td><td>cell cycle progression at S phase</td><td>A_33_P3299590</td><td>2.60</td><td>3.19E-04</td></tr><tr><td>ALMS1</td><td>Alstrom Syndrome 1</td><td>intracellular transport control of cell cycle and action of tumor suppressor proteins</td><td>A_33_P3332724</td><td>2.39</td><td>1.76E-04</td></tr><tr><td>E2F3</td><td>E2F Transcription Factor 3</td><td>cell cycle progression</td><td>A_33_P3418516</td><td>2.14</td><td>3.46E-04</td></tr><tr><td>CHTOP</td><td>Chromatin Target Of PRMT1</td><td></td><td>A_33_P3243957</td><td>3.35</td><td>6.94E-05</td></tr></table>

Proliferation

<table><tr><td>AMD1</td><td>Adenosylmethionine Decarboxylase 1 Dual-Specificity Tyrosine-(Y)- Phosphorylation Regulated Kinase 1A</td><td>cellular proliferation and tumor promotion</td><td>A_33_P3395369</td><td>2.40</td><td>3.29E-04</td></tr><tr><td>DYRK1A</td><td>Mitogen-Activated Protein Kinase 9</td><td>cell proliferation proliferation,differentiation, transcription regulation and development</td><td>A_33_P3406998</td><td>2.23</td><td>6.46E-04</td></tr><tr><td>MAPK9</td><td>Ribosomal Protein S6 Kinase, 70kDa, Polypeptide 1</td><td>cell proliferation, cell growth and cell cycle progression</td><td>A_23_P167692</td><td>2.00</td><td>2.35E-04</td></tr><tr><td>RPS6KB1</td><td>Fibroblast Growth Factor Receptor 1</td><td>cell proliferation, differentiation and migration</td><td>A_21_P0011529</td><td>2.16</td><td>7.55E-04</td></tr><tr><td>FGFR1</td><td></td><td></td><td>A_23_P372923</td><td>2.25</td><td>5.36E-04</td></tr></table>

Induction of apoptosis (GO:0006917)

<table><tr><td>PLEKHM1</td><td>Pleckstrin Homology Domain Containing, Family M (With RUN Domain) Member 1</td><td>transduction of apoptotic signals</td><td>A_33_P3405897</td><td>2.46</td><td>5.05E-04</td></tr></table>

Endocytosis

<table><tr><td>MYOF</td><td>myoferlin</td><td>plasma and nuclear membranes, involved in endocytic recycling</td><td>A_24_P282309</td><td>2.64</td><td>1.06E-04</td></tr><tr><td>MYO5A</td><td>Myosin VA (Heavy Chain 12, Myoxin)</td><td>transport of vesicles involved in endosomal recycling and in phagocytosis</td><td>A_24_P255218</td><td>2.38</td><td>3.56E-05</td></tr><tr><td>RAB11FIP1</td><td>RAB11 Family Interacting Protein 1 (Class I)</td><td></td><td>A_33_P3232798</td><td>3.43</td><td>1.86E-05</td></tr></table>

Oxidative stress

<table><tr><td>APP</td><td>Amyloid Beta (A4) Precursor Protein</td><td>Inflammatory response, involved in oxydative stress</td><td>A_33_P3296479</td><td>3.08</td><td>4.25E-04</td></tr></table>

Wnt signaling

<table><tr><td>DISC1</td><td>Disrupted In Schizophrenia 1</td><td>Wnt-mediated proliferation, inhibits activation of AKT-mTOR signaling</td><td>A_21_P0000047</td><td>3.09</td><td>2.07E-04</td></tr></table>
<table><caption>**Table S6.** Down-regulated genes identified in microarray analysis in response to 4-h exposure with 200 µg/mL of empty ENPs.</caption><thead><tr><th>Gene</th><th>Description</th><th>Function</th><th>AGILENT_ID</th><th>FC</th><th>p value</th></tr></thead><tbody><tr><td colspan="6"><strong>Metabolic process</strong></td></tr><tr><td colspan="6"><strong>Glycosylation</strong></td></tr><tr><td>NPL</td><td>N-Acetylneuraminate<br/>Pyruvate Lyase<br/>(Dihydrodipicolinate<br/>Synthase)</td><td>N-glycolylneuraminic acid<br/>degradation pathway<br/>glycoprotein, inhibit the<br/>growth of tumors</td><td>A_33_P3414903</td><td>0.50</td><td>1.63E-04</td></tr><tr><td>AMH</td><td>Anti-Mullerian Hormone</td><td></td><td>A_23_P78944</td><td>0.45</td><td>3.51E-04</td></tr><tr><td colspan="6"><strong>Cellular metabolism</strong></td></tr><tr><td>THRSP</td><td>Thyroid Hormone Responsive<br/>ADAM Metallopeptidase With<br/>Thrombospondin Type 1</td><td>regulation of lipogenesis</td><td>A_23_P363954</td><td>0.20</td><td>8.79E-04</td></tr><tr><td>ADAMTS7</td><td>Motif, 7<br/>ATP-Binding Cassette, Sub-<br/>Family G (WHITE), Member 4</td><td>metalloprotease (degradation<br/>of COMP)<br/>in macrophage lipid<br/>homeostasis</td><td>A_33_P3210762</td><td>0.30</td><td>7.39E-04</td></tr><tr><td>ABCG4</td><td>Eukaryotic Translation<br/>Elongation Factor 1 Alpha 1</td><td>promotes GTP-dependent<br/>binding of ribosomes during<br/>protein biosynthesis</td><td>A_24_P355626</td><td>0.25</td><td>6.16E-06</td></tr><tr><td>EEF1A1</td><td></td><td></td><td>A_19_P00809682</td><td>0.31</td><td>1.32E-06</td></tr><tr><td colspan="6"><strong>Proliferation</strong></td></tr><tr><td>ANGPT2</td><td>Angiopoietin 2</td><td>cell apoptosis, facilitate with<br/>VEGFcell migration and<br/>proliferation</td><td>A_23_P60079</td><td>0.37</td><td>3.32E-04</td></tr><tr><td colspan="6"><strong>Cell structure</strong></td></tr><tr><td colspan="6"><strong>Cytoskeletal part (GO:0044430)</strong></td></tr><tr><td>SHROOM4</td><td>Shroom Family Member 4</td><td>regulator of cytoskeletal<br/>architecture<br/>component of ciliary and<br/>flagellar microtubules</td><td>A_33_P3310047</td><td>0.45</td><td>8.07E-05</td></tr><tr><td>TEKT4</td><td>Tektin 4</td><td>flagellar microtubules<br/>formation and regulation of<br/>the tight junction</td><td>A_32_P231179</td><td>0.36</td><td>1.78E-04</td></tr><tr><td>CGN</td><td>Cingulin</td><td></td><td>A_33_P3329974</td><td>0.27</td><td>2.42E-05</td></tr><tr><td colspan="6"><strong>Notch signaling pathway</strong></td></tr><tr><td>SBNO2</td><td>Strawberry Notch Homolog 2<br/>(Drosophila)</td><td>transcriptional repression<br/>activity in macrophages</td><td>A_33_P3220152</td><td>0.49</td><td>3.80E-04</td></tr></tbody></table>
<table><caption>**Table S7.** Up-regulated genes identified in microarray analysis in response to 4-h exposure with 200 µg/mL of GSNO-loaded ENPs.</caption><thead><tr><th>Gene</th><th>Description</th><th>Function</th><th>AGILENT_ID</th><th>FC</th><th>p value</th></tr></thead><tbody><tr><td colspan="6"><strong>Transcription (GO:0006350)</strong></td></tr><tr><td>MAFK</td><td>V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog K</td><td>transcriptional activator or repressor transcriptional activator</td><td>A_23_P373598</td><td>2.02</td><td>5.99E-04</td></tr><tr><td>ZNF521</td><td>Zinc Finger Protein 521 Cbp/P300-Interacting Transactivator, With Glu/Asp-Rich Carboxy-Terminal Domain, 2</td><td>transcriptional activator transcriptional regulator transcriptional regulator transcriptional regulator</td><td>A_33_P3219398</td><td>2.04</td><td>6.06E-04</td></tr><tr><td>CITED2</td><td>2</td><td>transcriptional activator transcriptional regulator transcriptional regulator transcriptional regulator</td><td>A_33_P3213374</td><td>2.30</td><td>6.25E-04</td></tr><tr><td>EGR1</td><td>Early Growth Response 1</td><td>transcriptional regulator transcriptional regulator transcriptional regulator</td><td>A_23_P214080</td><td>3.97</td><td>2.69E-04</td></tr><tr><td>PRDM8</td><td>PR Domain Containing 8</td><td>transcriptional regulator transcriptional regulator transcriptional regulator</td><td>A_33_P3342126</td><td>2.65</td><td>2.30E-04</td></tr><tr><td>ZNF511</td><td>Zinc Finger Protein 511 Wolf-Hirschhorn Syndrome Candidate 1</td><td>transcriptional regulator transcriptional regulator transcriptional regulator</td><td>A_33_P3384885</td><td>2.12</td><td>1.51E-04</td></tr><tr><td>WHSC1</td><td>WHSC1 Candidate 1</td><td>transcriptional regulator transcriptional inhibitor transcription, promotes cell differentiation</td><td>A_33_P3380346</td><td>2.08</td><td>4.31E-06</td></tr><tr><td>E2F6</td><td>E2F Transcription Factor 6</td><td>transcriptional inhibitor transcription, promotes cell differentiation</td><td>A_32_P12610</td><td>2.18</td><td>1.67E-05</td></tr><tr><td>HES6</td><td>Hairy And Enhancer Of Split 6 (Drosophila)</td><td>transcriptional inhibitor transcription, promotes cell differentiation</td><td>A_23_P131202</td><td>2.30</td><td>1.78E-04</td></tr><tr><td>XRN2</td><td>5'-3' Exoribonuclease 2</td><td>transcriptional inhibitor transcription, termination by RNA polymerase II transcription regulation, DNA repair and replication, chromosomal stability</td><td>A_23_P166135</td><td>2.28</td><td>8.30E-04</td></tr><tr><td>HIST1H2AL</td><td>Histone Cluster 1, H2al</td><td>transcriptional stability transcription regulation, DNA repair and replication, chromosomal stability</td><td>A_23_P363174</td><td>4.20</td><td>8.70E-04</td></tr><tr><td>HIST1H4J</td><td>Histone Cluster 1, H4j</td><td>transcriptional stability transcription regulation, chromatin remodeling</td><td>A_23_P30805</td><td>2.88</td><td>3.94E-04</td></tr><tr><td>BRD9</td><td>Bromodomain Containing 9</td><td>chromosome condensation and segregation during mitosis</td><td>A_23_P19004</td><td>2.06</td><td>6.57E-04</td></tr><tr><td colspan="6"><strong>Nucleus</strong></td></tr><tr><td>SETDB2</td><td>SET Domain, Bifurcated 2</td><td>chromosome condensation and segregation during mitosis integrity of mitotic centrosomes</td><td>A_23_P341532</td><td>2.08</td><td>2.15E-04</td></tr><tr><td>FRY</td><td>Furry Homolog (Drosophila)</td><td>chromosome condensation and segregation during mitosis integrity of mitotic centrosomes role in epigenetic chromatin</td><td>A_33_P3223503</td><td>2.28</td><td>9.57E-04</td></tr><tr><td>TET3</td><td>Tet Methylcytosine Dioxygenase 3</td><td>chromosome condensation and segregation during mitosis integrity of mitotic centrosomes role in epigenetic chromatin reprogramming mediates actin nuclear export</td><td>A_33_P3235177</td><td>2.67</td><td>1.23E-04</td></tr><tr><td>XPO6</td><td>Exportin 6</td><td>chromosome condensation and segregation during mitosis integrity of mitotic centrosomes role in epigenetic chromatin reprogramming mediates actin nuclear export</td><td>A_23_P325438</td><td>2.16</td><td>9.56E-04</td></tr><tr><td colspan="6"><strong>Oncogene</strong></td></tr><tr><td>PVT1</td><td>Pvt1 Oncogene (Non-Protein Coding)</td><td>oncogene</td><td>A_19_P00317872</td><td>2.20</td><td>5.76E-04</td></tr></tbody></table>
<table><tbody><tr><td>RAB44</td><td>RAB44, Member RAS Oncogene Family</td><td>-</td><td>A_21_P0005115</td><td>2.06</td><td>4.28E-04</td></tr><tr><td>PSMD10</td><td>Proteasome (Prosome, Macropain) 26S Subunit, Non-ATPase, 10</td><td>proto-oncoprotein</td><td>A_33_P3335621</td><td>2.34</td><td>6.98E-04</td></tr><tr><td colspan="6">Regulation of Ras protein signal transduction (GO:0046578)</td></tr><tr><td>RABGAP1L</td><td>RAB GTPase Activating Protein 1-Like</td><td>-</td><td>A_24_P316005</td><td>2.77</td><td>5.81E-04</td></tr><tr><td>RSU1</td><td>Ras Suppressor Protein 1</td><td>role in the Ras signal transduction pathway</td><td>A_33_P3422614</td><td>2.33</td><td>8.40E-04</td></tr><tr><td>SOS1</td><td>son of sevenless homolog 1 (Drosophila)</td><td>promotes the exchange of Ras-bound GDP by GTP capable of binding</td><td>A_23_P343808</td><td>2.09</td><td>5.78E-04</td></tr><tr><td>GTPBP2</td><td>GTP Binding Protein 2</td><td>GTP or GDP</td><td>A_24_P298013</td><td>2.40</td><td>2.79E-04</td></tr><tr><td colspan="6">Ankyrin (IPR002110)</td></tr><tr><td>AGAP7</td><td>ArfGAP With GTPase Domain, Ankyrin Repeat And PH Domain 7</td><td>GTPase-activating protein</td><td>A_32_P41526</td><td>2.06</td><td>2.17E-04</td></tr><tr><td>POTEF</td><td>POTE Ankyrin Domain Family, Member F</td><td>cytoskeleton</td><td>A_24_P59220</td><td>2.07</td><td>2.23E-05</td></tr><tr><td colspan="6">Zinc and metal ion binding</td></tr><tr><td colspan="6">Zinc ion binding (GO:0008270)</td></tr><tr><td>PHF7</td><td>PHD Finger Protein 7 solute Carrier Family 30 (Zinc Transporter), Member 1</td><td>role in spermatogenesis zinc transport out of the cell</td><td>A_23_P80684</td><td>2.21</td><td>4.25E-04</td></tr><tr><td>SLC30A1</td><td>5'-Nucleotidase Domain Containing 3</td><td>-</td><td>A_23_P23815</td><td>2.44</td><td>1.63E-04</td></tr><tr><td colspan="6">Metal ion binding (GO:0046872)</td></tr><tr><td>NT5DC3</td><td>StAR-Related Lipid Transfer (START) Domain Containing 13</td><td>-</td><td>A_32_P76720</td><td>2.38</td><td>2.12E-04</td></tr><tr><td colspan="6">Cell structure</td></tr><tr><td colspan="6">Integral to membrane (GO:0016021)</td></tr><tr><td>AVL9</td><td>AVL9 Homolog (S. Cerevisiase)</td><td>cell migration mucolipin cation channel proteins</td><td>A_24_P272967</td><td>2.10</td><td>7.42E-05</td></tr><tr><td>MCOLN3</td><td>Mucolipin 3 Regulator Of G Protein Signaling</td><td>phototransduction targeting of connexins to the plasma membrane</td><td>A_23_P12241</td><td>2.16</td><td>8.58E-04</td></tr><tr><td>RGS9BP</td><td>9 Binding Protein</td><td>-</td><td>A_32_P227605</td><td>2.80</td><td>6.70E-04</td></tr><tr><td>CNST</td><td>Consortin, Connexin Sorting Protein</td><td>-</td><td>A_33_P3424462</td><td>2.37</td><td>4.79E-04</td></tr><tr><td colspan="6">Cytoskeletal part (GO:0044430)</td></tr><tr><td>GJD3</td><td>Gap Junction Protein, Delta 3, 31.9kDa</td><td>formation of gap junctions cytoskeletal reorganization, cell proliferation, tumor suppressor cytoskeletal organization</td><td>A_33_P3367301</td><td>3.47</td><td>1.84E-04</td></tr><tr><td>STARD13</td><td>StAR-Related Lipid Transfer (START) Domain Containing 13</td><td>-</td><td>A_33_P3382143</td><td>4.85</td><td>2.58E-04</td></tr><tr><td>PARVG</td><td>Parvin, Gamma</td><td>-</td><td>A_33_P3223338</td><td>2.02</td><td>3.09E-04</td></tr><tr><td>PDLIM1</td><td>PDZ And LIM Domain 1 WAS/WASL Interacting Protein</td><td>cytoskeletal protein activity on the actin cytoskeleton</td><td>A_23_P149992</td><td>2.20</td><td>6.42E-04</td></tr><tr><td>WIPF1</td><td>Family, Member 1</td><td>-</td><td>A_33_P3339376</td><td>2.24</td><td>2.30E-04</td></tr></tbody></table>
## Metabolic process

### Glycosylation

<table><tr><td>CLEC12A</td><td>C-Type Lectin Domain Family 12, Member A</td><td>mediates tyrosine phosphorylation of MAP kinases calcium regulation and bone homeostasis</td><td>A_23_P128470</td><td>2.43</td><td>5.41E-04</td></tr><tr><td>BMP8B</td><td>Bone Morphogenetic Protein 8b Phosphatidylinositol Glycan</td><td> GPI-anchor biosynthesis</td><td>A_32_P780862</td><td>2.45</td><td>7.79E-04</td></tr><tr><td>PIGS</td><td>Anchor Biosynthesis, Class S Glucuronidase, Beta</td><td></td><td>A_23_P66715</td><td>2.14</td><td>1.27E-05</td></tr><tr><td>GUSBP11</td><td>Pseudogene 11 Chondroitin Sulfate N- Acetylgalactosaminyltransferase</td><td>-</td><td>A_33_P3374559</td><td>2.03</td><td>2.59E-05</td></tr><tr><td>CSGALNACT2</td><td>2</td><td>addition of the first GalNAc synthesis and degradation of fructose 2,6- bisphosphate</td><td>A_33_P3266255</td><td>3.00</td><td>6.03E-05</td></tr><tr><td>PFKFB3</td><td>6-Phosphofructo-2- Kinase/Fructose-2,6- Biphosphatase 3</td><td></td><td>A_24_P206604</td><td>2.51</td><td>8.80E-04</td></tr></table>

### Regulation of cellular protein metabolic process (GO:0032268)

<table><tr><td>FXR2</td><td>fragile X mental retardation, autosomal homolog 2</td><td>RNA-binding protein</td><td>A_23_P4007</td><td>2.10</td><td>7.37E-04</td></tr><tr><td>RRP12</td><td>Ribosomal RNA Processing 12</td><td>-</td><td>A_23_P24192</td><td>2.14</td><td>1.08E-04</td></tr><tr><td>EPB41L2</td><td>Homolog Erythrocyte Membrane Protein Band 4.1-Like 2</td><td>-</td><td>A_23_P134109</td><td>2.18</td><td>4.19E-04</td></tr><tr><td>LSM14B</td><td>LSM14B, SCD6 Homolog B</td><td>control of mRNA translation</td><td>A_33_P3349774</td><td>2.24</td><td>1.85E-04</td></tr><tr><td>SBDS</td><td>Shwachman-Bodian-Diamond Syndrome</td><td>ribosome biogenesis nucleolar processing of pre-18S ribosomal RNA</td><td>A_33_P3238866</td><td>2.02</td><td>7.78E-04</td></tr><tr><td>NOP14</td><td>NOP14 Nucleolar Protein</td><td>in ubiquitinated proteins degradation, binds to TNFR1</td><td>A_32_P22622</td><td>2.07</td><td>1.61E-05</td></tr><tr><td>PSMD2</td><td>ATPase, 2</td><td>intracellular domain regulatory processes of mRNA metabolism</td><td>A_24_P42681</td><td>2.24</td><td>5.59E-05</td></tr><tr><td>PABPC4</td><td>Poly(A) Binding Protein, Cytoplasmic 4</td><td>constitute part of the spliceosome (pre-mRNA splicing)</td><td>A_33_P3271105</td><td>2.62</td><td>6.17E-04</td></tr><tr><td>SRSF2</td><td>Serine/Arginine-Rich Splicing Factor 2</td><td>pre-mRNA splicing</td><td>A_33_P3412945</td><td>2.70</td><td>7.06E-04</td></tr><tr><td>CWC25</td><td>CWC25 Spliceosome-Associated Protein Homolog</td><td>pre-mRNA splicing</td><td>A_24_P48318</td><td>2.12</td><td>1.46E-04</td></tr><tr><td>THOC3</td><td>THO Complex 3</td><td>pre-mRNA splicing</td><td>A_33_P3336642</td><td>2.83</td><td>6.26E-04</td></tr><tr><td>SF3B2</td><td>145kDa</td><td>pre-mRNA splicing</td><td>A_33_P3702906</td><td>2.15</td><td>6.85E-04</td></tr><tr><td>DHX38</td><td>Polypeptide 38</td><td>pre-mRNA splicing</td><td>A_23_P38011</td><td>2.01</td><td>5.80E-05</td></tr></table>

### Cellular metabolism

<table><tr><td>CCZ1</td><td>CCZ1 Vacuolar Protein Trafficking And Biogenesis Associated Homolog</td><td>protein trafficking and biogenesis activates acetoacetate to acetoacetyl-CoA</td><td>A_33_P3333863</td><td>2.40</td><td>9.78E-04</td></tr><tr><td>AACS</td><td>Acetoacetyl-CoA Synthetase</td><td></td><td>A_33_P3403392</td><td>2.13</td><td>5.65E-05</td></tr></table>
<table><tbody><tr><td>VPS35</td><td>Vacuolar Protein Sorting 35<br>Homolog</td><td>proteins transport<br>from endosomes to<br>Golgi network</td><td>A_21_P0011439</td><td>2.25</td><td>5.54E-04</td></tr><tr><td>IPCEF1</td><td>Interaction Protein For<br>Cytohesin Exchange Factors 1</td><td>promotion of guanine-nucleotide exchange</td><td>A_33_P3397995</td><td>2.03</td><td>5.60E-04</td></tr><tr><td>SLC17A5</td><td>Solute Carrier Family 17<br>(Anion/Sugar Transporter),<br>Member 5</td><td>transports glucuronic<br>acid out of the<br>lysosome</td><td>A_33_P3416321</td><td>2.40</td><td>8.38E-04</td></tr><tr><td colspan="6"><strong>Secreted</strong></td></tr><tr><td>INSL4</td><td>Insulin-Like 4 (Placenta)</td><td>trophoblast<br>development,<br>regulation of bone<br>formation</td><td>A_23_P112220</td><td>2.98</td><td>2.92E-05</td></tr><tr><td colspan="6"><strong>Signal</strong></td></tr><tr><td>METTL7B</td><td>Methyltransferase Like 7B</td><td>methyltransferase</td><td>A_24_P64653</td><td>2.92</td><td>4.92E-04</td></tr><tr><td colspan="6"><strong>Catabolic process</strong></td></tr><tr><td colspan="6"><strong>Cellular macromolecule catabolic process<br>(GO:0044265~)</strong></td></tr><tr><td>FBXO18</td><td>F-Box Protein, Helicase, 18</td><td>protein ubiquitination<br>and degradation<br>protein ubiquitination,<br>proteasome-dependent<br>degradation</td><td>A_23_P12849</td><td>2.27</td><td>2.66E-04</td></tr><tr><td>VPRBP</td><td>Vpr (HIV-1) Binding Protein</td><td>protein ubiquitination</td><td>A_23_P212595</td><td>2.24</td><td>2.11E-06</td></tr><tr><td>USP31</td><td>Ubiquitin Specific Peptidase 31<br>DDB1 And CUL4 Associated</td><td>protein ubiquitination</td><td>A_23_P206612</td><td>2.15</td><td>8.95E-04</td></tr><tr><td>DCAF4</td><td>Factor 4<br>STE20-Related Kinase Adaptor</td><td>protein ubiquitination</td><td>A_33_P3419234</td><td>2.15</td><td>3.56E-04</td></tr><tr><td>STRADA</td><td>Alpha<br>Phosphoglycerate Mutase 1<br>(Brain)</td><td>catalytic activity</td><td>A_33_P3224157</td><td>2.01</td><td>2.38E-05</td></tr><tr><td>PGAM1</td><td>Phosphoglycerate Mutase 1<br>(Brain)</td><td>glycolytic pathway</td><td>A_33_P3268334</td><td>2.95</td><td>2.03E-04</td></tr><tr><td colspan="6"><strong>Proteolysis (GO:0006508)</strong></td></tr><tr><td>CTSC</td><td>Cathepsin C<br>Plasminogen Activator,</td><td>exopeptidase and<br>endopeptidase<br>cleaves plasminogen<br>to active form plasmin</td><td>A_23_P1552</td><td>2.23</td><td>2.19E-04</td></tr><tr><td>PLAU</td><td>Urokinase</td><td></td><td>A_23_P24104</td><td>2.28</td><td>5.46E-04</td></tr><tr><td colspan="6"><strong>Cell cycle (GO:0007049)</strong></td></tr><tr><td>MCM7</td><td>Minichromosome Maintenance<br>Complex Component 7</td><td>initiation of genome<br>replication<br>regulates mitogenic<br>signal transduction</td><td>A_33_P3258223</td><td>2.10</td><td>7.88E-05</td></tr><tr><td>DUSP2</td><td>Dual Specificity Phosphatase 2<br>NudE Nuclear Distribution E</td><td>required for mitosis<br>role in mitosis by<br>dephosphorylating<br>mitotic substrates<br>nucleosome complex,<br>chromosome<br>organization, normal<br>mitosis progress</td><td>A_24_P37409</td><td>2.57</td><td>7.10E-05</td></tr><tr><td>NDEL1</td><td>Homolog (A. Nidulans)-Like 1<br>CTD (Carboxy-Terminal Domain,<br>RNA Polymerase II, Polypeptide<br>A) Phosphatase, Subunit 1</td><td>required for mitosis<br>role in mitosis by<br>dephosphorylating<br>mitotic substrates<br>nucleosome complex,<br>chromosome<br>organization, normal<br>mitosis progress</td><td>A_33_P3327852</td><td>2.03</td><td>2.75E-04</td></tr><tr><td>CTDP1</td><td>Phosphatase, Subunit 1</td><td>required for mitosis<br>role in mitosis by<br>dephosphorylating<br>mitotic substrates<br>nucleosome complex,<br>chromosome<br>organization, normal<br>mitosis progress</td><td>A_23_P89727</td><td>2.27</td><td>5.73E-05</td></tr><tr><td>CENPT</td><td>Centromere Protein T</td><td>actin cytoskeleton<br>organization, required<br>for normal progress<br>through mitosis</td><td>A_23_P163580</td><td>4.06</td><td>4.75E-04</td></tr><tr><td>SEPT7</td><td>Septin 7</td><td></td><td>A_24_P291973</td><td>2.02</td><td>2.41E-04</td></tr></tbody></table>
Induction of apoptosis (GO:0006917)

<table><tr><td>PHLPP2</td><td>PH Domain And Leucine Rich Repeat Protein Phosphatase 2</td><td>triggers apoptosis, decreases cell proliferation in BCR-induced</td><td>A_33_P3271810</td><td>2.14</td><td>1.64E-04</td></tr><tr><td>SH3BP5</td><td>SH3-Domain Binding Protein 5 (BTK-Associated)</td><td>apoptotic cell death in apoptosis role in MAPK cascade, proliferation, differentiation,</td><td>A_24_P148750</td><td>2.62</td><td>7.05E-04</td></tr><tr><td>CAPRIN2</td><td>Caprin Family Member 2</td><td>proliferation, differentiation, apoptosis, survival... putative tumor suppressor gene</td><td>A_24_P56281</td><td>2.10</td><td>7.64E-04</td></tr><tr><td>RAF1</td><td>V-Raf-1 Murine Leukemia Viral Oncogene Homolog 1</td><td>apoptosis, survival... putative tumor suppressor gene</td><td>A_23_P40952</td><td>2.15</td><td>9.48E-04</td></tr><tr><td>FRMD3</td><td>FERM domain containing 3</td><td></td><td>A_23_P135132</td><td>4.44</td><td>6.18E-05</td></tr></table>

Immune response (GO:0006955) and cell activation (GO:0001775)

<table><tr><td>HS1BP3</td><td>HCLS1 Binding Protein 3</td><td>modulator of IL-2 signaling (lymphocyte activation)</td><td>A_33_P3344277</td><td>2.04</td><td>6.87E-04</td></tr></table>

Mitochondrion (GO:0005739)

<table><tr><td>OXCT2</td><td>3-Oxoacid CoA Transferase 2 Succinate Dehydrogenase Complex, Subunit A,</td><td>transfers CoA moiety from succinate to acetoacetate</td><td>A_23_P137896</td><td>2.74</td><td>2.96E-04</td></tr><tr><td>SDHAP1</td><td>Flavoprotein Pseudogene 1 RNA Component Of Mitochondrial RNA Processing Endoribonuclease</td><td>-</td><td>A_33_P3325280</td><td>2.29</td><td>1.29E-04</td></tr><tr><td>RMRP</td><td>Electron-Transfer-Flavoprotein, Alpha Polypeptide</td><td>cleaves mitochondrial RNA electron acceptor for several dehydrogenases to mitochondrial respiratory chain in complex II</td><td>A_33_P3401284</td><td>2.36</td><td>4.72E-04</td></tr><tr><td>ETFA</td><td>Succinate Dehydrogenase Complex, Subunit A, Flavoprotein (Fp)</td><td>mitochondrial electron transport chain, tumor suppressor ATP-independent protease, degrades mitochondrial transit peptides</td><td>A_32_P119616</td><td>2.47</td><td>6.97E-04</td></tr><tr><td>SDHA</td><td>Pitrilysin Metallopeptidase 1</td><td></td><td>A_23_P250035</td><td>2.23</td><td>4.43E-05</td></tr><tr><td>PITRM1</td><td>Pitrilysin Metallopeptidase 1</td><td></td><td>A_33_P3265104</td><td>2.13</td><td>2.34E-04</td></tr></table>

Endocytosis

<table><tr><td>FLOT2</td><td>Flotillin 2 N-Ethylmaleimide-Sensitive Factor</td><td>formation of caveolae vesicles vesicle-mediated transport</td><td>A_24_P253818</td><td>2.12</td><td>6.48E-04</td></tr><tr><td>NSF</td><td></td><td>mediate exit of cholesterol from endosomes/lysosomes</td><td>A_21_P0000605</td><td>2.09</td><td>3.83E-04</td></tr><tr><td>OSBPL5</td><td>Oxysterol Binding Protein-Like 5</td><td></td><td>A_33_P3394972</td><td>3.15</td><td>3.23E-04</td></tr></table>

Oxidative stress

<table><tr><td>CTBP2</td><td>C-Terminal Binding Protein 2</td><td>corepressor targeting diverse transcription regulators</td><td>A_24_P103922</td><td>2.12</td><td>9.55E-04</td></tr><tr><td>GFOD1</td><td>Glucose-Fructose</td><td>-</td><td>A_23_P134058</td><td>2.71</td><td>3.36E-04</td></tr></table>
Oxidoreductase Domain
Containing 1

Wnt
signaling

frequently Rearranged In
Advanced T-Cell Lymphomas

Transducin-Like Enhancer Of
Split 1

activates Wnt
signaling pathway by
stabilizing beta-
catenin
inhibits the
transcriptional
activation in Wnt
signaling

A_33_P3261173

A_19_P00804020

3.04

5.18E-04

2.71

2.05E-04
<table><caption>**Table S8.** Down-regulated genes identified in microarray analysis in response to 4-h exposure with 200 µg/mL of GSNO-loaded ENPs.</caption><thead><tr><th>Gene</th><th>Description</th><th>Function</th><th>AGILENT_ID</th><th>FC</th><th>p value</th></tr></thead><tbody><tr><td>METTL11B</td><td>Methyltransferase Like 11B</td><td>methyltransferase</td><td>A_33_P3405936</td><td>0.39</td><td>6.82E-04</td></tr><tr><td>IQCF6</td><td>IQ Motif Containing F6</td><td>-</td><td>A_32_P13756</td><td>0.47</td><td>6.26E-04</td></tr></tbody></table>
## 2.4. Résultats et conclusion

Nous avons observé une augmentation de l'activité métabolique mitochondriale aussi bien avec les NP vides (NP-) que chargées en GSNO (NP+). Cependant, seule l'exposition aux NP- a conduit à une augmentation de la croissance cellulaire. L'internalisation des NP a été visualisée. Elle est énergie dépendante car inhibée à + 4°C et en présence d'azide de sodium. Cette internalisation se fait par endocytose, selon deux mécanismes principaux : la voie clathrine dépendante et celle des cavéolines.

Une première analyse des transcriptomes des THP-1 exposés aux NP+, aux NP- ainsi qu'au GSNO seul, a montré, selon le traitement, une différence des profils d'expression des gènes impliqués dans la réponse cellulaire. L'analyse des données des transcriptomes s'est faite en regroupant les gènes en clusters. Après exposition aux NP-, une surexpression des gènes a été observée principalement dans les clusters « chromosomal part » et « prolifération », alors que les NP+ induisent une activation importante au sein des clusters « metabolic process » et « mitochondrion ». Donc l'expression des gènes varie grandement en fonction du traitement. Le GSNO encapsulé a une activité qui peut cependant différer de celle du GSNO libre peut-être du fait d'une libération ralentie par l'insertion dans les NP, mais aussi d'une induction de réponses cellulaires qui leur sont propres. Cependant, les réponses génomiques sont cohérentes avec les tests cellulaires. L'étude génomique devient un outil indispensable pour mieux comprendre les interactions entre NP et cellules et a ainsi toute sa place en toxicologie.

Les données de transcriptomes ont donné lieu à des analyses complémentaires qui font l'objet de l'article 4 qui est en préparation.
### 3. Article 3. Comment on: S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated Leishmania major or Leishmania braziliensis skin lesions

Dans le cadre de l'immunité, le •NO est un effecteur, mis en jeu dans la lutte contre les agents microbiens (parasites, bactéries et virus) et les cellules tumorales. Il est produit par les macrophages et agit contre les agents intra- et extracellulaires [205]. A titre d'exemple, les lésions cutanées à *Leishmania* chez l'homme ont reçu des traitements topique avec des donneurs de •NO mais ceux-ci ont donné des résultats contradictoires positifs [206] ou non [207]. De même, au cours d'un essai clinique, des applications de patchs cutanés contenant des nitrites et de l'acide ascorbique et libérant du •NO n'ont pas été jugées efficace [208]. Ces résultats contradictoires pourraient éventuellement être expliqués par la mauvaise maîtrise de la libération de •NO à partir de ces formulations [209]. Les RSNOs synthétiques S-nitroso-N-acétylpénicillamine (SNAP) se sont avérés toxiques pour des formes de *Leishmania infantum* promastigotes [210]. Plus récemment, GSNO et S-nitroso-N-acétylcystéine ont montré une puissante activité inhibitrice de la croissance des promastigotes de *Leishmania amazonensis* et de *Leishmania major in vitro* [211]. L'étude de l'activité cytotoxique de GSNO sur les amastigotes intracellulaires de *Leishmania braziliensis* ou major a conclu que [209] : i) le GSNO réduit la taille des lésions cutanées ulcérées de souris infectées après application topique ; et ii) que le nombre d'amastigotes intracellulaires de *Leishmania major* est réduit dans les macrophages THP-1 exposées à 300 µM de GSNO *in vitro*. En conclusion, ce serait la nitrosation par GSNO des protéines des parasites et de la cellule hôte qui serait responsable de son activité leishmanicide.

Dans notre étude de transcriptome (cellules THP-1 exposées à 50 µM de GSNO libre, 4 h), au sein du cluster « Réponse immunitaire », les gènes CCL4, CCL3, CCL20, INHBA, VEGFA et IL23A impliqués dans la voie de signalisation « cytokine-cytokine interactions » sont surexprimés (base de données STRING). Cela nous a permis de proposer que, dans ce modèle cellulaire, le GSNO agit comme activateur de la réponse immunitaire en stimulant une voie de signalisation spécifique conduisant à la destruction du parasite.
Sir,

We read with great interest the letter by Köser et al.,¹ responding to our article on the characterization of the *embB* gene in the area of Barcelona.² The point that Köser et al.¹ raise is very interesting and deserves further comment. Codon 378 has indeed been described by several authors as a phylogenetic polymorphism not related to resistance.³,⁴ However, in our opinion the role of this codon remains unclear, since some studies describe the existence of ethambutol-resistant isolates with a mutation only in this codon.⁵,⁶ Even one work⁷ cited by Köser et al.¹ includes two isolates with a single Glu378Ala substitution and a decreased susceptibility to ethambutol.

The design of our microarray was based on the existing literature, taking into account all the possible *embB* codons that have been implicated in ethambutol resistance. We finally included the ones that were also found in our setting. The microarray system was not designed to determine the association of each mutation with the phenotypic resistance (allelic exchange experiments would be required for this purpose), but to reflect the variety of *embB* substitutions prevalent in our area. Moreover, the frequency of mutations in *embB*378 is low (<2% in our study), so we consider that the probability of misassigning a result of *embB*378 mutation to phenotypic resistance rather than to an epidemiological cause is negligible and does not compromise the effectiveness of the microarray.

Regarding the isolates, we included a collection of *Mycobacterium tuberculosis* complex (MTBC) clinical isolates (not identified to the species level). Isolate 233R has now been analysed and identified as *M. tuberculosis/Mycobacterium canettii*. It contains the *embB*378Glu variant; therefore, a homoplastic event may be present (experimental error was ruled out).

Likewise, the MIRU-VNTR genotyping was performed not for epidemiological purposes (i.e. for the identification of lineages), but to establish the real frequency of *embB* mutations among circulating MTBC isolates in our geographical area.

Finally, we stress that the main objective of this study was to highlight the relevance of mutations in *embB* codons apart from *embB*306, focusing on codon 406, which represents 20% of the *embB* mutations in our area. Our results show that this target (*embB*406) should be included in any genotypic method for rapid ethambutol resistance detection.

**Transparency declarations**

None to declare.

**References**

1. Köser CU, Bryant JM, Comas I et al. Comment on: Characterization of the *embB* gene in *Mycobacterium tuberculosis* isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array. *J Antimicrob Chemother* 2014; **69**: 2298-9.

2. Moure R, Español M, Tudó G et al. Characterization of the *embB* gene in *Mycobacterium tuberculosis* isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array. *J Antimicrob Chemother* 2014; **69**: 947-54.

3. Sekiguchi J, Miyoshi-Akiyama T, Augustynowicz-Kopeć E et al. Detection of multidrug resistance in *Mycobacterium tuberculosis*. *J Clin Microbiol* 2007; **45**: 179-92.

4. Feuerriegel S, Köser CU, Niemann S. Phylogenetic polymorphisms in antibiotic resistance genes of the *Mycobacterium tuberculosis* complex. *J Antimicrob Chemother* 2014; **69**: 1205-10.

5. Srivastava S, Garg A, Ayyagari A et al. Nucleotide polymorphism associated with ethambutol resistance in clinical isolates of *Mycobacterium tuberculosis*. *Curr Microbiol* 2006; **53**: 401-5.

6. Srivastava S, Ayyagari A, Dhole TN et al. *emb* nucleotide polymorphisms and the role of *embB*306 mutations in *Mycobacterium tuberculosis* resistance to ethambutol. *Int J Med Microbiol* 2009; **299**: 269-80.

7. Campbell PJ, Morlock GP, Sikes RD et al. Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of *Mycobacterium tuberculosis*. *Antimicrob Agents Chemother* 2011; **55**: 2032-41.

J Antimicrob Chemother 2014
doi:10.1093/jac/dku122

Advance Access publication 28 April 2014

**Comment on: S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated *Leishmania major* or *Leishmania braziliensis* skin lesions**

Carole Ronzani, Ramia Safar, Alain Le Faou, Bertrand H. Rihn and Olivier Joubert*

Université de Lorraine, Faculté de Pharmacie de Nancy, EA 3452
Cithéfor, France

*Corresponding author. Tel: (+33)-3-83-68-22-88;
E-mail: olivier.joubert@univ-lorraine.fr

**Keywords:** nitrosoglutathione, parasite, transcriptome, chemokines

Sir,

A previous study by Costa et al.¹ published in this journal reported the action of S-nitrosoglutathione (GSNO) (CAS number: 57564-91-7) on skin disease induced by *Leishmania* parasites. The major findings of this study were: (i) GSNO following topical application reduced ulcerated skin lesions of infected mice; and (ii) the number of intracellular *Leishmania major* amastigotes is reduced in THP-1 macrophages exposed to 300 µM GSNO. The authors assume that the nitrosation by GSNO of host cell and parasite proteins could explain the *in vitro* leishmanicidal activity of GSNO in THP-1 cell culture.

We suggest a complementary mechanism of GSNO action. We have recently analysed the transcriptome of THP-1 cells exposed to 50 µM GSNO for 4 h. Some of our results might be of high
<figure><img src="image_25.png" /><figcaption>**Figure 1.** Protein-protein interactions of 128 modified genes in THP-1 cells exposed to 50 µM GSNO for 4 h, as retrieved from the String database. Total RNA was extracted and analysed by microarray. One hundred and three of 128 genes whose expression was significantly altered and recognized by the String database were linked at the confidence, evidence or action level. Proteins involved in the cytokine-cytokine receptor interaction KEGG pathway are shown in red. Microarray results were confirmed by real-time PCR for CCL4 and VEGFA genes (up-regulated 21.9- and 3.4-fold, respectively, *P*<0.001, as determined by Student's t-test). The detailed protocol and the raw data of the microarrays are available on the GEO NCBI database using the accession number GSE51186. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.</figcaption></figure>

interest with regard to those obtained by Costa et al.:¹ analysis of
the protein-protein interaction of differentially expressed genes
(fold change ≤0.5 or ≥2, *P*<0.001) in response to GSNO shows

the involvement of the Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway called ‘cytokine-cytokine receptor
interaction’. This pathway links the human genes CCL4, CCL3,
CCL20, INHBA, VEGFA and IL23A, which are overexpressed 3- to 15-fold compared with control (Figure 1). Chemokines, which coordinate the recruitment of leucocytes involved in homeostasis and in innate and adaptive immune responses, play a crucial role in the regulation of immunity against *Leishmania*.<fnref n="2" /> In addition, CCL3 and CCL20 have been shown to display antiparasitic activity against the promastigote form of the *Leishmania mexicana* parasite by acting directly on plasma membranes on *Leishmania* parasites, causing their lysis.³ Evidence of an increase in phagocytosis and NO-mediated destruction of *Leishmania* by chemokines (including CCL3 and CCL4) was reported a decade ago.⁴ Moreover, we show here that IL-23A levels are induced by GSNO. Murray et al.⁵ showed that IL-23 displayed an antileishmanial effect, especially during late-stage parasitic infection. Thus, GSNO may be implicated in a cellular signalling pathway leading to macrophage activation and exhibiting immunostimulatory effects.

Our results indicate that GSNO not only exhibits microcidal activity but also acts as a macrophage activator. This activation was possible with the involvement of at least three chemokines (CCL4, CCL3 and CCL20) and one cytokine (IL23A). All of them are known to have antiparasitic activity, and thus could be effective against *Leishmania* infection. In conclusion, our results may be relevant to investigators working on GSNO antiparasitic activity *in vitro* and *in vivo*.

## Funding

This work was supported by the Agence Nationale de la Recherche (ANR NanoSNO). R. S. is the recipient of a PhD grant from the Ministry of Higher Education of Syria.

## Transparency declarations

None to declare.

## References

1. Costa ISF, de Souza GFP, de Oliveira MG et al. S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated *Leishmania major* or *Leishmania braziliensis* skin lesions. *J Antimicrob Chemother* 2013; **68**: 2561-8.

2. Oghumu S, Lezama-Davila CM, Isaac-Marquez AP et al. Role of chemokines in regulation of immunity against leishmaniasis. *Exp Parasitol* 2010; **126**: 389-96.

3. Söbirk SK, Mörgelin M, Egesten A et al. Human chemokines as antimicrobial peptides with direct parasiticidal effect on *Leishmania mexicana* in vitro. *PLoS One* 2013; **8**: e58129.

4. Brandonisio O, Panaro MA, Fumarola I et al. Macrophage chemotactic protein-1 and macrophage inflammatory protein-1α induce nitric oxide release and enhance parasite killing in *Leishmania infantum*-infected human macrophages. *Clin Exp Med* 2002; **2**: 125-9.

5. Murray HW, Tsai CW, Liu J et al. Responses to *Leishmania donovani* in mice deficient in interleukin-12 (IL-12), IL-12/IL-23, or IL-18. *Infect Immun* 2006; **74**: 4370-4.

J Antimicrob Chemother 2014
doi:10.1093/jac/dku223
Advance Access publication 19 June 2014

# S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated *Leishmania major* or *Leishmania braziliensis* skin lesions—authors' response

Inez Silva Fernandes Costa<fnref n="1" />, Gabriela Freitas Pereira de Souza<fnref n="2" />, Marcelo Ganzarolli de Oliveira<fnref n="2" /> and Ises de Almeida Abrahamsohn<fnref n="1" />*

**Keywords:** antiprotozoals, leishmaniasis, mode of action

Sir,

Thank you to Ronzani et al.<fnref n="1" /> for calling attention to our paper.<fnref n="2" /> The results reported by Ronzani et al.<fnref n="1" /> are certainly interesting as they indicate that expression of chemokines and cytokines in a human monocyte cell line (THP-1) can be up-regulated by S-nitrosoglutathione (GSNO). Although most studies have reported down-regulation of the synthesis of most proteins, up-regulation also occurs and the net result of GSNO action appears to be much dependent on the levels of nitrosoglutathione reductase expressed by each cell type.³,⁴ In addition, NO and GSNO also act post-transcriptionally and directly inhibit the phosphorylation of transcription factors such as STAT-3.⁵ Regarding the activities of chemokines and cytokines on *Leishmania* infection, these molecules are secreted by several host cells of the innate or acquired immunity systems. They are secreted to the extracellular milieu and act by recruiting and/or activating the respective cell targets, e.g. neutrophils, lymphocytes and macrophages (after interaction with target-cell membrane receptors).⁶ Thus, chemokines and cytokines are recognized as important regulators of the immune and inflammatory responses and because of this many participate in the control of *Leishmania* parasitism in the vertebrate host.⁷

Regarding a possible direct cytotoxic activity of chemokines/cytokines against *Leishmania*, it should be recalled that the study by Söbirk et al.⁸ described this cytotoxicity to occur against free promastigotes of *Leishmania*, at micromolar concentrations of these mediators in contrast to the nanomolar levels normally found in the inflammatory microenvironment.

However, in the cultures of *Leishmania*-infected THP-1 cells described in our paper,<fnref n="2" /> the parasites were amastigotes lying inside parasitophorous vacuoles in the cytoplasm. Therefore,

<footnote n="1">¹Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, USP, 05508-900 São Paulo, SP, Brazil;</footnote>

<footnote n="2">²Departamento de Fisico-Química, Instituto de Química, Universidade Estadual de Campinas, UNICAMP, 13083-970, CP 6154, Campinas, SP, Brazil</footnote>

<footnote>*Corresponding author. Tel: +55-11-3091-7327; Fax: +55-11-3091-7224; E-mail: iabraham@usp.br</footnote>
## 3.1. Résultats complémentaires:

### 3.1.1. Mise à jour des résultats :

La mise à jour de la base de données STRING le 12 avril 2015, nous a amené à refaire l'analyse des interactions entre protéines. Ce sont maintenant 17 gènes de la « pathway : cytokine-cytokine interactions » qui sont significativement régulés (Figure 12, Tableau 4).

<table><caption>Tableau 4. Gènes significativement impliqués dans la « pathway » "cytokine-cytokine interaction", 50 µM GSNO, 4 h</caption><thead><tr><td>Symbole du gène</td><td>Fold Change</td><td>Nom du gène</td></tr></thead><tbody><tr><td>Il6ST</td><td>+ 2.17</td><td>interleukin 6 signal transducer</td></tr><tr><td>IL23A</td><td>+ 4.19</td><td>interleukin 23, alpha subunit p19</td></tr><tr><td>CSF2RA</td><td>+ 2.01</td><td>colony stimulating factor 2 receptor, alpha</td></tr><tr><td>OSM</td><td>+ 4.80</td><td>oncostatin M</td></tr><tr><td>TNFRSF1A</td><td>- 2.09</td><td>tumor necrosis factor receptor superfamily, member 1A</td></tr><tr><td>FLT1</td><td>+ 2.44</td><td>fms-related tyrosine kinase 1</td></tr><tr><td>VEGFA</td><td>+ 5.75</td><td>vascular endothelial growth factor A</td></tr><tr><td>TNF</td><td>+ 6.45</td><td>tumor necrosis factor</td></tr><tr><td>CCL4</td><td>+ 24.53</td><td>chemokine (C-C motif) ligand 4</td></tr><tr><td>TGFBR2</td><td>+ 2.46</td><td>transforming growth factor, beta receptor II</td></tr><tr><td>CCL3</td><td>+ 5.32</td><td>chemokine (C-C motif) ligand 3</td></tr><tr><td>INHBA</td><td>+ 10.35</td><td>inhibin, beta A</td></tr><tr><td>CCR6</td><td>- 2.28</td><td>chemokine (C-C motif) receptor 6</td></tr><tr><td>CXCL5</td><td>- 3.44</td><td>chemokine (C-X-C motif) ligand 5</td></tr><tr><td>CCL20</td><td>+ 9.62</td><td>chemokine (C-C motif) ligand 20</td></tr><tr><td>CXCL2</td><td>+ 11.88</td><td>chemokine (C-X-C motif) ligand 2</td></tr><tr><td>CXCL3</td><td>+ 13.35</td><td>chemokine (C-X-C motif) ligand 3</td></tr></tbody></table>

En gras, figurent les 6 gènes précédemment cités dans l'article #3. En rouge sont les gènes surexprimés et en vert les gènes réprimés.
<figure><img src="image_26.png" /><figcaption>Figure 12. Réseau de protéines correspondantes aux gènes de THP-1 dont l'expression est significativement modifiée (STRING database) après exposition de GSNO à 50µM, 4 h</figcaption></figure>

En rouge : les gènes de « pathways cytokine-cytokine interactions »

### 3.1.2. RT-PCR :

Les résultats des puces à ADN ont été confirmés par PCR en temps réel pour les gènes surexprimés CCL4 et VEGFA (FC= + 21,9 et + 3,4, respectivement) (Figure 13).
<figure><img src="image_27.png" /><figcaption>Figure 13. Quantification de l'expression des gènes CCL4 et VEGFA par RT-PCR après 4 h d'exposition des THP-1 à 50 µM de GSNO</figcaption></figure>
**4. Article 4 (en préparation). Dose- and time- dependent responses of THP-1
cells exposed to free or encapsulated S-nitrosoglutathione. A
transcriptomic comparison**

**4.1. Introduction**

Les puces à ADN « DNA microarrays » permettent l'analyse de l'expression de l'ensemble du génome cellulaire en une seule manipulation, rapidement par l'analyse quantitative des ARNm produits. Cette technique produit une grande quantité de données qui doivent respecter un niveau de qualité et être notamment normalisées afin d'en retirer une information pertinente [212]. L'analyse des données conduit à identifier les gènes différentiellement exprimés en fonction des conditions. Ainsi, chacune d'entre-elles dispose de sa propre signature génique. Ensuite, l'utilisation de bases de données « data mining » permet de situer ces gènes au sein des « pathways » [213].

La technique de mise en œuvre des puces à ADN se décompose en sept étapes : choix d'un plan expérimental, extraction des ARNm, synthèse des ADNc, synthèse et marquage des ARNc, hybridation, mesure des fluorescences et enfin l'analyse des données.

L'analyse des données se décompose en 3 parties :

• Normalisation:

C'est le processus d'élimination des variations expérimentales. Celles-ci sont nombreuses et se produisent dans toutes les manipulations de puces à ADN. Elles affectent les niveaux d'expression des gènes mesurés comme par exemple les biais associés aux différents fluorochromes. Il est effectivement rare d'avoir des intensités de fluorochromes égales dans tous les spots de deux mêmes échantillons. Même si ces biais systématiques n'ont souvent que peu d'impact, ils peuvent être sources de confusion lors de la recherche de différences minimes d'expression. Ces biais peuvent provenir de différents facteurs, comme les propriétés physiques des fluorochromes (sensibilité à la lumière, à la chaleur, à l'ozone, faisant varier la demi-vie), l'efficacité de leur incorporation, la variabilité expérimentale dans les procédures d'hybridation et de traitement, ou les paramètres choisis du scanner [214]. Plusieurs types de normalisation peuvent être utilisés, comme les méthodes des « lowess » ou des « quantiles ».
* Détection de gènes différentiellement exprimés :

Les gènes exprimés de manière différentielle sont ceux dont les niveaux d'expression sont statistiquement significativement différents entre deux groupes d'expériences [215]. Ensuite, la fixation d'un facteur de variation ou Fold Change (FC) permet de déterminer la significativité biologique de l'observation (classiquement $FC > |2|$). Cependant, la significativité biologique peut également reposer sur des petites variations reproductibles qui peuvent être aussi informatives que les grandes [216].

Il existe plusieurs méthodes statistiques pour déterminer une variation d'expression significative d'un gène à partir des données de puces d'expression normalisées, le t-test de Student est le plus utilisé. Pour comparer plusieurs conditions, des analyses de variance (ANOVA ou MANOVA) sont utilisées [213]. Plusieurs logiciels sont disponibles pour identifier les variations d'expression en utilisant des analyses statistiques comme SOLO et Genespring®.

* Interprétation biologique :

Le but de cette étape est de donner un sens biologique aux observations, c'est à dire de lier les variations des expressions de gènes observées à des fonctions cellulaires. Différentes approches complémentaires sont possibles :

* Approche globale : par regroupement, ou « clustering », les gènes sont regroupés par fonction et cet ensemble de gènes « cluster » est étudié à l'aide des bases de données, libres ou payantes, comme DAVID (Database for Annotation, Visualization and Integrated Discovery), PANTHER (Classification system / Geneontology ; http://pantherdb.org/), Genespring®.

* Approche ciblée : en analysant les données gène par gène. Le diagramme de Venn est classiquement utilisé pour faciliter la visualisation de nombre des gènes communs entre deux ou plusieurs conditions.

* Analyse des interactions protéine-protéine : elle est utile pour déterminer les fonctions cellulaires des produits des gènes [217]. Plusieurs bases de données ont été ainsi développées pour étudier les interactions entre les protéines, telle la base de données STRING [http://string.embl.de] des interactions protéine-protéine connues et possibles. Cette base a été utilisé pour déterminer les interactions directes et indirectes entre les protéines codées par les gènes sur ou sous exprimés.
* Analyse des « pathways » : cette méthode semble la plus intéressante car elle étudie les interactions entre les gènes d'une même « pathway » et peut conduire à un niveau plus élevé de compréhension du mécanisme d'action des NP, par exemple, auxquelles les cellules ont été exposées [218]. Des bases des données ont été développées pour ce type d'analyse comme Ingenuity Pathway Analysis (IPA QIAGEN) et Genespring® (Agilent).

Il est important de noter qu'il n'existe pas une stratégie universelle pour l'analyse des données de puces à ADN, celle adoptée dépend des questions biologiques posées.

## 4.2. Objectif :

L'article 2 présente en partie l'analyse de transcriptomes des cellules THP-1 exposées à 200 µg/mL de NP vides (NP-) ou chargées de 6 µM de GSNO (NP+), ou à une dose de GSNO 50 µM pendant 4 h. Des conditions complémentaires ont été étudiées, toujours avec le même modèle cellulaire :

* 50 µg/mL de (NP-) ou (NP+), 4 h d'exposition : pour identifier la réponse précoce à une concentration de NP induisant une croissance cellulaire significative par rapport au contrôle (Figure 1, Article 2)

* 50 µg/mL de (NP-) ou (NP+), 24 h d'exposition : pour identifier la réponse tardive à cette même dose.

* 1,4 µM de GSNO libre, 4 h d'exposition, concentration correspondant à la quantité de GSNO encapsulée dans 50 µg/mL de NP. Le GSNO ayant une demi-vie courte, les temps tardifs n'ont pas été étudiés.

* 6 µM de GSNO libre, 4 h d'exposition, concentration correspondant à la quantité de GSNO encapsulée dans 200 µg/mL de NP.

Dans l'article 2, nous avons fait une analyse par clusters de gènes. Dans cette partie, nous avons poursuivi ce travail afin d'identifier les gènes et les « pathways » communes entre les différentes conditions d'exposition des cellules THP-1. Le but était d'identifier des biomarqueurs invariants d'exposition aux NP. Pour ce faire, nous avons bénéficié, pour une semaine, de la version de démonstration du logiciel IPA, auquel il manque plusieurs fonctions d'analyse.
## 4.3. Résultats et discussion:

Les données ont été normalisées par Genespring® (version 13.1).

### 4.3.1. Sélection des gènes différentiellement exprimés

Après la normalisation des données, l'analyse statistique (t-test, Genespring®) a fourni un sous-ensemble de gènes dont l'expression est statistiquement significativement modifiée. La valeur de significativité statistique de t-test (P) choisi était 0,001, en raison d'un trop grand nombre de gènes pour P= 0,05 (> 9000 pour certaines conditions) (Tableau 5). Nous avons fixé une valeur seuil pour le Fold Change à 2 pour les gènes sur-exprimés et -2 pour les gènes sous-exprimés ce qui est classiquement utilisé.

<table><caption>Tableau 5. Nombre de gènes dont l'expression est modifiées pour deux valeurs de P pour chacune des conditions testées</caption><thead><tr><td>Condition</td><td>P &lt; 0.001</td><td>P &lt; 0.05</td></tr></thead><tbody><tr><td>GSNO 1,4 µM / 4h</td><td>937</td><td>9616</td></tr><tr><td>GSNO 6 µM / 4h</td><td>477</td><td>6678</td></tr><tr><td>GSNO 50 µM / 4h</td><td>729</td><td>6858</td></tr><tr><td>NP- 50 µg/mL / 4h</td><td>9</td><td>329</td></tr><tr><td>NP+ 50 µg/mL / 4h</td><td>76</td><td>4127</td></tr><tr><td>NP- 200 µg/mL / 4h</td><td>258</td><td>5476</td></tr><tr><td>NP+ 200 µg/mL / 4h</td><td>275</td><td>6316</td></tr><tr><td>NP- 50 µg/mL / 24h</td><td>14</td><td>617</td></tr><tr><td>NP+ 50 µg/mL / 24h</td><td>434</td><td>5772</td></tr></tbody></table>

La variation du facteur « temps d'incubation » (de 4 h à 24 h) avec les NP- induit une modification de l'expression d'un petit nombre de gènes (9 et 14 gènes, respectivement), alors qu'avec les NP+, ce nombre est beaucoup plus important (76 et 434 gènes, respectivement). La variation du facteur « dose » (de 50 à 200 µg/mL) pour les NP- induit une augmentation de l'ordre de 28 fois du nombre de gènes différentiellement exprimés (9 et 258 gènes, respectivement). Celle-ci n'est que de 3 fois pour les NP+ (76 et 275 gènes, respectivement) (Tableau 5). Tout se passe comme si l'activation par GSNO n'était que peu ou pas dose-dépendante.
### 4.3.2. Gènes et « pathways » communs

En croisant les listes des gènes différentiellement exprimés des deux ou trois conditions en utilisant le logiciel Venny 2.0.2 (Oliveros, J.C. 2007-2015), nous avons obtenu les résultats suivants :

#### 4.3.2.1. Effet du temps d'exposition aux NP- ou NP+

##### ○ Gènes communs

Nous avons croisé les listes des gènes choisis dans l'étape précédente résultants d'une exposition des cellules à 50 µg/mL de NP- ou de NP+ après 4 et 24h. Les résultats montrent que : un seul et même gène a une expression modifiée pour les deux conditions que les NP soient vides ou chargées. Il correspond au locus XLOC-014512 (Figure 14 A et B), avec une valeur de FC de -3,24 (NP-, 4 h), de -2,46 (NP-, 24 h), de -3,29 (NP+, 4 h) et de -4,1 (NP+, 24h). Sa fonction n'est pas connue et il n'est pas lié à la présence de GSNO, mais plutôt à l'interaction NP / cellules.

A

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /><figcaption>Figure 14. Comparaison de l'effet du temps d'exposition (4 et 24 h) à des NP vides ou chargées en GSNO sur le nombre des gènes significativement régulés par diagramme de Venn
A : NP- 50 µg/mL, B : NP+ 50 µg/mL</figcaption></figure>

##### ○ Pathways communes

L'étude des voies de signalisation par le logiciel IPA n'a pas permis d'identifier de « pathways » activée commune après incubation des cellules avec des NP- à 4 et 24 h. En
revanche, bien qu'il n'y ait pas de gènes connus communs, il existe une « pathway » commune entre les gènes différemment régulés par l'exposition aux NP+ à 4 et 24 h : *regulation of IL-2 expression in activated and anergic T lymphocytes* qui comprend 76 gènes. Ceci serait la résultante d'une libération continue de GSNO. En fait, au sein de cette « pathway » et après 4 h d'exposition aux NP+, l'expression d'un gène : *ZAP70* est diminuée (-2,887). Après 24 h ce sont 5 autres gènes qui ont leur activité modifiée [VAV2 (-2,984) / TGFBR2 (+2,915) / SOS1 (+2,139) / MAPK9 (+2,902) / MAPK8 (+2,473)]. Le logiciel ne nous a pas permis de conclure sur l'activation ou l'inhibition de cette « pathway ». Néanmoins, la diminution de l'expression de *ZAP70* et l'augmentation de l'expression de *TGFBR2* seraient en faveur de son inhibition et donc d'une diminution de l'activation des monocytes. Une production de •NO concomitante à une diminution de l'expression de l'IL-2 a déjà été décrite [219].

#### 4.3.2.2. Effet de la dose des NP- ou NP+

##### ○ Gènes communs

Parmi les différences d'expression en réponse à l'exposition à 50 µg/mL ou 200 µg/mL de NP- pendant 4 h, seule celle de XLOC-014512 (-3.24, -3.98 respectivement) varie selon les critères d'analyse retenus (Figure 15 A). Par contre, la comparaison entre les conditions 50 µg/mL et 200 µg/mL des NP+ a fourni des modifications d'expression de 26 gènes (Figure 15 B, liste en Annexe 1) parmi lesquels 15 ne sont pas caractérisés, sont non codants, ou considérés comme pseudogènes. Ces quinze gènes sont tous régulés de la même manière aux deux doses de NP+ pour 50 et 200 µg/mL. Sur les 11 gènes caractérisés, deux sont sur-exprimés *JMJD1C* (+2.22 / +2.17 respectivement), *DLX2* (+2.57 / +2.09 respectivement) et 9 gènes sous-exprimés. Parmi ces derniers, deux sont impliqués dans la formation du cytosquelette et dans la polymérisation de l'actine *KANK4* (-4.81 / -2.26 respectivement), *PTPN20B* (-3.49 / -3.14 respectivement). Un résultat similaire a été observé sur des polynucléaires neutrophiles exposés à un flux de •NO provoquant une S-nitrosylation de l'actine cytosolique et sa polymérisation [220]. On peut noter également la sous-expression de *ATG9B* (-4.10 / -2.60 respectivement) car ce gène est non seulement impliqué dans la voie de l'autophagie, mais aussi dans la régulation de *eNOS3* (endothelial nitric oxide synthase 3).
A

<figure><img src="image_30.png" /></figure>

B

<figure><img src="image_31.png" /><figcaption>Figure 15. Comparaison de l'effet de deux doses des NP vides ou chargées en GSNO sur le nombre des gènes significativement régulés après 4 h d'exposition, par diagramme de Venn</figcaption></figure>

A : NP-, B : NP+

o *"Pathways" communes*

Les études des « pathways » montrent qu’il n’en existe pas de communes après
exposition aux NP- aux deux doses à 4 h alors que pour les NP+, à 4 h, il en existe 2 principales :

*   *G-as signaling*, signalisation par les protéines G (108 gènes) : l'exposition à 50 µg/mL induit une diminution d'expression du gène *VIPR2* (-4.93), alors qu'à la dose de 200 µg/mL, l'expression de 4 gènes ont été modifiés [*VIPR2* (-4.09) / *PTGR* (-2.30) / *GPER1* (-2.69) / *CREB5* (-3.41)]. L'inhibition de cette voie plus significative à 200 µg/mL est donc liée à l'apport de •NO par les NP.

*   *cAMP-mediated signaling* (216 gènes) : de même, l'expression d'un seul gène a été modifiée en cas d'exposition à 50 µg/mL des NP+ *VIPR2* (-4.93), alors que celle de 5 gènes a été modifiée avec 200 µg/mL [*VIPR2* (-4,09) / *AKAP2* (2,466) / *CREB5* (-3,41) / *GPER1* (-2,69) / *PTGIR* (-2,30)], correspondant à une inhibition de cette voie de signalisation. Trois de ces gènes sont également présents dans la voie *G-as signaling*. Ceci n'est pas étonnant puisque ces deux voies concernent la même cascade de signalisation cellulaire, G-as étant en amont de la signalisation par l'AMPc.
4.3.2.3. Effet du GSNO

1) NP 50 µg/mL, GSNO 1,4 µM

○ Gènes communs

Le locus XLOC-014512 est commun aux trois conditions d'exposition des cellules THP-1 pendant 4 h (Figure 16 A). Il existe également un autre gène commun pour les NP- et le GSNO libre, RAD9B (RAD9 Homolog B (S. Pombe) (-2.31 et +2.02 respectivement), impliqué dans le cycle cellulaire. En revanche, on observe 17 gènes communs avec les NP+ et la dose équivalente de GSNO libre (liste en Annexe 2). Parmi ces gènes, 12, qui ont des fonctions inconnues, sont tous surexprimés à l'exception de LOC101927787 sur-exprimé avec NP+ mais sous-exprimé pour le GSNO. Parmi les cinq autres gènes, deux impliqués dans l'immunité sont sur-exprimés : IGSF9B (+3.41 / +3.69 respectivement), IRGC (+3.46 / +3.79 respectivement). Par contre, un gène de la voie de l'AMPc est sous-exprimé : VIPR2 (-4.93 / -3.71 respectivement).

A

<figure><img src="image_32.png" /></figure>

B

<figure><img src="image_33.png" /><figcaption>Figure 16. Comparaison de l'effet (en nombre de gènes à expression modifiée) de deux concentrations de NP chargées ou non en GSNO et de la concentration du GSNO libre corrrespondante sur les cellules THP-1 par diagrammes de Venn</figcaption></figure>

A : 50 µg/mL de NP et 1,4 µM de GSNO, B : 200 µg/mL de NP et 6 µM de GSNO
○ « Pathways » communes

Alors qu'aucune « pathway » commune entre les NP- et NP+, ni entre le NP- et le GSNO libre n'a été identifiée, il en a été observées trois après exposition aux NP+ et au GSNO libre :

* *Allograft rejection signaling* (39 gènes) avec un gène *FAS* (+3.57) sur-exprimé après exposition aux NP+ et 4 gènes [*CD80* (-2,407) / *HLA-A* (-3,302) / *IFNG* (+2,336) / *IL2* (+2,327)] après exposition au GSNO.

* *Graft-versus-host disease signaling*, voie pour laquelle nous n'avons pas les informations.

* *T-cell receptor signaling* (94 gènes) dont un gène *ZAP70* (-2.887) est réprimé en réponse à l'exposition aux NP+, et 3 gènes [*PPP3R2* (+5.006) / *CTLA4* (+4.466) / *CD8B* (+5.047)] sont surexprimés en réponse au GSNO libre.

Le GSNO libre ou encapsulé induit l'expression de gènes impliqués dans les réactions immunitaires corroborant nos travaux antérieurs (articles 3).

2) NP 200 µg/mL, GSNO 6 µM

○ Gènes communs

Le nombre de gènes communs dont l'expression est modifiée pour les trois conditions d'exposition est de 28 (Figure 16 B, liste en Annexe 3). Douze de ces gènes sont soit non caractérisés, soit non codant, soit des pseudogènes. Pour les 16 autres, les fonctions sont variées, par exemple, *OR2A7* récepteur olfactif, *VIPR2* impliqué dans l'inhibition de la voie de l'IL-2, *RASSF6* inducteur d'apoptose, etc...

En plus des 28 précités, 52 gènes sont retrouvés dans la réponse aux NP+ et au GSNO libre (liste en **Annexe 4**). Parmi ceux-ci, 28 ont une fonction inconnue. Par contre, on observe une augmentation de l'expression de gènes, impliqués dans la régulation redox cellulaire *PRDX3* (+2.88 / +2.76 respectivement) qui est sous le contrôle de •NO et H₂O₂, et la régulation de l'expression de la cytokine TGF-β *TGFBR3* (+2.30 / +2.45 respectivement) qui module la réponse immunitaire notamment en inhibant la voie de l'IL-2. *HSPA1A* (-2.15 / -2.18 respectivement), sous exprimé, joue un rôle dans les mécanismes d'activation du CFTR par GSNO. On peut conclure que cette réponse cellulaire est liée au GSNO.
Les modifications d'expression de 35 gènes sont communes lors d'exposition aux NP+ et aux NP- (liste en **Annexe 5**) dont 16 gènes inconnus. Parmi les gènes sur-exprimés i) **CCZ1** (+2.02 / +2.57 respectivement) participe à la circulation des protéines et au transport médié par les vésicules ; ii) **STARD13** (+2.79 / +5.46 respectivement) et **KLHL15** (+2.22 / +2.58 respectivement) induisent la régulation de la réorganisation du cytosquelette ; et iii) **MYOF** (+2.49 / +3.13 respectivement) impliqué dans la fusion membranaire et la régénération / réparation de la membrane. Par contre, les gènes **CYTH1** (-2.05 / -2.53 respectivement) qui joue un rôle dans la circulation des membranes, **KANK4** (-2.26 / -3.32 respectivement) impliqué dans le contrôle de la formation de cytosquelette en régulant la polymérisation de l'actine, et **SYCN** (-2.58 / -3.28 respectivement) qui régule l'exocytose sont sous-exprimés. Ces deux types de réponse suggèrent que les produits de ces gènes participent au transport intracellulaire des NP.

L'expression de 14 gènes est modifiée après exposition aux NP- et au GSNO libre (liste en **Annexe 6**) dont 10 ne possèdent pas de fonctions connues ou sont non codants.

o « Pathways » communes

Cinq « pathways » sont communes aux 3 conditions du traitement (**Tableau 6**):

<table><caption>**Tableau 6.** « Pathways » communes et principaux gènes mis en jeux pour les 3 conditions d'expositions (200 µg/mL de NP- ou NP+ ou GSNO 6 µM) 4 h</caption><thead><tr><th>Pathway</th><th>n*</th><th>GSNO</th><th>NP+</th><th>NP-</th></tr></thead><tbody><tr><td>Gas signaling</td><td>109</td><td>CREB5 / VIPR2</td><td>CREB5 / VIPR2<br>PTGIR/GPER1</td><td>VIPR2<br>ADD1</td></tr><tr><td>Chondroitin sulfate biosynthesis (late stages)</td><td>46</td><td>NDST3</td><td>CSGALNACT2<br>SULT1C2</td><td>CSGALNACT2</td></tr><tr><td>Triacylglycerol biosynthesis</td><td>33</td><td>GPAT2</td><td>GPAT2<br>ELOVL6</td><td>GPAT2</td></tr><tr><td>Autoimmune thyroid disease signaling</td><td>34</td><td>CD80</td><td>CD80<br>TPO</td><td>CD86</td></tr><tr><td>Circadian rhythm signaling</td><td>33</td><td>CREB5/VIPR2</td><td>CREB5/VIPR2</td><td>VIPR2 /<br>PER3</td></tr></tbody></table>

En comparant l'exposition aux NP+ et au GSNO libre (6 µM) (**Tableau 7**), 6 « pathways » sont communes dont une est inhibée (cAMP-mediated signaling) (**Figure 17**), en plus des 5 communes aux 3 conditions. A la plus forte dose de NP+ et de GSNO libre, cette « pathway » est significativement réprimée. On avait déjà identifié la voie de l'AMPc pour les

<footnote>* n : Nombre total des gènes composant les pathways</footnote>
NP+ à 50 µg/mL, mais pas pour la dose correspondante de GSNO libre. Cela pourrait être un argument en faveur d'une libération prolongée du •NO encapsulé. La voie de l'IL-17F est impliquée dans l'activation de la voie de l'IL-2, du TGF-β et de l'activation des monocytes. Deux voies (agranulocytes adhesion and diapedesis et granulocytes adhesion and diapedesis) suggèrent une activation de la réponse immunitaire par la mobilisation des éléments figurés blancs.

<figure><img src="image_34.png" /><figcaption>**Figure 17. Mécanisme d'inhibition de la "cAMP-mediated signaling Pathway"**
Schéma fourni par IPA, après fusion des résultats obtenus après exposition des cellules THP-1 à 200 µg/mL de NP+ et à 6 µM de GSNO libre</figcaption></figure>

En vert : les gènes sous-exprimés, en rouge : les gènes sur-exprimés
<table><caption>Tableau 7. « Pathways » communes et principaux gènes mis en jeux pour les conditions d'expositions (200 µg/mL de NP+ ou GSNO 6 µM) 4 h</caption><thead><tr><th>Pathway</th><th>n*</th><th>GSNO</th><th>NP+</th></tr></thead><tbody><tr><td>Agranulocytes adhesion and diapedesis</td><td>173</td><td>CLDN9 / CXCL5 / IL33</td><td>CCL19/CXCL9/ITGA3/ PECAM1/SELP</td></tr><tr><td>cAMP-mediated signaling</td><td>216</td><td>AKAP12 / CREB5 / VIPR2 / GABBR1 / HTR1B / PDE1C</td><td>AKAP12/CREB5/ VIPR2/ GPER1/PTGIR</td></tr><tr><td>Granulocytes adhesion and diapedesis</td><td>163</td><td>--</td><td>SLEP/PECAM1/ITGA3/ CXCL9 / CCL19</td></tr><tr><td>G-protein coupled receptor signaling</td><td>254</td><td>CREB5 / VIPR2 / GABBR1 / HTR1B / PDE1C</td><td>CREB5/ VIPR2 / GPER1 / PTGIR</td></tr><tr><td>Role of IL-17F in allergic inflammatory airway diseases</td><td>41</td><td>CREB5 / CXCL5</td><td>CREB5</td></tr><tr><td>GABA receptor signaling</td><td>66</td><td>GABBR1 / GABRR2 / KCNQ2</td><td>GABRE</td></tr></tbody></table>

Sur les 12 « pathways » communes activées après l'exposition aux NP- ou aux NP+, 7 d'entre-elles sont pour ces deux conditions (Tableau 8). On peut citer la voie APRIL (qui fait partie de la superfamille du TNF) qui a un rôle proinflammatoire et d'activateur de monocytes [221] et la 4-1BB des lymphocytes T.

<table><caption>Tableau 8. « Pathways » communes et principaux gènes mis en jeux pour les conditions d'expositions (200 µg/mL de NP+ ou NP-) 4 h</caption><thead><tr><th>Pathway</th><th>n*</th><th>NP+</th><th>NP-</th></tr></thead><tbody><tr><td>April mediated signaling</td><td>38</td><td>TRAF1</td><td>TRAF1 / MAPK9</td></tr><tr><td>Chondroitin and dermatan biosynthesis</td><td>6</td><td>CSGALNACT2</td><td>CSGALNACT2</td></tr><tr><td>FGF signaling</td><td>85</td><td>CREB5/FGF1/FGF5</td><td>FGFR1</td></tr><tr><td>ID-myo-inositol hexakisphosphate biosynthesis V (from Ins (1,3,4)p3)</td><td>3</td><td>IPMK</td><td>IPMK</td></tr><tr><td>Pyridoxal 5'-phosphate salvage pathway</td><td>63</td><td>PRPF4B*</td><td>PRPF4B* DYRK1A / MAPK9</td></tr><tr><td>Salvage pathways of pyrimidine ribonucleotides</td><td>91</td><td>PRPF4B*</td><td>CMPK1 / DYRK1A / MAPK 9 / PRPF4B*</td></tr><tr><td>4-1BB signaling in T lymphocytes</td><td>31</td><td>TRAF1</td><td>MAPK9 / TRAF1</td></tr></tbody></table>

Après exposition aux NP- et au GSNO libre, une seule « pathway » est modifiée : granzyme A signaling avec un gène commun HIST1H1T et deux uniquement avec les NP-HIST1H1E et SET.

<footnote>* n : Nombre total des gènes composant les pathways</footnote>

<footnote>* n : Nombre total des gènes composant les pathways</footnote>
# Discussion générale
L'utilisation des NP est en progression exponentielle, et ses domaines d'application touchent
tous les secteurs industriels. Un des intérêts de ces objets est que, du fait de leur taille
nanométrique, leurs composants acquièrent des propriétés physico-chimiques nouvelles. Pour le
vivant, un matériau de taille nanométrique peut, entre autres, devenir plus toxique, passer plus
facilement les barrières anatomiques, et être plus biopersistant, qu'à une taille micrométrique
[53]. Ainsi, les risques posés par ces nouveaux toxiques doivent être identifiés. Pour ce faire, les
tests classiques de toxicité ne sont pas forcément adaptés, notamment du fait d'interactions entre
les réactifs utilisés et les NP étudiées. Dans notre laboratoire, l'évaluation de la toxicité des NP
se fait en combinant des approches classiques de biologie cellulaire (tests au MTT, au WST-1 ou
au Bleu trypan) et plus récentes de biologie moléculaire (qRT-PCR, puces à ADN ou
« microarray »), dans le but d'identifier des marqueurs de toxicité lors d'exposition à des NP.

L'EA 3452, CITHEFOR, développe différentes outils (nano- et microparticules, implants,...) et différentes formulations (liposomes, NP polymériques ou métalliques) afin de vectoriser des donneurs de S-nitrosothiols à visées thérapeutiques cardiovasculaires. Au sein de cette unité, notre laboratoire avait développé un modèle de monocytes/macrophages de rat (NR8383) qui a permis de montrer que des NP d'Eudragit® RS de 55 nm présentaient une forte cytotoxicité à une concentration de 200 µg/mL après 24 heures d'exposition, mais, même à 50 µg/mL, elles induisaient la mort cellulaire par autophagie [143], [103]. Le mécanisme de cette toxicité était lié, après endocytose des NP, à une atteinte mitochondriale, avec déstabilisation de leur structure, et dégradation par mitophagie. Afin d'utiliser un modèle de macrophage humains pour confirmer, cette étude a été poursuivie avec des monocytes primaires CD14+ provenant de volontaires. Ceci a fait l'objet de mon master 2. La grande variabilité des résultats obtenus avec ces cellules, d'un patient à l'autre, n'a pas permis d'obtenir de conclusion. Aussi, a-t'il été décidé d'utiliser une lignée monocytaire humaine THP-1 avec laquelle des résultats préliminaires avaient montré que les NP d'Eudragit® induisaient une activation métabolique se traduisant par une augmentation de la viabilité cellulaire. En parallèle, un travail mené en collaboration avec le laboratoire du Pr Brooks à Berkeley (USA) a montré les mêmes résultats avec une lignée de cellules épithéliales mammaires humaines HMEC 184. Les NP induisent une augmentation de la
fusion de mitochondries, et donc leur activation [168]. Afin de garder le modèle macrophagique, ces cellules étant souvent les premières en contact avec les particules étrangères au sein de l'organisme, les études toxicologiques se sont poursuivies en se limitant à la lignée de THP-1.

L'objectif principal de ce travail était donc de mieux comprendre l'effet, sur ces cellules, de NP d'Eudragit® RL, chargées ou non en GSNO, choisi comme PA, et de cette molécule seule. En premier, il a été tenté de décrypter les mécanismes biologiques induits après une exposition à des NP d'Eudragit®. L'utilisation des outils de la toxicogénomique avait pour but d'étudier la réponse adaptative des cellules aux trois conditions d'exposition. Le premier article a confirmé les différences entre les deux lignées cellulaires de rat et humaine. En effet, la sur-expression des gènes *ATG16L*, *BCL2*, and *TNFA* dans les cellules NR8383, permet d'expliquer l'induction de l'autophagie et des voies conduisant à l'inflammation. Par contre, la sous-expression de *NCF1*, *NFKB*, et *IL1B* dans les THP-1, peut expliquer l'augmentation de la viabilité cellulaire. Ces observations ont montré l'importance du choix du modèle quand il s'agit d'étudier les réponses cellulaires à un agent toxique du fait des résultats opposés obtenus avec des cellules possédant les mêmes fonctions mais provenant l'une de rongeurs et l'autre d'Homme. Le fait que des résultats proches de ceux de THP-1 aient été obtenus avec une lignée épithéliale humaine tend à montrer que c'est principalement l'espèce animale choisie qui gouverne la réponse cellulaire.

L'internalisation des NP d'Eudragit® apparaît comme la première étape menant à la réponse cellulaire. Comme déjà rapporté, cette pénétration s'effectue selon plusieurs mécanismes concomitamment : voies cavéolines et clathrine dépendantes, principalement, mais aussi d'autres voies énergie dépendante ou indépendante, peu définies. L'étude transcriptomique, désormais considérée comme une technique consensus pour l'évaluation de la réponse cellulaire aux NP, a été menée en parallèle. Ses résultats ont été ajoutés à la base des données Nanominer (http://compbio.uta.fi/estools/nanommune/index.php/) qui regroupe toutes les études utilisant cette approche. Avec cette technique, le nombre de gènes dont l'expression est modifiée est très important et pour pouvoir interpréter nos résultats, nous avons systématiquement utilisé une limite de *P* à 0,001. L'article 2 présente les résultats de l'étude après analyse par cluster pour une seule condition de dose et de temps. Il nous a paru intéressant de reprendre ce travail en étudiants, dans les mêmes conditions expérimentales, plusieurs doses et plusieurs temps. Pour l'analyse nous avons utilisé un logiciel plus performant (IPA) mais dans une configuration de démonstration et donc limité en possibilité. Il a été ainsi possible d'étudier les relations entre les
gènes et l'analyse des « pathways » dans lesquelles un ou plusieurs gènes on leur expression modifiée. Ces résultats complémentaires sont exposés dans la 4ème partie. Du fait de conditions d'analyse adoptées, seuls quelques gènes impliqués dans les voies d'endocytose et le trafic intracellulaire ont été identifiés. Le logiciel en démonstration n'était pas complet et il ne nous a pas été possible d'étudier spécifiquement certaines « pathways » qui n'ont pas été mises en évidence dans cette étude. Cependant elle a été intéressante puisqu'elle a montré, en comparant deux à deux les trois conditions d'exposition, qu'il existait des « pathways » spécifiquement liées au GSNO ainsi que d'autres liées aux NP. L'acquisition de ce logiciel nous permettra de compléter et d'affiner ces résultats en vue d'une publication.

L'article 3 est une application directe de notre étude transcriptomique. L'organisme animal met en jeu le •NO pour lutter contre les agents infectieux, et en particulier les parasites comme les *Leishmania*. Cette molécule, effectrice de la réponse immunitaire mais pourrait aussi être utilisée en thérapeutique. Nous avons proposé un mécanisme d'activation de la réponse du macrophage au •NO par une mise en jeu de cytokines pro-inflammatoires. La réponse au GSNO libre a également montré que 29 gènes de la famille des récepteurs olfactifs étaient activés (Annexe 7). L'expression des protéines de cette famille, qui seraient impliquée dans l'induction du chimiotactisme des monocytes [222] n'est pas restreinte aux cellules spécialisées dans l'olfaction mais survient également dans d'autres tissus, notamment intestinal [223]. Dans cette hypothèse, l'activation des gènes correspondant, participerait au processus inflammatoire. Ainsi l'étude transcriptomique apporte beaucoup d'informations, parfois difficile à décrypter mais permet d'expliquer les phénomènes observées lors de contact entre NP et cellule et si elle confirme certaines données, elle permet également de mettre en évidence des « pathways » jusque-là ignorés.
# Conclusions générales et perspectives
Malgré l'intérêt de la transcriptomique pour l'élucidation des réponses cellulaires aux NP, celui-ci reste limité par le fait que seule une analyse globale de l'expression des gènes est réalisée et elle ne dépasse pas l'étape pré traductionnelle. Cependant pour les gènes intéressants une étude complémentaire spécifique peut être menée par qRT-PCR. L'étude de l'expression des protéines, sans doute plus précise, peut venir en complément en utilisant la protéomique qui prend en charge l'étape post traductionnelle ainsi que les modifications qui peuvent en découler (glycosylation par exemple).

Pour mieux comprendre la réponse cellulaire aux NP, il est envisagé d'utiliser une deuxième modèle qui s'appliquerait aux NP d'Eudragit® qui sont destinées à des voies orales. La mise en œuvre d'un modèle de cellules intestinales (Caco-2) exposées aux mêmes traitements que THP-1 est déjà faite. Il permettra tout d'abord de comparer les réponses cellulaires obtenues avec les deux modèles afin d'identifier des invariants qui serviront de biomarqueurs d'exposition. Par la suite des cocultures seront développées avec des Caco-2, des HT29-MTX cellules sécrétrices de mucus, et des THP-1. Il sera alors possible de se rapprocher des conditions physiologiques, dans le cadre d'une administration orale, tout en s'affranchissant de l'expérimentation animale. Ce type d'approche est actuellement en plein développement et représente, pour l'étude de la toxicologie des NP, une voie d'avenir.
Annexes
# Annexe 1

Liste des 26 gènes communs dont l'expression est modifiée après exposition aux NP+ 50 et 200 µg/mL pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ...).

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 50 µg/mL</th><th>FC / NP+ 200 µg/mL</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4" style="text-align: center; font-weight: bold;">Gènes connus</td></tr><tr><td>OR2A7</td><td>- 4.06</td><td>- 3.45</td><td><b>Olfactory Receptor, Family 2, Subfamily A, Member 7.</b><br>Odorant receptor.</td></tr><tr><td>VIPR2</td><td>- 4.93</td><td>- 4.09</td><td><b>Vasoactive Intestinal Peptide Receptor 2</b>. This gene encodes a receptor for vasoactive intestinal peptide, a small neuropeptide. Vasoactive intestinal peptide is involved in smooth muscle relaxation, exocrine and endocrine secretion, and water and ion flux in lung and intestinal epithelia. Its actions are effected through integral membrane receptors associated with a guanine nucleotide binding protein which activates adenylate cyclase.</td></tr><tr><td>MAATS1</td><td>- 2.66</td><td>- 2.59</td><td><b>MYCBP-Associated</b>. Testis Expressed 1, may play a role in spermatogenesis.</td></tr><tr><td>ANKDD1B</td><td>- 2.86</td><td>- 2.88</td><td><b>Ankyrin Repeat And Death Domain Containing 1B</b>. An important paralog of this gene is MIB2</td></tr><tr><td>JMJD1C</td><td>+ 2.22</td><td>+ 2.17</td><td><b>Jumonji Domain Containing 1C</b>. Probable histone demethylase that specifically demethylates Lys-9 of histone H3, thereby playing a central role in histone code. Demethylation of Lys residue generates formaldehyde and succinate. May be involved in hormone-dependent transcriptional activation, by participating in recruitment to androgen-receptor target genes</td></tr><tr><td>HOXB2</td><td>- 4.86</td><td>- 3.97</td><td><b>Homeobox B2</b>. Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis.</td></tr><tr><td>DLX2</td><td>+ 2.57</td><td>+ 2.09</td><td><b>Distal-Less Homeobox 2</b>. Likely to play a regulatory role in the development of the ventral forebrain. May play a role in craniofacial patterning and morphogenesis.</td></tr><tr><td>ATG9B</td><td>- 4.10</td><td>- 2.60</td><td><b>Autophagy Related 9B</b>. This gene functions in the regulation of autophagy, a lysosomal degradation pathway. This gene also functions as an antisense transcript in the posttranscriptional regulation of the endothelial nitric oxide synthase 3 gene, which has 3' overlap with this gene on the opposite strand. Mutations in this gene and disruption of the autophagy process have been associated with multiple cancers. Alternative splicing results in multiple transcript variants.</td></tr><tr><td>CD164L2</td><td>- 3.07</td><td>- 2.85</td><td><b>CD164 Sialomucin-Like 2</b>. An important paralog of this gene is CD164.</td></tr><tr><td>PTPN20B</td><td>- 3.49</td><td>- 3.14</td><td><b>Protein Tyrosine Phosphatase, Non-Receptor Type 20B</b>. Tyrosine-protein phosphatase targeted to sites of actin polymerization in response of varied extracellular stimuli. Has tyrosine phosphatase activity towards various tyrosyl phosphorylated substrates</td></tr><tr><td>KANK4</td><td>- 4.81</td><td>- 2.26</td><td><b>KN Motif And Ankyrin Repeat Domains 4</b>. May be involved in the control of cytoskeleton formation by regulating actin polymerization.</td></tr></tbody></table>
<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 50 µg/mL</th><th>FC / NP+ 200 µg/mL</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4">Gènes inconnus, pseudogènes ou non codants</td></tr><tr><td>FAM83E</td><td>- 3.18</td><td>- 2.10</td><td>Family With Sequence Similarity 83, Member E.</td></tr><tr><td>C3orf36</td><td>+ 2.72</td><td>+ 2.69</td><td>Chromosome 3 Open Reading Frame 36. Uncharacterized protein C3orf36</td></tr><tr><td>TMEM52B</td><td>- 6.58</td><td>- 5.41</td><td>Transmembrane Protein 52B.</td></tr><tr><td>XLOC_007808</td><td>+ 2.32</td><td>+ 2.46</td><td>No data available</td></tr><tr><td>XLOC_002176</td><td>- 2.46</td><td>- 2.25</td><td>No data available</td></tr><tr><td>LOC285638</td><td>- 2.30</td><td>- 2.20</td><td>No data available</td></tr><tr><td>XLOC_011507</td><td>- 3.54</td><td>- 3.30</td><td>No data available</td></tr><tr><td>XLOC_010915</td><td>- 2.57</td><td>- 2.24</td><td>No data available</td></tr><tr><td>XLOC_007305</td><td>- 3.43</td><td>- 2.69</td><td>No data available</td></tr><tr><td>LOC102723617</td><td>- 3.31</td><td>- 4.14</td><td>No data available</td></tr><tr><td>XLOC_012572</td><td>- 2.58</td><td>- 2.92</td><td>No data available</td></tr><tr><td>XLOC_009927</td><td>- 4.32</td><td>- 5.55</td><td>No data available</td></tr><tr><td>XLOC_012733</td><td>- 2.72</td><td>- 2.48</td><td>No data available</td></tr><tr><td>XLOC_013612</td><td>- 4.25</td><td>- 4.13</td><td>No data available</td></tr><tr><td>XLOC_014512</td><td>- 3.29</td><td>- 2.98</td><td>No data available</td></tr></tbody></table>
Annexe 2

Liste des 17 gènes communs dont l'expression est modifiée après exposition aux NP+ 50 µg/mL ou GSNO 1,4 µM pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ..)

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 50 µg/mL</th><th>FC / GSNO 1,4 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4" style="text-align: center; font-weight: bold;">Gènes connus</td></tr><tr><td>VIPR2</td><td>- 4.93</td><td>- 3.71</td><td>Vasoactive Intestinal Peptide Receptor 2. This gene encodes a receptor for vasoactive intestinal peptide, a small neuropeptide. This peptide is involved in smooth muscle relaxation, exocrine and endocrine secretion, and water and ion flux in lung and intestinal epithelia. Its actions are effected through integral membrane receptors associated with a guanine nucleotide binding protein which activates adenylate cyclase.</td></tr><tr><td>FARP1</td><td>+ 2.50</td><td>+ 2.89</td><td>FERM, RhoGEF (ARHGEF) And Pleckstrin Domain Protein 1 (Chondrocyte-Derived), Functions as guanine nucleotide exchange factor for RAC1. May play a role in semaphorin signaling. Plays a role in the assembly and disassembly of dendritic filopodia, the formation of dendritic spines, regulation of dendrite length and ultimately the formation of synapses</td></tr><tr><td>NUGGC</td><td>+ 2.41</td><td>+ 4.93</td><td>Nuclear GTPase, Germinal Center Associated. Plays a role as replication-related GTPase protein in germinal center B-cell</td></tr><tr><td>IRGC</td><td>+ 3.46</td><td>+ 5.79</td><td>Immunity-Related GTPase Family Cinema.</td></tr><tr><td>IGSF9B</td><td>+ 3.41</td><td>+ 3.69</td><td>Immunoglobulin Superfamily, Member 9B.</td></tr><tr><td colspan="4" style="text-align: center; font-weight: bold;">Gènes inconnus, pseudogènes ou non codants</td></tr><tr><td>FAM83E</td><td>- 3.18</td><td>- 3.02</td><td>Family With Sequence Similarity 83, Member E.</td></tr><tr><td>TMEM52B</td><td>- 6.58</td><td>- 3.53</td><td>Transmembrane Protein 52B</td></tr><tr><td>HOXA-AS3</td><td>+ 3.02</td><td>+ 2.38</td><td>HOXA Cluster Antisense RNA 3. Non-Protein Coding.</td></tr><tr><td>GAS6-AS1</td><td>+ 2.61</td><td>+ 3.56</td><td>GAS6 Antisense RNA 1. Non-Protein Coding</td></tr><tr><td>C3orf36</td><td>+ 2.72</td><td>+ 4.2</td><td>Chromosome 3 Open Reading Frame 36. Uncharacterized protein C3orf36</td></tr><tr><td>LINC00993</td><td>+ 2.96</td><td>+ 7.00</td><td>Long Intergenic. Non-Protein Coding RNA 993</td></tr><tr><td>XLOC_011971</td><td>+ 4.01</td><td>+ 4.28</td><td>No data available</td></tr><tr><td>LOC100128164</td><td>+ 2.76</td><td>+ 2.31</td><td>No data available</td></tr><tr><td>XLOC_003721</td><td>+ 7.17</td><td>+ 4.95</td><td>No data available</td></tr><tr><td>XLOC_011382</td><td>+ 3.08</td><td>+ 4.85</td><td>No data available</td></tr><tr><td>XLOC_002732</td><td>+ 2.43</td><td>+ 2.77</td><td>No data available</td></tr><tr><td>LOC101927787</td><td>+ 2.63</td><td>- 4.50</td><td>No data available</td></tr></tbody></table>
Annexe 3

Liste des 28 gènes communs dont l'expression est modifiée après exposition aux NP+ ou NP- 200 µg/mL ou GSNO 6 µM pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ..)

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP-200 µg/mL</th><th>FC / NP+200 µg/mL</th><th>FC / GSNO 6 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="5" style="text-align: center; font-weight: bold;">Gènes connus</td></tr><tr><td>KRT3</td><td>-2.71</td><td>-2.70</td><td>-2.21</td><td><strong>Keratin 3</strong>. differentiation of simple and stratified epithelial tissues.</td></tr><tr><td>GPAT2</td><td>-2.50</td><td>-2.57</td><td>-2.37</td><td><strong>Glycerol-3-phosphate acyltransferase 2</strong>. Mitochondrial, Play an essential step in glycerolipid biosynthesis.</td></tr><tr><td>ELFN1</td><td>-3.38</td><td>-2.82</td><td>-3.79</td><td><strong>Extracellular leucine-rich repeat and fibronectin.</strong> Aregulator of presynaptic release probability to direct the formation of highly facilitating pyramidal-OLM synapses (By similarity). Inhibits phosphatase activity of protein phosphatase 1 (PP1) complexes.</td></tr><tr><td>HOXB2</td><td>-3.86</td><td>-3.97</td><td>-5.83</td><td><strong>Homeobox B2</strong>. A sequence-specific transcription factor that is involved in development</td></tr><tr><td>MIR221</td><td>+3.57</td><td>+3.78</td><td>+3.02</td><td><strong>MicroRNA 221 (MIR221), microRNA</strong>. Involved in post-transcriptional regulation of gene expression by affecting both the stability and translation of mRNAs.</td></tr><tr><td>ETV2</td><td>-2.70</td><td>-2.43</td><td>-2.87</td><td><strong>Ets variant 2</strong>. Binds to DNA sequences containing the consensus pentanucleotide, regulation of transcription.</td></tr><tr><td>PTPN20B</td><td>-3.41</td><td>-3.14</td><td>-7.72</td><td><strong>Protein tyrosine phosphatase, non-receptor type 20B.</strong> Tyrosine-protein phosphatase targeted to sites of actin polymerization in response of varied extracellular stimulation.</td></tr><tr><td>SCTR</td><td>-2.67</td><td>-2.86</td><td>-3.41</td><td><strong>Secretin receptor</strong>. A member of the glucagon receptor family. They are classically localized to the epithelial cells within the pancreatic and biliary ducts, where they stimulate the release of alkaline bicarbonate-rich fluid to neutralize acidic chyme from the stomach. This protects the duodenal mucosa and provides an optimal intraluminal environment for digestion to take place. Secretin receptors are also found in the CNS, where they are thought to play a role in the regulation of the hypothalamic-pituitary-axis (</td></tr><tr><td>SELP</td><td>-3.10</td><td>-2.64</td><td>-2.55</td><td><strong>Selectin P (granule membrane protein antigen CD62)</strong>. A calcium-dependent receptor that binds to sialylated forms of Lewis blood group carbohydrate antigens on neutrophils and monocytes, platelet activation.</td></tr><tr><td>GPR52</td><td>+2.89</td><td>+2.91</td><td>+2.42</td><td><strong>G protein-coupled receptor 52</strong>. Signal transduction from the external to inside of the cell</td></tr><tr><td>TUSC1</td><td>-3.55</td><td>-2.94</td><td>-4.03</td><td><strong>Tumor suppressor candidate 1</strong>. Tumor suppressor gene</td></tr><tr><td>HIST1H2AL</td><td>+4.91</td><td>+3.49</td><td>+3.44</td><td><strong>Histone cluster 1</strong>. Chromatin organization, transcription regulation, DNA repairs, DNA replication and chromosomal stability.</td></tr><tr><td>RASSF6</td><td>-2.42</td><td>-2.08</td><td>-2.65</td><td><strong>Ras association (RalGDS/AF-6) domain family.</strong> Induction of apoptosis, through both caspase-dependent and caspase-independent pathways</td></tr></tbody></table>
<table><thead><tr><th>Symbole du gène</th><th>FC / NP-200 µg/mL</th><th>FC / NP+200 µg/mL</th><th>FC / GSNO 6 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td>ZNF551</td><td>-3.78</td><td>-3.95</td><td>+2.09</td><td>Zinc finger protein 551. Transcriptional regulation.</td></tr><tr><td>VIPR2</td><td>-4.57</td><td>-4.09</td><td>-3.22</td><td>Vasoactive intestinal peptide receptor 2</td></tr><tr><td>OR2A7</td><td>-4.55</td><td>-3.45</td><td>-4.36</td><td>Olfactory receptor, family 2, subfamily A, member 7. A odorant receptor.</td></tr><tr><td colspan="5" style="text-align: center;">Gènes inconnus, pseudogènes ou non codants</td></tr><tr><td>TMEM151B</td><td>-5.60</td><td>-5.07</td><td>-4.89</td><td>Transmembrane protein 151B</td></tr><tr><td>TMEM52B</td><td>-4.87</td><td>-5.41</td><td>-4.82</td><td>Transmembrane protein 52B</td></tr><tr><td>GOLGA2P7</td><td>-3.85</td><td>-3.45</td><td>-4.07</td><td>Golgin A2 Pseudogene 7. A Pseudogene.</td></tr><tr><td>AOX2P</td><td>-4.01</td><td>-4.01</td><td>-3.08</td><td>Aldehyde oxidase 2. A pseudogene.</td></tr><tr><td>LOC100130430</td><td>-4.69</td><td>-3.72</td><td>-5.22</td><td>No data available</td></tr><tr><td>LOC100507547</td><td>-7.21</td><td>-4.00</td><td>-13.39</td><td>No data available</td></tr><tr><td>LOC102723617</td><td>-3.36</td><td>-4.14</td><td>-3.66</td><td>No data available</td></tr><tr><td>XLOC_014512</td><td></td><td></td><td></td><td>No data available</td></tr><tr><td>XLOC_005203</td><td>-3.29</td><td>-4.28</td><td>-2.95</td><td>No data available</td></tr><tr><td>XLOC_012572</td><td>-2.43</td><td>-2.92</td><td>-3.17</td><td>No data available</td></tr><tr><td>XLOC_011429</td><td>-2.60</td><td>-2.47</td><td>-2.34</td><td>No data available</td></tr><tr><td>XLOC_013612</td><td>-4.48</td><td>-4.13</td><td>-4.22</td><td>No data available</td></tr></tbody></table>
# Annexe 4

Liste des 52 gènes communs dont l'expression est modifiée après exposition aux NP+ 200 µg/mL ou GSNO 6 µM pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ..)

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 200 µg/mL</th><th>FC / GSNO 6 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4">Gènes connus</td></tr><tr><td>HSPA1A</td><td>-2.15</td><td>-2.18</td><td>Heat shock 70kDa protein 1A. Stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. (Mechanisms of CFTR activation by s-nitroglutathione)</td></tr><tr><td>HYDIN</td><td>-3.80</td><td>-4.05</td><td>Axonemal central pair apparatus protein. Involved in cilia motility</td></tr><tr><td>MAL</td><td>-4.62</td><td>-4.03</td><td>Mal, T-cell differentiation protein. Could be an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane</td></tr><tr><td>SLC5A1</td><td>-5.02</td><td>-4.01</td><td>Solute carrier family 5 (sodium/glucose cotransporter). Sodium-dependent glucose transporter, primary mediator of dietary glucose and galactose uptake from the intestinal lumen. Actively transports glucose into cells by Na(+) cotransport with a Na(+) to glucose coupling ratio of 2:1.</td></tr><tr><td>PRDX3</td><td>+2.88</td><td>+2.76</td><td>Peroxiredoxin 3. A mitochondrial protein with antioxidant function. Involved in redox regulation of the cell. Protects radical-sensitive enzymes from oxidative damage by a radical-generating system. Acts synergistically with MAP3K13 to regulate the activation of NF-kappa-B in the cytosol.</td></tr><tr><td>TGFBR3</td><td>+2.30</td><td>+2.45</td><td>Transforming growth factor, beta receptor II. Binds to TGF-beta, involved in capturing and retaining TGF-beta for presentation to the signaling receptors. (La cytokine TGF-β joue un rôle intégral dans la régulation des réponses immunitaires. Le TGF-β a des effets pléiotropiques sur l'immunité adaptative)</td></tr><tr><td>PNMA1</td><td>+2.09</td><td>+2.05</td><td>Paraneoplastic Ma antigen 1. This gene encodes a neuron- and testis-specific protein that is also expressed in some paraneoplastic syndromes affecting the nervous system.</td></tr><tr><td>ANKRD44</td><td>-2.90</td><td>-3.54</td><td>Ankyrin repeat domain 44. Putative regulatory subunit of protein phosphatase 6 (PP6) that may be involved in the recognition of phosphoprotein substrates.</td></tr><tr><td>KRT85</td><td>-3.44</td><td>-3.68</td><td>Keratin 85. A member of the keratin gene family</td></tr><tr><td>ARHGAP40</td><td>-2.84</td><td>-6.70</td><td>Rho GTPase activating protein. GTPase activator for the Rho-type GTPases by converting them to an inactive GDP-bound state.</td></tr><tr><td>FPGT-TNNI3K</td><td>-2.73</td><td>-2.77</td><td>FPGT-TNNI3K readthrough (FPGT-TNNI3K). This locus represents naturally occurring read-through transcription from the neighboring fucose-1-phosphate guanylyltransferase (FPGT) and TNNI3 interacting kinase (TNNI3K) genes. (protein kinase activity, protein tyrosine kinase activity, protein binding, ATP binding, transferase activity, transferring phosphorus-containing groups).</td></tr></tbody></table>
<table><thead><tr><td>Symbole du gène</td><td>FC / NP+ 200 µg/mL</td><td>FC / GSNO 6 µM</td><td>Nom et fonction du gène</td></tr></thead><tbody><tr><td>CD80</td><td>-2.21</td><td>-2.61</td><td>CD80 molecule. A membrane receptor that is activated by the binding of CD28 or CTLA-4. The activated protein induces T-cell proliferation and cytokine production. (Involved in the costimulatory signal essential for T-lymphocyte activation. T-cell proliferation and cytokine production is induced by the binding of CD28, binding to CTLA-4 has opposite effects and inhibits T-cell activation)</td></tr><tr><td>DCAF13</td><td>+2.41</td><td>+2.38</td><td>DDB1 and CUL4 associated factor 13. Possible role in ribosomal RNA processing (By similarity). May function as a substrate receptor for CUL4-DDB1 E3 ubiquitin-protein ligase complex.</td></tr><tr><td>ASIC5</td><td>-2.50</td><td>-2.41</td><td>Acid-sensing (proton-gated) ion channel family. Cation channel that gives rise to very low constitutive currents in the absence of activation. The activated channel exhibits selectivity for sodium, and is inhibited by amiloride. The amiloride-sensitive Na(+) channel encoded by this gene is primarily expressed in the small intestine, however, its exact function is not known.</td></tr><tr><td>KRTAP20-3</td><td>-3.30</td><td>-3.28</td><td>Keratin associated protein 20-3. In the hair cortex, hair keratin intermediate filaments are embedded in an interfilamentous matrix, consisting of hair keratin-associated proteins (KRTAP), which are essential for the formation of a rigid and resistant hair shaft through their extensive disulfide bond cross-linking with abundant cysteine residues of hair keratins. The matrix proteins include the high-sulfur and high-glycine-tyrosine keratins.</td></tr><tr><td>ETFA</td><td>+2.10</td><td>+2.42</td><td>Electron-transfer-flavoprotein, alpha polypeptide. The electron transfer flavoprotein serves as a specific electron acceptor for several dehydrogenases, including five acyl-CoA dehydrogenases, glutaryl-CoA and sarcosine dehydrogenase. It transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF dehydrogenase). ETFA participates in catalyzing the initial step of the mitochondrial fatty acid beta-oxidation</td></tr><tr><td>CREB5</td><td>-3.41</td><td>-3.17</td><td>cAMP responsive element binding protein 5. It binds to the cAMP response element and activates transcription. specifically binds to CRE (cAMP response element) as a homodimer or a heterodimer and functions as a CRE-dependent trans-activator</td></tr><tr><td>CACNA1B</td><td>-5.30</td><td>-6.05</td><td>Calcium channel, voltage-dependent, N type, alpha 1B subunit. Voltage-gated calcium channels (CaV) are present in the membrane of most excitable cells and mediate calcium influx in response to depolarisation. They regulate intracellular processes such as contraction, secretion, neurotransmission and gene expression. Voltage-gated calcium channels are formed from four or five distinct subunits; the alpha-subunit is the largest subunit and incorporates the voltage sensor, conduction pore and gating apparatus.</td></tr><tr><td>LAPTM4B</td><td>+2.08</td><td>+2.09</td><td>Lysosomal protein transmembrane 4 beta. A potential protooncogene, whose overexpression is involved in carcinogenesis and progression of HCC. In normal cells, it may also play important roles such as regulation of cell proliferation and survival</td></tr><tr><td>GJA9</td><td>-4.57</td><td>-4.39</td><td>Gap junction protein, alpha 9. Connexins, such as GJA9, are involved in the formation of gap junctions, intercellular conduits that directly connect the cytoplasms of contacting cells.</td></tr></tbody></table>
<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 200 µg/mL</th><th>FC / GSNO 6 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td>SLC9A2</td><td>-2.50</td><td>-2.33</td><td>Solute carrier family 9, subfamily A (NHE2, cation proton antiporter 2). Involved in pH regulation to eliminate acids generated by active metabolism or to counter adverse environmental conditions</td></tr><tr><td>PANX2</td><td>-5.91</td><td>-5.61</td><td>Pannexin 2 (PANX2). Structural component of the gap junctions and the hemichannels</td></tr><tr><td>PSMD10</td><td>+2.02</td><td>+2.07</td><td>Proteasome (prosome, macropain) 26S subunit, non-ATPase. This gene encodes a subunit of the PA700/19S complex, which is the regulatory component of the 26S proteasome. The 26S proteosome complex is required for ubiquitin-dependent protein degradation. This protein is a non-ATPase subunit that may be involved in protein-protein interactions</td></tr><tr><td>HOXC5</td><td>-4.31</td><td>-3.27</td><td>Homeobox C5. Sequence-specific transcription factor, which is part of a developmental regulatory system.</td></tr><tr><td colspan="4" style="text-align: center;">Gènes inconnus, pseudogènes ou non codants</td></tr><tr><td>FAM83E</td><td>-2.10</td><td>-2.38</td><td>Family With Sequence Similarity 83, Member E. No data available</td></tr><tr><td>FAM45A</td><td>+2.31</td><td>+2.32</td><td>Family With Sequence Similarity 45, Member A. No data available</td></tr><tr><td>FAM230B</td><td>+2.26</td><td>+2.06</td><td>Family With Sequence Similarity 230, Member B, Non-Protein Coding.</td></tr><tr><td>FAM74A1</td><td>-4.85</td><td>-5.12</td><td>Family With Sequence Similarity 74, Member A1. lncRNA</td></tr><tr><td>SNORD16</td><td>-2.14</td><td>-2.20</td><td>Small Nucleolar RNA, C/D Box 16, snoRNA.</td></tr><tr><td>SNORD37</td><td>-2.09</td><td>-2.43</td><td>Small Nucleolar RNA, C/D Box 37, snoRNA.</td></tr><tr><td>MIR7-3HG</td><td>-3.63</td><td>-5.90</td><td>MIR7-3 Host Gene, lncRNA.</td></tr><tr><td>LINC00162</td><td>-4.31</td><td>-3.27</td><td>No data available</td></tr><tr><td>XLOC_005147</td><td>-4.49</td><td>-3.15</td><td>No data available</td></tr><tr><td>LOC101929542</td><td>-2.15</td><td>-2.91</td><td>No data available</td></tr><tr><td>XLOC_011179</td><td>-5.55</td><td>-4.09</td><td>No data available</td></tr><tr><td>LOC387810</td><td>-5.47</td><td>-4.96</td><td>No data available</td></tr><tr><td>XLOC_007305</td><td>-2.69</td><td>-2.52</td><td>No data available</td></tr><tr><td>LINC01204</td><td>-2.69</td><td>-2.52</td><td>No data available</td></tr><tr><td>XLOC_007585</td><td>-2.62</td><td>-2.07</td><td>No data available</td></tr><tr><td>XLOC_002597</td><td>+10.25</td><td>+3.16</td><td>No data available</td></tr><tr><td>LOC729159</td><td>-2.06</td><td>+2.89</td><td>No data available</td></tr><tr><td>XLOC_012733</td><td>-2.48</td><td>-2.42</td><td>No data available</td></tr><tr><td>LOC221814</td><td>-3.08</td><td>-2.31</td><td>No data available</td></tr><tr><td>XLOC_011701</td><td>-5.48</td><td>-4.64</td><td>No data available</td></tr><tr><td>LOC100996291</td><td>-2.79</td><td>-3.32</td><td>No data available</td></tr><tr><td>LOC100134040</td><td>-2.48</td><td>-3.98</td><td>No data available</td></tr><tr><td>XLOC_009927</td><td>-5.54</td><td>-5.62</td><td>No data available</td></tr><tr><td>LOC102725453</td><td>+2.27</td><td>+2.03</td><td>No data available</td></tr><tr><td>XLOC_001891</td><td>-3.12</td><td>-2.89</td><td>No data available</td></tr><tr><td>XLOC_009986</td><td>-3.78</td><td>-5.42</td><td>No data available</td></tr><tr><td>LOC101929514</td><td>-3.46</td><td>-3.61</td><td>No data available</td></tr><tr><td>XLOC_011339</td><td>+3.80</td><td>+3.36</td><td>No data available</td></tr></tbody></table>
Annexe 5

Liste des 35 gènes communs dont l'expression est modifiée après exposition aux NP+ ou NP- 200 µg/mL pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ..)

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 200 µg/mL</th><th>FC / NP- 200 µg/mL</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4">Gènes connus</td></tr><tr><td>LRRC10</td><td>-2.54</td><td>-3.02</td><td>Leucine rich repeat containing 10. A cardiac-specific factor that is crucial for proper cardiac development and deletion of Lrrc10 in mice results in dilated cardiomyopathy.</td></tr><tr><td>CCZ1</td><td>+2.02</td><td>+2.57</td><td>CCZ1 vacuolar protein trafficking and biogenesis associated homolog (S. cerevisiae). vacuolar protein trafficking vesicle-mediated transport</td></tr><tr><td>MAGEB5</td><td>-3.78</td><td>-5.98</td><td>Melanoma antigen family B 5. The four MAGE genes (MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3 are not expressed in normal tissues, except for testis, and are expressed in tumors of different histological origins.</td></tr><tr><td>ACKR3</td><td>-2.44</td><td>-2.80</td><td>Atypical chemokine receptor 3 (ACKR3). It controls chemokine levels and localization via high-affinity chemokine binding that is uncoupled from classic ligand-driven signal transduction cascades, resulting instead in chemokine sequestration, degradation, or transcytosis.</td></tr><tr><td>STARD13</td><td>+2.79</td><td>+5.46</td><td>STAR-related lipid transfer (START) domain containing 13. GTPase-activating protein for RhoA, and perhaps for Cdc42. May be involved in regulation of cytoskeletal reorganization, cell proliferation and cell motility. Acts a tumor suppressor in hepatocellular carcinoma cells.</td></tr><tr><td>CYTH1</td><td>-2.05</td><td>-2.53</td><td>Cytohesin 1. It mediates the regulation of protein sorting and membrane trafficking. This gene is highly expressed in natural killer and peripheral T cells, and regulates the adhesiveness of integrins at the plasma membrane of lymphocytes.</td></tr><tr><td>SYCN</td><td>-2.85</td><td>-3.28</td><td>Syncollin. Functions in exocytosis in pancreatic acinar cells regulating the fusion of zymogen granules with each other. May have a pore-forming activity on membranes and regulate exocytosis in other exocrine tissues</td></tr><tr><td>KLHL15</td><td>+2.22</td><td>+2.58</td><td>Kelch-like family member 15. Involved in protein ubiquitination and cytoskeletal organization.</td></tr><tr><td>TNFRSF14</td><td>-2.72</td><td>-2.77</td><td>Tumor necrosis factor receptor superfamily. The encoded protein functions in signal transduction pathways that activate inflammatory and inhibitory T-cell immune response</td></tr><tr><td>CSGALNACT2</td><td>+2.54</td><td>+3.19</td><td>Chondroitin sulfate N-acetylgalactosaminyltransferase 2. The encoded protein is involved in elongation during chondroitin sulfate synthesis.</td></tr><tr><td>KANK4</td><td>-2.26</td><td>-3.32</td><td>KN motif and ankyrin repeat domains 4. May be involved in the control of cytoskeleton formation by regulating actin polymerization</td></tr><tr><td>MECOM</td><td>-4.43</td><td>-3.53</td><td>MDS1 and EVI1 complex locus (MECOM). Functions as a transcriptional regulator binding to DNA sequences in the promoter region of target genes and regulating positively or negatively their expression. Oncogene which plays a role in development, cell proliferation and differentiation. May also play</td></tr></tbody></table>
<table><thead><tr><th>Symbole du gène</th><th>FC / NP+ 200 µg/mL</th><th>FC / NP- 200 µg/mL</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td>ATG9B</td><td>-2.60</td><td>-2.99</td><td>a role in apoptosis through regulation of the JNK and TGF-beta signaling. Involved in hematopoiesis.<br><b>Autophagy related 9B.</b> Involved in autophagy and cytoplasm to vacuole transport (Cvt) vesicle formation. This gene also functions as an antisense transcript in the posttranscriptional regulation of the endothelial nitric oxide synthase 3 gene, which has 3' overlap with this gene on the opposite strand.</td></tr><tr><td>IPMK</td><td>+2.81</td><td>+3.31</td><td><b>Inositol polyphosphate multikinase.</b> Plays an important role in the biosynthesis of inositol 1,3,4,5,6-pentakisphosphate, and has a preferred 5-kinase activity. This gene may play a role in nuclear mRNA export.</td></tr><tr><td>ANGPT4</td><td>-3.29</td><td>-3.25</td><td><b>Angiopoietin 4.</b> Binds to TEK/TIE2, modulating ANGPT1 signaling. Can induce tyrosine phosphorylation of TEK/TIE2. Promotes endothelial cell survival, migration and angiogenesis.</td></tr><tr><td>TCTE1</td><td>-2.72</td><td>-3.19</td><td><b>t-complex-associated-testis-expressed 1.</b></td></tr><tr><td>AFF4</td><td>+2.02</td><td>+2.38</td><td><b>AF4/FMR2 family, member 4.</b> Key component of the super elongation complex (SEC), a complex required to increase the catalytic rate of RNA polymerase II transcription</td></tr><tr><td>TRAF1</td><td>-2.41</td><td>-2.77</td><td><b>TNF receptor-associated factor 1.</b> Associate with, and mediate the signal transduction from various receptors of the TNFR superfamily. Regulates the activation of NF-kappa-B and JNK. Plays a role in the regulation of cell survival and apoptosis</td></tr><tr><td>MYOF</td><td>+2.49</td><td>+3.13</td><td><b>Myoferlin.</b> Play a role in calcium-mediated membrane fusion events, suggesting that it may be involved in membrane regeneration and repair.</td></tr></tbody></table>

Gènes inconnus, pseudogènes ou non codants

<table><tr><td>EIF1B-AS1</td><td>-3.10</td><td>-2.13</td><td><b>EIF1B Antisense RNA 1.</b> lncRNA. No data available</td></tr><tr><td>FAM83A</td><td>-3.58</td><td>-3.61</td><td><b>Family With Sequence Similarity 83, Member A.</b> No data available</td></tr><tr><td>FAM107A</td><td>-2.80</td><td>-3.92</td><td><b>Family With Sequence Similarity 107, Member A.</b> No data available</td></tr><tr><td>MIR4458HG</td><td>-2.36</td><td>-3.12</td><td><b>MIR4458 Host Gene.</b> Non-Protein Coding.</td></tr><tr><td>MT1IP</td><td>-3.11</td><td>-6.12</td><td><b>Metallothionein 1I.</b> A Pseudogene, lncRNA.</td></tr><tr><td>DUX4L2</td><td>-2.71</td><td>-3.44</td><td><b>Double Homeobox 4 Like 2.</b> No data available</td></tr><tr><td>XLOC_000099</td><td>-7.30</td><td>-5.36</td><td>No data available</td></tr><tr><td>XLOC_009926</td><td>-2.67</td><td>-2.69</td><td>No data available</td></tr><tr><td>XLOC_005621</td><td>-2.08</td><td>-3.51</td><td>No data available</td></tr><tr><td>LOC345051</td><td>-2.41</td><td>-3.96</td><td>No data available</td></tr><tr><td>XLOC_011507</td><td>-3.30</td><td>-4.80</td><td>No data available</td></tr><tr><td>XLOC_009604</td><td>-4.01</td><td>-3.59</td><td>No data available</td></tr><tr><td>XLOC_007011</td><td>-2.58</td><td>-3.02</td><td>No data available</td></tr><tr><td>LOC101927354</td><td>-3.45</td><td>-3.82</td><td>No data available</td></tr><tr><td>XLOC_000294</td><td>-2.62</td><td>-2.88</td><td>No data available</td></tr><tr><td>XLOC_002616</td><td>-3.13</td><td>-3.23</td><td>No data available</td></tr></table>
# Annexe 6

Liste des 14 gènes communs dont l'expression est modifiée après exposition aux NP- 200 µg/mL ou au GSNO 6 µM pendant 4 h avec les fold change (FC) correspondants et les fonctions (GeneCards, Pubmed, ..)

En vert : gènes sousexprimés, en rouge : gènes surexprimés

<table><thead><tr><th>Symbole du gène</th><th>FC / NP-200 µg/mL</th><th>FC / GSNO 6 µM</th><th>Nom et fonction du gène</th></tr></thead><tbody><tr><td colspan="4" style="text-align: center; font-weight: bold;">Gènes connus</td></tr><tr><td>HIST1H1T</td><td>+3.76</td><td>+4.13</td><td><strong>Histone cluster 1, H1t.</strong> Histones H1 is necessary for the condensation of nucleosome chains into higher-order structures.</td></tr><tr><td>LHX3</td><td>-4.17</td><td>-4.86</td><td><strong>LIM homeobox 3.</strong> This gene encodes a member a large protein family which carry the LIM domain, a unique cysteine-rich zinc-binding domain. The encoded protein is a transcription factor that is required for pituitary development and motor neuron specification.</td></tr><tr><td>LAT2</td><td>-2.80</td><td>-2.52</td><td><strong>Linker for activation of T cells family, member 2.</strong> Involved in FCER1 (high affinity immunoglobulin epsilon receptor)-mediated signaling in mast cells. May also be involved in BCR (B-cell antigen receptor)-mediated signaling in B-cells and FCGR1 (high affinity immunoglobulin gamma Fc receptor I)-mediated signaling in myeloid cells</td></tr><tr><td>ZNF763</td><td>-2.87</td><td>-2.31</td><td><strong>Zinc finger protein 763.</strong> May be involved in transcriptional regulation</td></tr><tr><td colspan="4" style="text-align: center; font-weight: bold;">Gènes inconnus, pseudogènes ou non codants</td></tr><tr><td>FAM177B</td><td>-16.18</td><td>-33.41</td><td><strong>Family With Sequence Similarity 177, Member B.</strong> No data available</td></tr><tr><td>LKAAEAR1</td><td>-3.73</td><td>-3.73</td><td><strong>LKAAEAR Motif Containing 1.</strong> No data available</td></tr><tr><td>GAS6-AS1</td><td>+2.76</td><td>+3.53</td><td><strong>GAS6 Antisense RNA 1.</strong> Non-Protein Coding.</td></tr><tr><td>RNA5-8S5</td><td>+3.86</td><td>+2.13</td><td><strong>RNA, 5.8S Ribosomal 5.</strong></td></tr><tr><td>XLOC_006625</td><td>-3.06</td><td>-2.48</td><td>No data available</td></tr><tr><td>LOC440157</td><td>+2.38</td><td>+4.56</td><td>No data available</td></tr><tr><td>XLOC_006291</td><td>-3.70</td><td>-3.61</td><td>No data available</td></tr><tr><td>XLOC_002588</td><td>-2.56</td><td>-2.38</td><td>No data available</td></tr><tr><td>LOC101927637</td><td>-3.06</td><td>-2.48</td><td>No data available</td></tr><tr><td>XLOC_000918</td><td>-2.34</td><td>-2.05</td><td>No data available</td></tr></tbody></table>
Annexe 7

<figure><img src="image_35.png" /><figcaption>Figure 18. Réseau de protéines correspondantes aux gènes de THP-1 dont l'expression est significativement modifiée (STRING database) après exposition à 1,4 µM de GSNO, 4 h (29 gènes de la famille des récepteurs olfactifs)</figcaption></figure>
# Annexe 8

## Uptake of Eudragit Retard L (Eudragit® RL) Nanoparticles by Human THP-1 Cell Line and Its Effects on Hematology and Erythrocyte Damage in Rats

### Résumé :

Le but de cette étude était de préparer les NP d'Eudragit® RL et de déterminer leurs propriétés, leur internalisation dans les THP-1 *in vitro* et leur effet sur les paramètres hématologiques chez le rat. Les NP ont été de taille moyenne de 329,0 ± 18,5 nm, et portent une charge positive 57,5 ± 5,47 mV. Ces NP ont été internalisées dans les THP-1 après 2 h d'incubation. Dans l'étude *in vivo*, des rats mâles Sprague-Dawley ont été exposés par voie orale ou par voie intrapéritonéale (IP) à une dose unique de NP (50 mg / kg de masse corporelle). Des échantillons de sang ont été prélevés au bout de 4 h, 48 h, une semaine et trois semaines pour l'analyse hématologique. Les NP induisent des perturbations hématologiques significatives dans les plaquettes, les globules rouges (RBC) totales et les numérations différentielles de globules blancs (WBC) après 4 h, 48 h et une semaine. Les NP augmentent les activités de dérivés des Met-Hb et Hb et diminuent l'activité de met-Hb réductase. Ces paramètres étaient comparables au contrôle après trois semaines après administration par voie orale. Il pourrait être conclu que la voie d'administration a un effet majeur sur l'induction de troubles hématologiques et doit être envisagée lorsque les NP sont utilisées comme systèmes de délivrance de médicaments.
Article

# Uptake of Eudragit Retard L (Eudragit® RL) Nanoparticles by Human THP-1 Cell Line and Its Effects on Hematology and Erythrocyte Damage in Rats

Mosaad A. Abdel-Wahhab <fnref n="1" />*, Khaled G. Abdel-Wahhab <fnref n="2" />, Fathia A. Mannaa <fnref n="2" />,

Nabila S. Hassan <fnref n="3" />, Ramia Safar <fnref n="4" />, Roudayna Diab <fnref n="4" />, Bernard Foliguet <fnref n="4" />, Luc Ferrari <fnref n="4" /> and
Bertrand H. Rihn <fnref n="4" />*

Received: 18 October 2013; in revised form: 18 November 2013 / Accepted: 18 February 2014 /
Published: 28 February 2014

**Abstract:** The aim of this study was to prepare Eudragit Retard L (Eudragit RL) nanoparticles (ENPs) and to determine their properties, their uptake by the human THP-1 cell line *in vitro* and their effect on the hematological parameters and erythrocyte damage in rats. ENPs showed an average size of 329.0 ± 18.5 nm, a positive zeta potential value of +57.5 ± 5.47 mV and nearly spherical shape with a smooth surface. THP-1 cell lines could phagocyte ENPs after 2 h of incubation. In the *in vivo* study, male Sprague-Dawley rats were exposed orally or intraperitoneally (IP) with a single dose of ENP (50 mg/kg body weight). Blood samples were collected after 4 h, 48 h, one week and three weeks for hematological and erythrocytes analysis. ENPs induced significant hematological disturbances in platelets, red blood cell (RBC) total and differential counts of white blood

<footnote n="1">¹ Food Toxicology & Contaminants Department, National Research Center, Dokki, Cairo 12311, Egypt</footnote>

<footnote n="2">² Medical Physiology Department, National Research Center, Dokki, Cairo 12311, Egypt;
E-Mails: kgm194@yahoo.com (K.G.A.-W.); fathia_98@yahoo.com (F.A.M.)</footnote>

<footnote n="3">³ Pathology Department, National Research Center, Dokki, Cairo 12311, Egypt;
E-Mail: nabilas.hassan@yahoo.com</footnote>

<footnote n="4">⁴ Faculty of Pharmacy, EA 3452 CITHEFOR (Cibles thérapeutiques, formulation et expertise préclinique du médicament), Lorraine University, Nancy Cedex 54001, France;
E-Mails: ramia.safar@univ-lorraine.fr (R.S.); roudayna.diab@univ-lorraine.fr (R.D.);
bernard.foliguet@univ-lorraine.fr (B.F.); luc.ferrari@univ-lorraine.fr (L.F.)</footnote>

<footnote>* Authors to whom correspondence should be addressed;
E-Mails: mosaad_abdelwahhab@yahoo.com (M.A.A.-W.);
bertrandRIHN@pharma.uhp-nancy.fr (B.H.R.); Tel.: +2-228-319-43 (M.A.A.-W.);
+33-0383-682-301 (B.H.R.); Fax: +2-333-709-31 (M.A.A.-W.); +33-0383-682-301 (B.H.R.).</footnote>
cells (WBCs) after 4 h, 48 h and one week. ENP increased met-Hb and Co-Hb derivatives and decreased met-Hb reductase activity. These parameters were comparable to the control after three weeks when administrated orally. It could be concluded that the route of administration has a major effect on the induction of hematological disturbances and should be considered when ENPs are applied for drug delivery systems.

**Keywords:** Eudragit; nanoparticles; nanotoxicity; hematology; erythrocytes damage; oxidative stress

# 1. Introduction

The quantity of nanoparticles (NPs) produced annually is rapidly increasing [1], fueled by growing markets for products that incorporate these materials. This trend will likely lead to the appearance of human-made NPs in air, water, soils and organisms [2]. The unique physicochemical properties of NPs that have given rise to applications in many fields, including drug delivery [3], cancer therapy [4], biosensors [5], food additives and cosmetics [6]. Possible toxicological risks associated with NP exposure may arise during material fabrication, handling, usage and waste disposal [7,8]. Consequently, the risk of toxicity to humans or the environment will increase [9,10].

Because of their small size, NPs have many physicochemical properties that differ from those of their bulk forms. Recent studies have indicated that the physicochemical characteristics of NPs mainly, the size, shape, surface area, solubility, chemical composition and dispersion factor, play critical roles in determining their biological responses [9,11,12]. For example, NPs of a smaller size can enter the mitochondria of cells through various pathways, subsequently inducing oxidative stress and cell death via apoptosis [13] or autolysis [14].

Polymeric NPs have been used as a preferred nanoscale drug delivery vehicle, especially for their excellent endocytosis efficiency, passive tumor-targeting, high encapsulation efficiency and delivery of a wide range of therapeutic agents [15]. Eudragit is a model polymeric NP, and it is a copolymer that has been widely used to improve the solubility of poorly water soluble drugs [16]. Eudragit RS (ERS), a non-biodegradable, positively-charged copolymer, has been licensed for clinical use by the major health authorities of Europe, Japan and the USA [17] as an efficient nano-drug delivery system (NDDS). ERS NPs prepared by nanoprecipitation (NP) or by double emulsion (DE) techniques containing ibuprofen and cyclosporin [18], indomethacin [19], melatonin [20], DNA plasmid [21] or low-molecular weight heparin [22] (LMWH) have been obtained and been suggested for therapeutic usage. On the other hand, Eudragit Retard L (Eudragit RL) polymer NPs have been investigated as carrier systems for the ophthalmic release of non-steroidal anti-inflammatory drugs, such as ibuprofen or flurbiprofen [18,23]. Moreover, Eudragit L 100-55 (EL 100-55 methacrylic acid-ethyl acrylate copolymer type A, 1:1) has been commonly used for the preparation of enteric solid dosage forms as a good coating and skeleton material [24,25]. The aim of the current study was to develop Eudragit RL NPs as a delivery for several drugs and to evaluate their properties, uptake by the human THP-1 cell line *in vitro* and effect on the hematological parameters *in vivo* using an animal model. In a previous study, Eudragit RL nanoparticles (ENPs) were used to encapsulate heparin for oral administration and
were tested *in vivo* on rabbit [22]. Subsequently, it was necessary to assess their short-term toxicity and to evaluate the intraperitoneal route, because it is widely used for chemotherapy in patients with gastrointestinal and gynecological cancers [26], compared to the oral route.

**2. Results**

We first established that the characteristics of the prepared ENPs were an average size of 329.0 ± 18.5 nm and positive zeta potential values of 57.5 ± 5.47 mV. The prepared ENPs did not show any changes in their size in neutral or acidic conditions, indicating that no aggregation had occurred, except when serum was added to the media. The transmission electron microscope (TEM) images showed that the ENPs were nearly spherical in shape with a smooth surface (Figure 1). Moreover, in the *in vitro* study, the TEM observations revealed that the uptake of ENPs by THP-1 cell lines occurred after 2 h of incubation with a concentration of 200 µg ENP/mL (Figure 2a,b). The ENPs were observed in the cells after 24 h without inducing any changes in the cell structure (Figure 2c).

<figure><img src="image_36.png" /><figcaption>**Figure 2.** TEM of ENP uptake by the human THP-1 cell line. Monocytes were exposed to 200 µg/mL for 2 h; the arrows indicate (a) internal; (b) external nanoparticles; and (c) that no changes were observed in the cell structure after 24 h.</figcaption></figure>

<figure><img src="image_37.png" /></figure>
<figure><img src="image_38.png" /><figcaption>Figure 2. Cont.</figcaption></figure>

The results presented in Table 1 illustrated the effect of the route (oral and intraperitoneally; i.p) of administration on animals' total and differential count of white blood cells (WBCs), as well as the platelet count at different time intervals as compared to the control group. These results revealed that after 4 h of ENP treatment, the platelet count recorded a significant decrease regarding IP injection and still close to normal in the case of oral administration. The total WBC count was not affected, while its differential count was changed, as the granulocyte percentage was significantly elevated after IP injection. This elevation could be on the expanse of the lymphocyte percentage, which was insignificantly decreased. The percentage of monocytes did not significantly change by comparison of the routes of treatment to each other, as well as with the control group.

After 48 h of IP injection of ENPs, the platelet count significantly increased compared to its count after 4 h and became close to that of the normal group or its corresponding value of those treated orally. The total WBC count of both the IP and oral groups was significantly increased. Their differential count was changed, as the granulocyte percentage was significantly elevated in both the IP and oral cases. This elevation could be on the expanse of the lymphocyte percentage, which decreased insignificantly. On the other hand, the percentage of monocytes did not significantly change, either by comparing the routes of treatment to each other or with the control group.

One and three weeks after either oral or IP treatment with ENPs, the obtained data showed that the platelet count was slightly elevated over the control, while the total count of WBCs decreased gradually towards the control range. However, the decline was slower regarding IP injection. In addition, the WBC differential count showed a reduction in the percentage of granulocytes accompanied with an elevation in lymphocyte percentage; however, monocytes were again more or less unaffected significantly.

The results of blood values presented in Table 2 revealed that both oral administration and IP injection with ENPs showed a time-dependent gradual change, but not an administration-route-dependent change. The data illustrated that animals either orally administrated or IP injected with ENPs showed a marked decrease in red blood cell (RBC) count and HCT percentage after four or 48 h and returned to the control range by the other intervals. No sharp difference was
observed between both routes at the matched time intervals. Similarly, Hb, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) levels recorded a time-dependent gradual decrease at all tested intervals, although this decrease was slightly pronounced after 4 h and significant after seven days for Hb concentration only. Instead, no significant differences were found in MCHC between the control and the ENP-treated groups. In contrast, the mean corpuscular volume (MCV) level showed a time-transit elevation in all groups, either orally administrated or IP injected with ENPs. This increase was markedly pronounced after one week for both treatments with ENPs. At the end of the third week, all blood values were carried up or down, more or less, towards the control values.

The data of both functional (O₂-Hb) and nonfunctional (Met-Hb, S-Hb and Co-Hb) hemoglobin derivatives of different ligands are illustrated in Table 3. The current results showed that after IP or oral administration of ENPs, the functional derivative (oxy-Hb) or the nonfunctional one (S-Hb) were not affected over all the tested time intervals, while the nonfunctional fraction Co-Hb was found to be elevated significantly after 4 h, 48 h and one week, whatever the exposure route, and to fall down near the control range. Furthermore, the nonfunctional met-Hb derivative showed a time/route gradual elevation, which was significant after three weeks of IP injection (Table 3).

In contrast, the obtained data illustrated that the activity of met-HbR was comparable to that of the control after four and 48 h, while a significant decrease was pronounced after the first and third weeks in both routes of treatments (Figure 3).

<figure><img src="image_39.png" /><figcaption>**Figure 3.** The effect of oral administration and intraperitoneal injection of ENPs on the erythrocyte methemoglobin reductase (metHbR) activity of rats at different time intervals.</figcaption></figure>
<table><caption>**Table 1.** The effect of oral administration and intraperitoneally (i.p.) injection of ENPs on white blood cells (WBCs) and differential counts in rats. PLT, platelet.</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="2">PLT (10<sup>3</sup>/cc)</th><th colspan="2">WBCs (10<sup>3</sup>/cc)</th><th colspan="2">Lymph (%)</th><th colspan="2">Mono (%)</th><th colspan="2">Gran (%)</th></tr><tr><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th></tr></thead><tbody><tr><td>Control</td><td>341 ± 28<sup>aA</sup></td><td>389 ± 16<sup>abA</sup></td><td>5.8 ± 0.55<sup>bA</sup></td><td>5.77 ± 0.26<sup>aA</sup></td><td>85.8 ± 1.66<sup>aA</sup></td><td>85.3 ± 1.81<sup>aA</sup></td><td>6.73 ± 0.32<sup>aA</sup></td><td>6.40 ± 0.41<sup>aA</sup></td><td>8.10 ± 0.95<sup>aA</sup></td><td>8.30 ± 1.5<sup>aA</sup></td></tr><tr><td>4 h</td><td>408 ± 19<sup>aA</sup></td><td>222 ± 7.4<sup>bB</sup></td><td>4.8 ± 1.12<sup>bA</sup></td><td>5.76 ± 0.45<sup>aA</sup></td><td>84.2 ± 4.1<sup>aA</sup></td><td>76.46 ± 6.62<sup>aA</sup></td><td>6.73 ± 0.55<sup>aA</sup></td><td>6.37 ± 0.70<sup>aA</sup></td><td>9.06 ± 1.19<sup>aA</sup></td><td>17.16 ± 2.63<sup>aB</sup></td></tr><tr><td>48 h</td><td>492 ± 39<sup>aA</sup></td><td>437 ± 67<sup>aA</sup></td><td>7.27 ± 1.11<sup>aA</sup></td><td>8.76 ± 0.64<sup>aB</sup></td><td>78.10 ± 3.0<sup>aA</sup></td><td>82.26 ± 4.0<sup>aA</sup></td><td>6.07 ± 0.81<sup>aA</sup></td><td>6.30 ± 1.05<sup>aA</sup></td><td>15.83 ± 1.19<sup>aA</sup></td><td>11.43 ± 3.04<sup>aB</sup></td></tr><tr><td>One week</td><td>473 ± 42<sup>aA</sup></td><td>451 ± 56<sup>abA</sup></td><td>6.80 ± 0.31<sup>aA</sup></td><td>7.46 ± 0.75<sup>aB</sup></td><td>80.40 ± 2.95<sup>aA</sup></td><td>82.66 ± 1.61<sup>aA</sup></td><td>6.57 ± 0.27<sup>aA</sup></td><td>7.06 ± 0.36<sup>aA</sup></td><td>13.03 ± 2.69<sup>aA</sup></td><td>10.27 ± 1.97<sup>aB</sup></td></tr><tr><td>Three weeks</td><td>457 ± 55<sup>aA</sup></td><td>455 ± 56<sup>aA</sup></td><td>5.17 ± 0.51<sup>bA</sup></td><td>6.63 ± 1.66<sup>aB</sup></td><td>81.83 ± 2.57<sup>aA</sup></td><td>83.20 ± 2.65<sup>aA</sup></td><td>7.37 ± 0.53<sup>aA</sup></td><td>6.97 ± 1.07<sup>aA</sup></td><td>10.80 ± 0.15<sup>aA</sup></td><td>9.83 ± 1.60<sup>aA</sup></td></tr></tbody></table>

All data are expressed as the mean ± standard error; means with different superscript letters (a, b) are significantly different (p ≤ 0.05); lowercase letters are for groups within the same column, while uppercase letters (A, B) to compare oral treatment with IP treatment within the same row.

<table><caption>**Table 2.** The effect of oral administration and i.p. injection of ENPs on red blood cells (RBCs) and hematological parameters in rats. Hct, hematocrite; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration.</caption><thead><tr><th rowspan="2">Parameter<br>group</th><th colspan="2">RBCs (10<sup>6</sup>)</th><th colspan="2">Hb (g/dL)</th><th colspan="2">Hct (%)</th><th colspan="2">MCV (fL)</th><th colspan="2">MCH (pg)</th><th colspan="2">MCHC (g/dL)</th></tr><tr><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th></tr></thead><tbody><tr><td>Control</td><td>5.42 ± 0.46<sup>aA</sup></td><td>5.77 ± 0.55<sup>aA</sup></td><td>14.60 ± 0.58<sup>aA</sup></td><td>13.01 ± 1.58<sup>aA</sup></td><td>42.4 ± 1.3<sup>aA</sup></td><td>36.7 ± 3.13<sup>aA</sup></td><td>47.73 ± 3.13<sup>bA</sup></td><td>51.3 ± 1.52<sup>bA</sup></td><td>29.6 ± 1.17<sup>aA</sup></td><td>28.3 ± 3.98<sup>aA</sup></td><td>34.4 ± 1.96<sup>aA</sup></td><td>31.7 ± 4.40<sup>bA</sup></td></tr><tr><td>4 h</td><td>5.41 ± 0.26<sup>aA</sup></td><td>4.43 ± 0.90<sup>bA</sup></td><td>14.93 ± 0.88<sup>aA</sup></td><td>13.73 ± 1.45<sup>aA</sup></td><td>42.37 ± 1.25<sup>aA</sup></td><td>30.4 ± 4.43<sup>bA</sup></td><td>45.33 ± 1.88<sup>bA</sup></td><td>51.0 ± 3.85<sup>bA</sup></td><td>27.4 ± 0.56<sup>aA</sup></td><td>31.60 ± 3.30<sup>aA</sup></td><td>33.4 ± 1.56<sup>aA</sup></td><td>32.3 ± 1.69<sup>abA</sup></td></tr><tr><td>48 h</td><td>4.50 ± 0.26<sup>bA</sup></td><td>4.95 ± 0.33<sup>aA</sup></td><td>13.46 ± 1.21<sup>abA</sup></td><td>13.8 ± 0.87<sup>aA</sup></td><td>34.66 ± 1.23<sup>aA</sup></td><td>32.4 ± 3.83<sup>bA</sup></td><td>57.66 ± 4.84<sup>bA</sup></td><td>62.33 ± 6.99<sup>bA</sup></td><td>31.03 ± 1.84<sup>aA</sup></td><td>34.83 ± 2.45<sup>aA</sup></td><td>36.13 ± 3.08<sup>aA</sup></td><td>41.2 ± 3.71<sup>abA</sup></td></tr><tr><td>One week</td><td>6.18 ± 0.72<sup>aA</sup></td><td>5.48 ± 0.26<sup>aA</sup></td><td>10.80 ± 1.50<sup>bA</sup></td><td>9.47 ± 0.41<sup>aA</sup></td><td>41.20 ± 2.37<sup>aA</sup></td><td>35.33 ± 1.56<sup>aA</sup></td><td>133.6 ± 2.02<sup>aA</sup></td><td>129.0 ± 1.73<sup>aA</sup></td><td>24.60 ± 1.10<sup>aA</sup></td><td>24.53 ± 1.41<sup>aA</sup></td><td>51.9 ± 2.36<sup>aA</sup></td><td>35.5 ± 1.27<sup>aA</sup></td></tr><tr><td>Three weeks</td><td>5.75 ± 0.88<sup>aA</sup></td><td>6.41 ± 0.49<sup>aA</sup></td><td>11.86 ± 0.87<sup>bA</sup></td><td>11.40 ± 1.51<sup>aA</sup></td><td>38.13 ± 1.98<sup>aA</sup></td><td>35.2 ± 2.11<sup>aA</sup></td><td>56.33 ± 1.33<sup>bA</sup></td><td>57.3 ± 1.53<sup>bA</sup></td><td>18.9 ± 1.14<sup>bA</sup></td><td>28.46 ± 1.57<sup>aA</sup></td><td>33.7 ± 1.32<sup>aA</sup></td><td>32.3 ± 1.95<sup>bA</sup></td></tr></tbody></table>

All data are expressed as the mean ± standard error; means with different superscript letters are significantly different (p ≤ 0.05); lowercase letters (a, b) are for groups within the same column, while uppercase letter (A) to compare oral treatment with IP treatment within the same row.

<table><caption>**Table 3.** The effect of oral administration and i.p. injection of ENPs on Hb derivatives (expressed as percent of total hemoglobin) in rats.</caption><thead><tr><th rowspan="2">parameter<br>group</th><th colspan="2">Oxy-Hb</th><th colspan="2">MetHb</th><th colspan="2">S-Hb</th><th colspan="2">Co-Hb</th></tr><tr><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th><th>Oral</th><th>IP</th></tr></thead><tbody><tr><td>Control</td><td>95.94 ± 0.87<sup>aA</sup></td><td>96.24 ± 1.05<sup>aA</sup></td><td>3.56 ± 0.74<sup>aA</sup></td><td>3.22 ± 0.74<sup>aA</sup></td><td>0.120 ± 0.01<sup>aA</sup></td><td>0.118 ± 0.03<sup>aA</sup></td><td>0.36 ± 0.18<sup>aA</sup></td><td>0.432 ± 0.14<sup>aA</sup></td></tr><tr><td>4 h</td><td>95.83 ± 1.75<sup>aA</sup></td><td>95.17 ± 0.74<sup>aA</sup></td><td>3.47 ± 0.80<sup>aA</sup></td><td>3.43 ± 0.79<sup>aA</sup></td><td>0.114 ± 0.03<sup>aA</sup></td><td>0.115 ± 0.04<sup>aA</sup></td><td>0.624 ± 0.20<sup>bA</sup></td><td>0.688 ± 0.19<sup>bA</sup></td></tr><tr><td>48 h</td><td>95.39 ± 1.13<sup>aA</sup></td><td>95.65 ± 1.11<sup>aA</sup></td><td>3.83 ± 0.67<sup>aA</sup></td><td>3.64 ± 0.69<sup>aA</sup></td><td>0.099 ± 0.02<sup>aA</sup></td><td>0.120 ± 0.03<sup>aA</sup></td><td>0.678 ± 0.19<sup>bA</sup></td><td>0.639 ± 0.21<sup>bA</sup></td></tr><tr><td>One week</td><td>95.72 ± 1.75<sup>aA</sup></td><td>95.42 ± 2.78<sup>aA</sup></td><td>3.88 ± 0.80<sup>aA</sup></td><td>3.82 ± 0.83<sup>aA</sup></td><td>0.117 ± 0.04<sup>aA</sup></td><td>0.122 ± 0.04<sup>aA</sup></td><td>0.682 ± 0.22<sup>bA</sup></td><td>0.709 ± 0.23<sup>bA</sup></td></tr><tr><td>Three weeks</td><td>95.49 ± 1.32<sup>aA</sup></td><td>95.05 ± 1.27<sup>aA</sup></td><td>3.99 ± 0.83<sup>aA</sup></td><td>4.41 ± 0.79<sup>bA</sup></td><td>0.156 ± 0.02<sup>aA</sup></td><td>0.128 ± 0.01<sup>aA</sup></td><td>0.400 ± 0.11<sup>aA</sup></td><td>0.404 ± 0.12<sup>aA</sup></td></tr></tbody></table>

All data are expressed as the mean ± standard error; means with different superscript letters are significantly different (p ≤ 0.05); lowercase letters (a, b) are for groups within the same column, while uppercase letter (A) to compare oral treatment with IP treatment within the same row.
### 3. Discussion

NPs (nanoparticles) can be transported around the body and be taken up by organ tissues and cell cultures, resulting in increased oxidative stress, inflammatory cytokine production or cell death. Unlike larger particles, NPs may be taken up by cell mitochondria, and cell studies demonstrated the potency of NPs to induce: (i) DNA mutation; (ii) major structural damage to mitochondria; and even (iii) cell death [27,28]. From this point of view, NPs used for medical applications should be evaluated *in vivo*, as well as *in vitro*. Polymeric NPs are biocompatible, surface-modifiable and capable of sustained drug release. They show potential applications in the treatment of various pulmonary conditions, such as asthma, chronic obstructive pulmonary disease (COPD), tuberculosis (TB) and lung cancer, as well as in diabetes [29–33].

In the current study, the average size of the developed Eudragit RL NP (ENP) was 329.0 ± 18.5 nm and the zeta potential was +57.5 ± 5.7 mV. From a pharmaceutical point of view, what is most important is the increase in saturation solubility and the adhesiveness to surfaces/membranes. According to the Kelvin equation, there is an increase in saturation solubility (Cs) below ~1 µm [34], this being more pronounced for poorly soluble than for soluble drugs [35]. The smaller the material is, the more adhesive it is. It is well known that the particle sizes of Eudragit RL and others polymers is affected by the preparation method. The double emulsion method was applied in the current study for the preparation of ENPs, which cannot produce a smaller size of NP (i.e., 30 nm). However, Eidi et al. [14] used the nanoprecipitation method to produce a smaller size of Eudragit RS NPs, which have reached 55 nm. On the other hand, particle size can be affected by the different pH and ionic strength used during administration. However, in the current study, the size of the nanoparticles was investigated in neutral (pH 7) and acidic (pH 3) solutions. The results showed that the preparation of ENPs with a size of 330 nm did not show any aggregation in the neutral or acidic conditions, except when the serum was added to the media.

Both pharmaceutically positive effects justify the definition of NPs with a size below 100 nm, and this size limit should be considered when defining a classification system. Although 100 nm is the second important size limit, the larger particles can enter the cell only by phagocytosis. Consequently, these particles can be taken up only by monocytes, a limited cell population in the body. In addition, many of them are difficult to access, e.g., to be taken up by liver and spleen monocytes, the particles need to enter the blood stream. Therefore, the developed ENPs possess a lower toxicity risk. However, particles below 100 nm can be internalized by certain cells by endocytosis or clathrin-dependent mechanism. Thus, these particles have a higher toxicity risk, and the 100 nm limit also has to be considered in the classification system [36].

Zeta potentials provide quantitative information on the stability of the particles. In the current study, ENPs had an absolute value of zeta potential higher than 30 mV (+ 57.5 ± 50.7 mV). It is well documented that the particles are more likely to remain dispersed if the absolute value of zeta potential is higher than 30 mV [37]. The current results demonstrated that ENPs possessed a uniform shape along with relatively favorable dispersibility. The uptake was performed within the concentration range and time period, when no damage to cell viability confirmed the low cytotoxicity of the assayed polymeric ENPs to THP-1 cells (data not shown). This could lead to a true understanding of the relationship between particle size, as well as surface charge and cellular uptake. The THP-1 cells
succeeded in inducing the uptake of ENPs (Figure 2). However, no difference was observed between the cells treated with a low or high dose of ENPs. On this concern, He et al. [38] reported that positively charged NPs showed a higher phagocytic uptake compared to negatively charged nanoparticles. However, Zauner et al. [39] suggested that the penetration of NPs into the cells might be attributed to the impairment of the cell membrane structure. It is well documented that the toxic effect of NPs is highly dependent on the organs and, more specifically, the type of cell encountered. This is due to the variation in cell physiology (e.g., epithelial or lymphoid), proliferation state (tumor or resting cells), membrane characteristics and phagocyte characteristics among different cell types [40]. The choice of the THP-1 cell line in the current study was based on the fact that these monocytes are the first line of innate immunity and, therefore, represent the first “barrier” or “target” of nanoparticulate delivery systems. On the other hand, the major objective of the current study was to investigate the effect of NPs on the hematological parameters, to assess the effect of the physical properties of ENPs on their toxicity and the possibility of NPs to penetrate the THP-1, as an example of blood cells.

The effect of ENPs on vascular connective tissue and hematological parameters was evaluated in vivo. NPs can translocate to both circulatory and lymphatic systems and, ultimately, to other body tissues and organs. However, translocation differs according to the route of exposure to nanoparticles [41]. In this study, both total and differential counts of WBCs were significantly changed at most tested time intervals. A significant elevation in both WBC count and granulocyte percentage was noticed after 48 h of oral or IP exposures. The elevation of the later was probably on the detriment of lymphocyte percentage, which decreased non-significantly. By the third week, the WBC of the oral group declined close to control values, while that of the IP group still elevated significantly compared to the PO exposed group.

Several reports suggested that high concentrations of NP can enter into the lymphatic system, inducing enlargement and inflammation in lymph nodes, which helps to increase the number of WBCs. However, after a given period, the activity of these glands decreased, and lymph node atrophy has been noted [42]. This finding is also supported by Choi et al. [43]. Some nanoparticles, depending on their composition and size, can produce irreversible damage to cells by oxidative stress or/and organelle injury [41]. ENPs can be taken up by various cell types, resulting in increased oxidative stress and inflammatory cytokine production. Regarding this concern, do Carmo et al. [44] reported that these effects trigger a chronic inflammatory condition that is associated with a high white blood cell count. Moreover, neutrophils, through NCF-1 and NADPH oxidase activation, increase phagocytosis and the formation of reactive oxygen compounds, as well as their own stimulation and migration to inflammatory sites.

Platelet count was decreased significantly after 4 h of ENP IP injection and returned to the control range by the other time intervals. As is known, NP charge plays an essential role in their uptake by platelets and their influence on blood clot formation [45]. Negatively charged NPs significantly inhibit thrombus formation, while positively charged NPs enhance platelet aggregation [46]. The interaction between platelets and positively charged particles seems to be due to the net negative charge that platelets carry on their surface [47]. In addition, the positively charged NPs interact with negatively charged platelets and reduce their surface charge, making them more prone to aggregation. This could be the possible reason for the thrombocytopenia occurring after 4 h of ENP injection. Until now, it was
thought that blood clots can be formed due to three main causes: when the blood flow is obstructed or slowed down, when the vascular endothelial cells are damaged or due to the blood biochemistry. Moreover, due to the large ENPs used in the current study, they failed to produce a thrombotic effect, suggesting that these NPs are insufficient for causing peripheral thrombosis [48].

In the current study, animals exposed to either oral or the IP treatment showed a marked decrease in RBC count and Hct percentage after four and 48 h, which returned to the control range by the other intervals with no valuable difference between both routes at the same time interval. Similarly, Hb, MCH and MCHC levels recorded a time-dependent gradual decreases at all tested times, although this decrease was slightly pronounced after 4 h (reflecting hypochromia) after seven days for Hb. In contrast, the MCV level showed a time-transit elevation (resulting in macrocytosis), especially after one week for both routes of treatment. These results suggested that the exposure route of ENPs is important and should be considered in the application of ENPs. Generally, the disturbances in erythrocytes and hemoglobin reported herein suggested that both bone marrow and erythrocytes may undergo a transient oxidative stress, as this damage was overcome by the third week. A high concentration of cerium oxide NPs reduced the number of blood cells due to: (i) inhibition of cell activity; (ii) antimitotic properties; (iii) stimulation of oxidative stress in cells; (iv) reduction of cellular antioxidants; and (v) activation of immune cell processes. Those effects may result in erythrocytopenia (low RBCs) and hypochromia via either a reduction of bone marrow activity, reducing, consequently, the RBC production rate, or the impairment of RBC membranes by making them more fragile and increasing their hemolytic rate. On this concern, Machiedo *et al.* [49] demonstrated that free radicals that were produced by NPs can be the main cause for the destruction of red blood cells by lipid peroxidation. Susan *et al.* [50] showed that their distribution in tissues and their effect change with respect to the diameter of the nanoparticles; if diameters are smaller, the influence of NPs on molecular mechanisms increases [45,51].

Both functional anemia (an elevation in nonfunctional Hb derivatives) and physiological anemia may evidence a weakness (erythrocytopenia, hypochromic-macrocytosis) in the oxygen supply of tissues (tissue hypoxia) at the intervals between the first and third week. Moreover, the current results showed that IP or PO administration did not affect either the functional derivative (O₂-Hb) or the nonfunctional derivative (S-Hb), while the nonfunctional derivative, Co-Hb, was found to be elevated significantly 4 h, 48 h and one week after the treatment through both routes, then falling down near the control range. Unfortunately, the nonfunctional met-Hb derivative recorded a time/route gradual elevation, which was significant after three weeks of IP injection with ENPs. Moreover, the data illustrated in Figure 3 revealed that the activity of met-HbR was comparable to the control after four and 48 h, while a significant inhibition was pronounced after the first and third weeks in both routes of treatment, as compared to the control values. It is well documented that in most cells, mitochondria are a major source of ROS [46]. Despite their lack of mitochondria, ROS are continuously produced in the erythrocytes, due to the high O₂ tension in arterial blood and their abundant heme iron content, inducing Fenton chemistry reactions [52]. The source of ROS in erythrocytes is the oxygen carrier protein hemoglobin, oxyhemoglobin (Oxy-Hb), which undergoes autoxidation to produce O₂⁻, which yields hydrogen peroxide [53]. Exposure of Hb to H₂O₂ leads to oxidative modifications that have been proposed as selective signals for proteolysis in erythrocytes [54]. The occasional reduction of O₂ to O₂⁻ is accompanied by the oxidation of the
functional ferrous-oxyhemoglobin (O₂-Hb) to the nonfunctional ferric-methemoglobin (Met-Hb), a “rust” brown-colored protein that cannot bind and transport O₂ [46]. This process is reversed by the presence of an efficient met-Hb reductase system in erythrocytes, comprising various enzymatic and non-enzymatic mechanisms. Moreover, the accumulation of a substantial amount of met-Hb in erythrocytes causes methemoglobinemia, clinically manifested by dyspnea and cyanosis [55]. Eventually, oxidative stress can lead to organelle injury and the formation of Hienz bodies, a form of methemoglobinemia, that precipitate on the inner side of the RBC membrane, triggering cell hemolysis [48]. Additionally, met-Hb can then react with the peroxides formed during the autoxidation process itself or elsewhere, resulting in hematological disorders [56–58].

Met-Hb is formed when the ferrous porphyrin complex of Hb is oxidized into the ferric form [59]. *In vivo*, Met-Hb is predominately reduced mainly by the NADH cytochrome b5-Met-Hb reductase system and to a lesser extent by the NADPH-dependent Met-Hb reductase enzyme [53]. It was suggested that intracellular NADPH concentration may be important in preventing Met-Hb generation. Loss of NADPH and glutathione (GSH) is thought to account for the enhanced rates of Met-Hb generation, as well as lipid peroxidation [52,60]. The free radicals may also induce configuration changes in the Hb molecule and make it susceptible to binding unfavorable ligands other than oxygen, such as carbon monoxide (CO) and sulfur (S). In addition to its inhibition of Met-Hb reductase activity, oxidative stress induced by NPs may also reduce GST, CAT and GPx, as well as GSH level in erythrocytes. Damodara and Venkaiah [55] suggested that the oxidative stress is normally challenged in erythrocytes by cellular defenses, including these antioxidant agents, which are important for dealing with the endogenous H₂O₂ produced by Hb autoxidation. The increased oxidative stress in erythrocytes could lead to the exhaustion of the antioxidant battery, which becomes insufficient to counteract the excessive production of ROS [61]. ROS was previously shown to inhibit RNA synthesis in liver and, therefore, affecting enzyme content [62]. In addition, metal interactions with hydrogen peroxide have been frequently implicated in catalyzing free-radical-mediated damage. Redox-active transition metals, such as iron, containing unpaired electrons in their d-orbitals are able to generate reactive species by Fenton Haber Weiss chemistry. In particular, free ferrous iron has been suggested to catalyze tissue damage by reacting with hydrogen peroxide to generate the highly reactive hydroxyl radical [63]. Taken together, these results revealed that ENPs could induce slight oxidative stress. However, the body succeeded in counteracting it after three weeks of treatment. The current study indicated that all blood cells of rats treated with ENPs showed slight changes in their physiological parameters. Consequently, we hope these studies can introduce a way to prevent and are aware of the toxicity effects of NPs in human health.

# 4. Experimental Section

## 4.1. Chemicals

Eudragit® RL PO (Mw = 150,000 Da (CAS number: 33434-24-1)), an acrylic polycationic copolymer of acrylic and methacrylic acid esters with a proportion of quaternary ammonium groups (0.5%-0.8%), was a generous gift from Evonik polymers (Darmstadt, Germany). Pluronic F68 (CAS
number: 11104-97-5) was used as a surfactant and was obtained from Sigma Aldrich (Saint-Quentin Fallavier, France). RPMI medium was purchased from GIBCO (Invitrogen, Cergy Pontoise, France).

## 4.2. Preparation of Eudragit® RL Nanoparticles

Eudragit® RL NPs (ENP) were prepared using the double emulsion/solvent evaporation technique, as described by Bodmeier *et al.* [64]. Briefly, 500 mg of Eudragit® RL were dissolved in 5 mL of an organic solution, dichloromethane (DCM, Laurylab, Saint Fons, France). One half milliliter of a surfactant aqueous solution at 0.1% w/v pluronic® F68 was emulsified into this organic phase by sonication (40 W for 1 min) using an ultrasonic homogenizer (Vibracell 75022, Bioblock, Illkirch, France) for the preparation of the primary w/o emulsion. This primary w/o emulsion was then dispersed by sonication (80 W for 30 s) into 20 mL of an aqueous solution of pluronic® F68 (0.1%, w/v), thus producing a secondary w/o/w emulsion. The resulting ENPs were obtained by the evaporation of the organic phase. The formed nanoparticles were separated by ultracentrifugation (Beckman, Miami, FL, USA) at 55,000 rpm for 30 min. The obtained nanoparticles were then re-suspended in an appropriate medium for further investigations. For the *in vitro* and *in vivo* studies, the weight of ENPs has been calculated based on the polymer concentration in the prepared NP suspension considering the density of the Eudragit polymer: 1 µg Eudragit polymer corresponds to $8.23 \times 10^7$ of ENPs.

## 4.3. Characterization of ENPs

### 4.3.1. Particle Size and Zeta Potential Analysis

Particle sizes were determined by photon correlation spectroscopy (PCS) using a Zetasizer™ 3000E (Malvern Instruments Worcestershire, UK). To avoid multi-scattering events, each sample was diluted with double distilled water until the appropriate concentration of particles; the concentration and dilution were kept constant for all samples. The particle size (z-average) and size distribution of equivalent hydrodynamic spheres were calculated using the Malvern associated software and an exponential sampling method. The size of the prepared ENPs was tested at neutral (pH 7) and acidic (pH 3) conditions, and each measurement was performed in triplicate. Zeta potential measurement was based on nanoparticle electrophoretic mobility and calculated from Smoluchowski's equation [65]. All measurements were performed in triplicate at 25 °C.

### 4.3.2. Morphological Determination

The morphological determination of ENPs was analyzed by transmission electron microscopy (Stereoscan 240 S/N Léo, Rueil-Malmaison, France). Briefly, a drop of the fresh ENP sample was placed onto a carbon-coated copper grid, forming a thin liquid film, which was negatively stained by the addition of a drop of uranyl acetate. The excess of the staining solution was removed with filter paper and then air-dried before the observation. Image acquisition was done with an Orius 1000 CCD camera (GATAN, Pleasanton, CA, USA).
## 4.4. *In Vitro* Study

### 4.4.1. Cell Culture

The human THP-1 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in RPMI 1640 medium (GIBCO, Invitrogen, Cergy Pontoise, France) supplemented with 10% of heat-inactivated fetal bovine serum (Eurobio, Les Ullis, France), as well as 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B (all from Sigma, Saint Quentin Fallavier, France). Cells were incubated at 37 °C under 5% CO₂ atmosphere and split every 3 days.

### 4.4.2. Cellular Uptake Visualization by Transmission Electronic Microscopy (TEM)

Briefly, THP-1 cells were seeded in 6-well plates at a density of $3 \times 10^5$ cell/well and treated on the following day with 200 µg/mL of ENPs for 2 h or 24 h. Then, they were immediately fixed with ice-cold 2.5% glutaraldehyde for 2 h. Cells were then post-fixed in 1% OsO₄ for 1 h at room temperature, progressively dehydrated by increasing concentrations of ethanol and, finally, treated with propylene oxide and included in resin. Semi-fine (2.0 µm) and ultra-fine (90 nm) sections were prepared with an ultra-microtome (Reichert-Yung) and examined with the electron microscope, Philips CM 12, operated at 80 kV.

## 4.5. *In Vivo* Study

### 4.5.1. Experimental Animals

Three-month-old male Sprague-Dawley rats (150–180 g, purchased from Animal House Colony, Giza, Egypt) were maintained on standard lab diet (protein: 160.4; fat: 36.3; fiber: 41 g/kg; and metabolizable energy: 12.08 MJ) purchased from Meladco Feed Co. (Aubor City, Cairo, Egypt). Animals were housed in filter-top polycarbonate cages in a room free from any source of chemical contamination, artificially illuminated and thermally controlled, at the Animal House Lab., National Research Center, Dokki, Cairo, Egypt. All animals received humane care in compliance with the guidelines of the Animal Care and Use Committee of the National Research Center, Dokki, Cairo, Egypt.

After an acclimatization period of one week, the animals were divided into two main groups (50 rats/group), and each group was divided into 5 subgroups (10 rats/subgroup). Ten rats from the first main group were injected intraperitoneally (i.p.) with saline solution and used as the control group, and the remaining rats were injected intraperitoneally (i.p.) with a single dose of ENPs (50 mg/kg body weight; b.w) suspended in saline. Ten rats from the second main group received a single oral dose of saline solution and were used as the control group, while the remaining rats received a single oral dose of ENPs (50 mg/kg b.w) using a stomach tube. Blood samples were collected from the two control groups after 4 h of saline treatment and after 4 h, 48 h, 1 wk and 3 wk of exposure from all ENP treated animals (10 rats/each time period) under diethyl ether anesthesia in two clean heparinized tubes; the first was used immediately for performing complete blood count (CBC), and the second part was used in the preparation of hemoglobin solution for the
determination of hemoglobin derivatives of different ligands, as well as methemoglobin reductase (Met-HbR) assessment.

A fully automatic blood cell counter with a double capillary instrument (Model PCE-210N, ERMA INC, Tokyo, Japan) was used for measuring complete blood count [red blood cell (RBC) count, total hemoglobin (Hb) concentration, hematocrite (Hct) percentage, platelet (PLT) count and white blood cells (WBCs) total and differential counts (lymphocytes, monocytes and granulocytes)] as well as blood indexes [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)].

### 4.5.2. Hemolysate Preparation and Determination of Hb Derivatives of Different Ligands

Hemolysate was prepared according to the method described by Silva *et al.* [66]. Briefly, whole blood was centrifuged at 3000 rpm for 15 min, and the buffy coat was removed. The packed red cells were washed three times with physiological saline, and the washed cells were lysed by suspending in hypotonic phosphate buffer and centrifuged at 7000 rpm for 30 min. The resulting pellet is the erythrocyte membrane, and the supernatant represents the hemolysate. The hemolysate obtained was further used for the assessment of both functional and nonfunctional hemoglobin derivatives of different ligands.

### 4.5.3. Hemoglobin Derivatives of Different Ligands Determination

Hemoglobin derivatives of different ligands were determined as a percent of total hemoglobin. The methemoglobin (Met-Hb) level was determined in the blood sample using the method described by Evelyn and Malloy [67]. Oxyhemoglobin (O₂-Hb), sulfhemoglobin (S-Hb) and carboxyhemoglobin (Co-Hb) levels in the blood sample were determined spectrophotometrically according to the method described by Van Kampen and Julstra [61].

### 4.5.4. Met-Hemoglobin (Met-Hb) Reductase Activity

Met-hemoglobin reductase (Met-HbR) activity was assayed in the washed packed erythrocytes according to the method of Hegesh *et al.* [62].

## 4.6. Statistical Analysis

All data were subjected to statistical analysis using the general linear model procedure of the statistical analysis system. Comparisons between means were carried out using a multiple one-way (Duncan) ANOVA test at the level of *p* ≤ 0.05 [63] using the statistical analysis system (SAS) program software; copyright© 1998 by SAS Institute Inc., Cary, NC, USA.

# 5. Conclusions

It could be concluded from the current study that the ENPs are internalized by the THP-1 cell line. ENPs induced moderate hematological disturbances, represented in platelet, total and differential counts of WBCs, as well as RBC counts and parameters. These resulted in some degree of physiological and functional anemia, likely through oxidative stress. However, most of these
disturbances were normalized after three weeks of treatment. More importantly, the route of administration has a major effect on the induction of hematological disturbances, and consequently, it should be considered when ENPs are applied as drug delivery systems.

## Acknowledgments

This work was supported by the National Research Centre, Dokki, Cairo, Egypt project # S90402 and ANR "NanoSNO", Faculty of Pharmacy, Lorraine University, Nancy Cedex, France. The authors especially thank Geoffroy Bontemps (IMCD France SAS) for its generous gift of Eudragit® RL polymer.

## Author Contributions

Authors Khaled G. Abdel-Wahhab, Fathia A. Mannaa and Nabila S. Hassan carried out the *in vivo* experiment and the hematological analysis. Authors Ramia Safar, Roudayna Diab, Bernard Foliguet carried out the preparation and characterization of ENPs as well as the *in vitro* studies. Author Luc Ferrari performed the statistical analysis and shared in writing the first draft of the manuscript. Authors Mosaad A. Abdel-Wahhab and Bertrand H. Rihn wrote the protocol, managed the project, managed the analyses of the study and wrote the final draft of the manuscript. All authors read and approved the final manuscript.

## Conflict of Interest

The authors declare no conflict of interest.

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# Annexe 9

## Neuronal Uptake and Neuroprotective Properties of Curcumin-Loaded Nanoparticles on SK-N-SH Cell Line: Role of Poly(lactide-co-glycolide) Polymeric Matrix Composition

### Résumé :

La curcumine, un agent neuroprotecteur d'approche thérapeutique prometteuse, a une faible biodisponibilité au niveau cérébral. Ici, nous avons montré que la curcumine encapsulée dans des NP de poly (lactide-co-glycolide) (PLGA) 50:50 (NPs-cur 50/50) sont en mesure d'empêcher la phosphorylation des protéines Akt et Tau induite par $H_2O_2$ dans les cellules SK-N-SH et de présenter des activités anti-inflammatoires et antioxydantes par rapport à la curcumine libre. Il existe différentes formulation de PLGA, avec différents ratio d'acide glycolique/polyglycolique. Ces différentes matrices présentent des caractéristiques physico-chimiques et de stabilité différentes. Nous avons montré que la libération de la curcumine piégée dans une matrice PLGA 50:50 (NPs-Cur 50:50) est plus rapide que dans la matrice PLGA 65:35. Nous avons étudié les effets de la matrice PLGA sur l'expression de certains gènes antioxidants et neuroprotecteurs tels que l'APOE, APOJ, TRX, GLRX et REST. NPs-Cur induisent une augmentation de l'expression de GLRX et TRX avec une diminution de l'expression d'APOJ, aucun effet sur l'expression d'APOE et REST. En présence de $H_2O_2$, les deux matrices NPs-Cur ont été plus efficaces que la curcumine libre pour empêcher l'induction de ces gènes. Une internalisation plus élevée a été trouvée avec les NPs-Cur 50:50 que NPs-Cur 65:35. En utilisant des nanoparticules de PLGA chargées avec le colorant fluorescent rouge Lumogen, nous avons montré que les NP de PLGA sont internalisées par les cellules neuronales. Ces données soulignent l'importance de la composition en polymère dans les propriétés thérapeutiques des nanosystèmes. Notre étude a montré que les NPs-Cur améliorent l'action de la curcumine sur plusieurs voies impliquées dans la physiopathologie de la maladie d'Alzheimer. Dans l'ensemble, ces résultats suggèrent que les nanoparticules PLGA sont une stratégie prometteuse pour la livraison de médicaments du cerveau pour le traitement de la maladie d'Alzheimer.