Epithelial to Mesenchymal transition, eIF2α phosphorylation and Hsp70 expression enable greater tolerance in A549 cells to TiO2 over ZnO nanoparticles

Type II alveolar cells are highly robust in nature, yet susceptible to aerosolized nanoparticles (NPs). Dysfunction in these specialized cells, can often lead to emphysema, edema, and pulmonary inflammation. Long-time exposure can also lead to dangerous epigenetic modifications and cancer. Among the manufactured nanomaterials, metal oxide nanoparticles are widely encountered owing to their wide range of applications. Scores of published literatures affirm ZnO NPs are more toxic to human alveolar cells than TiO2. However, signalling cascades deducing differences in human alveolar responses to their exposure is not well documented. With A549 cells, we have demonstrated that epithelial to mesenchymal transition and an increased duration of phosphorylation of eIF2α are crucial mechanisms routing better tolerance to TiO2 NP treatment over exposure to ZnO. The increased migratory capacity may help cells escape away from the zone of stress. Further, expression of chaperone such as Hsp70 is also enhanced during the same dose-time investigations. This is the first report of its kind. These novel findings could be successfully developed in the future to design relief strategies to alleviate metal oxide nanoparticle mediated stress.

We have organized our study by evaluating viability first and foremost. This investigation would substantiate the existing knowledge from literature on the degree of lethality between ZnO and TiO 2 treatment. Changes in the cellular morphology was evaluated thereafter. It is one of the first responses to any change in cellular environment and a key indicator of cellular stress 8 . Changes in cell and nuclear morphology as a dose and time dependent function of nanoparticle exposure was studied through Hoechst staining and characterized by the expression of Rho family members of Small GTPases primarily Rac, Rho and cdc42 9 . These proteins are widely known to regulate cytoskeletal organization 10 . They cycle between an inactive (GDP-bound) and an active (GTP-bound) conformation in which they interact with specific effector proteins 11 . Activation of Rho promotes the formation of stress fibers and focal adhesion complexes 12 , Rac promotes the polymerization of actin at the cell membrane, producing lamellipodia and membrane ruffles 13 and cdc42 promotes the formation of filopodia and microspikes at the cell periphery 14,15 . Thus, expression of Rho family of Small GTPases (Rac1, RhoA and cdc42) were evaluated both at mRNA and protein level to assess any differences in morphology attributed by changes in their expression.
Changes in protein expression owing to MeOx exposure was analyzed next by studying the inhibition of global protein synthesis, characterized by the phosphorylation of eIF2α 16 . The eukaryotic initiation factor (eIF2) is a well-known translation factor, and its phosphorylation is one of the first events to occur during Integrated Stress responses 17 . eIF2 is a multimeric protein consisting of 3 subunits; α, β and γ. Their sequences are greatly sustained across several species indicating possible roles crucial to cellular viability 18 . The eIF2α phosphorylation at Ser51 is also a highly conserved and adaptive response that can cause down regulation of translation initiation under several types of stresses and regulate gene expression 16 . It also routes in unfolded protein responses through PERK; PKR like endoplasmic reticulum kinase 19 . Human eIF2α accepts phosphate groups from kinases PKR (Double stranded RNA activated protein kinase); activated in response to viral infection 20 and interferons in mammalian cells 21 . Also, the expression of HRI (Heme regulated inhibitor of translation); activated in response to heme deprivation, heavy metals 22,23 and GCN2 in response to nutrient deprivation 24 results in phosphorylation of eIF2α at residue 51-serine. Phosphorylation of eIF2α was hence studied as a dose and time dependent function of MeOx NP toxicity to quantify the level of Integrated stress response.
As ER stress increases with increased accumulation of unfolded proteins, transcription factors ATF6, XBP1, ATF4 and ATF 5 are sequentially activated 17 . This is triggered by GRP78/Bip (78KDa glucose regulated protein or binding immunoglobulin protein) dissociation from the ER domains of ATF6, IRE-1 and PERK respectively, activating them in the process 25 . GRP78 or Hsp70 is a stress related chaperone which is crucial for activation of all ER transmembrane signalling molecules 26 and may also be expressed while eIF2α stays phosphorylated through activation 27 . Thus, studying the expression of Hsp70 enables analysis of the degree of unfolded protein response triggered to MeOx NP toxicity. To better understand such cellular responses, Hsp70 expression is evaluated at the protein level, for both ZnO and TiO 2 exposure on A549 cells.
Additionally, for further validation of some of our results, invasion assay was performed to test migratory potential and wound healing assay was carried out to evaluate proliferation capabilities. Cell movement is ordained by a series of signal transduction pathways that include small GTPases, cytoskeleton-modifying proteins, kinases, lipid secondary messengers and motor proteins. Cells achieve movement when different signalling cascades are consistently presented in specific locations within the cell while maintaining potency of response to extra cellular triggers. Both epithelial and mesenchymal cells can migrate, however, mesenchymal phenotype has increased invasive capabilities, combined with a greater resistance to cell death 28 . Further since, increased cellular migration is often a consequence to epithelial to mesenchymal transition 29 , therefore, MeOx NP treated samples were also tested for it.

Results
Where ever required, pictures were post processed for background reduction and contrast enhancement using PhotoScape, MOOII TECH, Korea. Analysis was performed by ImageJ, NIH. Standard deviation from 3 independent experiments has been annotated.
The trypan blue dye exclusion test. ZnO  Morphological Documentation. Enlargement of nucleus is often an indicator of cellular activity involving regulation of gene expression and chromatin organization 30 . Hoechst staining ( Fig. 2A) showed enlargement of nucleus (such as in i' , n' , o' , p' , q' and u' shown by orange arrows) and necrotic like cells more than apoptotic cells with increasing exposure to ZnO NPs. In comparison, response to TiO 2 NPs showed a reduction in cell number only after exposure to 50 µg/ml (0.62 mM) TiO 2 . Necrotic like cells with ruptured plasma membrane 31 32 were also visible in cells exposed to ZnO NPs (represented by yellow arrows in h' , j' , e' and f '). Nuclei characterized by increased fluorescence were commonly seen in cells exposed to ZnO NPs starting from 1.24 mM (as depicted by red arrows in k' and l'). Under constant Hoechst dose and UV exposure, increase in fluorescence might indicate a change in the structure of nucleus. Spherical morphology was seen in some TiO 2 exposed cells (blue arrows in t and u). This morphology is usually associated with detachment from basal lamina and often presents a lack of anchorage dependent growth 33 . Filopodial spikes often present in migrating cells 34 were seen more in response to TiO 2 treatment than to ZnO (highlighted by blue diamond arrows in c, e and g for ZnO treated samples and n, o, p, q, s, t, u, v, w and x in TiO 2 exposed cells). Figure 2B demonstrates the differences in morphological changes of a single cell following ZnO and TiO 2 exposure at 24 hours. The panel for ZnO is already published in Santimano et al. 35 . Morphological alterations for ZnO from 24 hours to 72 hours follows a dose dependent pattern. At 0.15 mM of exposure, stress fibres are visible in a cell enlarged as compared to the control (Fig. 2Ba), upon 0.31 mM (Fig. 2Bb) of treatment cells appear more flattened. Further stress up till 0.62 mM (Fig. 2Bc) is presented with spicules and vacuole like granules that seem to fill the cytoplasmic periphery. In the case of morphological response to TiO 2 NPs, Fig. 2Bd-f, we find increased number of elongated cell protoplasm as indicated by the arrows. These extensions are most likely filopodial spikes.  (Fig. 3C). At the highest dose evaluated; 0.62 mM, cdc42 expression increased 60% of control in TiO 2 exposed cells, while decreased 40% to ZnO treatment. Rac1 remains increased in expression from 0.15 to 0.62-mM in response TiO 2 while, ZnO exposed cells see upregulation till 0.31 mM and the expression is downregulated thereafter at 0.62 mM. Highest expression was noted at 0.62 mM for TiO 2 with 100% more than control, while the peak for ZnO was observed at 0.31 mM at 50% more than untreated samples. RhoA does not show any drastic increase in expression pattern, although the up regulation is seen more pronounced in TiO 2 exposure than ZnO. Highest expression of RhoA was seen at 0.31 mM in cases of both ZnO and TiO 2 treatment, again TiO 2 dictated 10% more expression than ZnO treated samples. Densitometric analysis is presented in Fig mRNA level expression analysis for EMT (epithelial to mesenchymal transition). E Cadherin upregulated in response to ZnO exposure along with EGFR. EGFR showed a significant upregulation, at 100% expression more than basal level for 1.24 mM of exposure (Fig. 5A). However, in response to TiO 2 , both E Cadherin and EGFR showed a marked downregulation. N Cadherin upregulated in response to both ZnO and TiO 2 , though it was more pronounced in response to TiO 2 . Clathrin, upregulated up till 0.31 mM in response to ZnO NPs, while it stayed elevated till 0.62 mM with TiO 2 exposure with 75% of control. A further dose treatment

Protein level expression analysis for EMT. EGFR and E Cadherin downregulated in response to TiO 2
NPs while they upregulated with ZnO exposure. ZnO treatment rendered 60% increase in E Cadherin expression and 70% increase in EGFR expression at 0.62 mM among all dose points evaluated (Fig. 5C). TiO 2 exposure resulted in a downregulation of E Cadherin to 40% of control, while EGFR to just 20% of basal expression. N Cadherin upregulated in response to both ZnO and TiO 2 exposure, though for the latter, the expression was more pronounced, with about 20% more than the basal expression. Densitometric analysis was represented in Fig. 5D.
Wound Healing Assay. The control healing potential of the original wound in an untreated sample, was recorded at 53.89% in 24 hours, while 58% for 48 hours. ZnO exposed cells showed wound healing only at 0.15 mM of exposure, with 13.7% for 24 hours (Fig. 6A) Fig. 6C,D. Transwell Invasion Assay. ZnO exposed cells showed a decline in the number of cells migrating with increase in dose of exposure till 0.62 mM at 24 and 48 hours. Upon further increase in dose, no migrated cells were observed. However, TiO 2 treated cells, showed an increase in migration with a maximum at 0.62 mM for 24 hours and 0.15 mM for 48 hours (Fig. 7A). The number of migrated cells is plotted against dose of exposure for TiO 2 and ZnO exposure in Fig. 7B.

Discussion
We selected two candidate nanoparticles of varying toxicities; ZnO and TiO 2, to draw out any differences in cellular responses of A549 cells to their exposure. A similar total viability response was observed of decrease in viability with increase of exposing dose. However, as expected ZnO was more lethal than TiO 2. With increased incubation periods, viability improves in case of both TiO 2 and ZnO exposure, especially at 72 hours. This might be because cells in question are alveolar type II cells (A549) which are known to display higher tolerance to stress 36 . Resazurin reduction assay was performed to gauge the metabolic activity and mitochondrial health of cells exposed to metal oxide nanoparticles. ZnO did confer higher lethality over TiO 2 , with LD 50 value of 2.26 mM as against 44.15 mM for the latter. Resazurin reduction values were significantly higher than total viability values, for respective doses evaluated, suggesting mitochondrial dysfunction may not be the only cause of death.
Morphological documentation reveals less budding typical of apoptosis and more cells with ruptured plasma membrane with increase in exposure, especially for ZnO treatment. This allied with analysis from trypan blue dye exclusion test and resazurin reduction assay suggest a majorly necrotic mode of death.
We had explored the phosphorylation status of eIF2α to understand if this well-studied unfolded protein stress response is an occurrence in nanoparticle mediated toxicity. A continued phosphorylation of eIF2α, particularly at 48 hours in response to TiO 2 as compared to ZnO nanoparticles was observed. This is the first report of its kind, discovered by us. Western blot analysis was undertaken and phosphorylation status of eIF2α was investigated with respect to total eIF2α as the control. This has been double checked with another internal control βActin (data not shown in the paper). The Integrated stress response (ISR) merges with the unfolded protein response (UPR) cascade at the PERK sensor (Protein kinase R like endoplasmic reticulum kinase). Sensors within the unfolded protein response cascade are present along the membrane endoplasmic reticulum (ER) membrane. Phosphorylation of eIF2α could thus be an adaptive response by providing the cell with an opportunity to limit deleterious effects of noxious agents and help conserve resources. Expression of specific repair agents such as Hsp70 during this period, can be regulated to aid stress recovery 37 . Over expression of Hsp70, is also known to reverse misfolded proteins including cytoplasmic aggregations such as the stress granules (SG) 38   The longer duration of phosphorylation of eIF2α observed with TiO 2 exposure along with an increase in Hsp70 expression is most crucial. This may provide the cell with systems to counter cellular damages such of misfolded proteins, ultimately increasing the potential for repair in TiO 2 treated cells. The phosphorylation status of eIF2α is also a translational regulation event 39 , which may account for the differences in expression profiles of small GTPases between the mRNA and the protein level. mRNA level expression of small GTPase for both ZnO 35 and TiO 2 exposure follow a similar pattern; upregulation of RhoA and Rac1 while a spiked expression for cdc42. The pattern varies however between ZnO and TiO 2 exposure at the protein level. With cdc42 upregulating upto 60% more than control at 0.62 mM in case of TiO 2 and downregulating to 40% of control expression at the same dose for ZnO. This can directly be co-related to the increased filopodial phenotypic expression to TiO 2 not observed with ZnO exposure.
We further, discovered E Cadherin downregulation along with N Cadherin upregulation in response to TiO 2 exposure, a hallmark for EMT 40 . This is a novel finding. This is not evident with ZnO NP treatment. This is supplemented with the allied EGFR expression that follows suit with E Cadherin 41 . Clathrin is crucial to EGFR internalization 42 . At mRNA level, Clathrin expression does stay elevated up till a dose of 0.62 mM at 78.34% more than control in TiO 2 exposure. Whereas, clathrin peaks at 0.31 mM with just 5% more than control to ZnO treatment. Thereafter, with further dose exposure, clathrin expression downregulates. These results suggest, with MeOx exposure on A549 cells, clathrin upregulation and its mediated internalization of EGFR, result in degradation of EGFR.
Increased proliferative and migrative capacity of TiO 2 NP treated cells over ZnO was also recorded through wound healing and transwell invasion assays. Wound healing assay shows a marked preservation in proliferation capacity with TiO 2 treatment as compared to ZnO. These cells may thus, migrate away from the zone of stress, enabling them to tolerate higher TiO 2 exposure as compared to ZnO. In toxicity assessments particularly, that of the in vitro set up, a zone of stress represents the layers of variant shear stress in a culture vessel 43 . We postulate that adherent cultures exhibit high stress on cells close to the basal membrane, with marked nutrient deprivation and increased steric hindrance through crowding of the cells. The density dependent depletion in cell growth and proliferation is widely documented for confluent cultures 44 . It is possible that epithelial to mesenchymal transition allows cells to float away from the lamina into a region more conducive for survival. Moreover, the regulated release of cytokines and chemokines by stressed cells also less affect the cells away from the high zone of stress at the basal lamina 43 . Though for all doses evaluated for both ZnO and TiO 2 exposure, mitotic capacities 45 lagged with the untreated control, suggesting, MeOx NP treatment does negatively affect cellular proliferation, irrespective of lethality.
Further epithelial to mesenchymal transition, is also known to phosphorylate eIF2α through Protein kinase RNA-like ER kinase (PERK) activating the unfolded protein response (UPR) in response to endoplasmic stress 46 . We have substantially proved that TiO 2 NP treated cells do have an increased duration for which eIF2α remains phosphorylated as compared to ZnO exposure. This may well be related to the onset of epithelial to mesenchymal transition observed in TiO 2 NP treated cells, not observed with ZnO exposure.
Our data sufficiently proves, A549 cells can withstand a greater dose of exposure from TiO 2 NPs as compared to ZnO (Fig. 8). LD 50 value for TiO 2 nanoparticle treatment on A549 cells is almost 20 times more than that with ZnO. This is due to the epithelial to mesenchymal transition that occurs at the molecular level along with cdc42 expression that renders filopodial extensions. Allied with increased Hsp70 expression and phosphorylation of There are already drugs being tested to increase chaperone expression such as Hsp70 47 . The capability of this protein to stabilize denatured protein complexes has developed as an attractive drug development concept. Members of this chaperone family are some of the most ubiquitous and conserved proteins making modelling for response accuracy more convenient along a wide range of systems. Recent research has already uncovered several potential drug molecules that could improve the expression of Hsp70. Derivatives of shikonin and echinochrome can upregulate Hsp70 and reduce cell mortality in response to heat stress, hydrogen peroxide and staurosporine treatment 48 . Exercise has also been implicated in increased Hsp70 expression 49 . Resvaratol, a common ingredient in wine, has proved to increase survivability in mice by upregulating Hsp70 50 . Foods rich in antioxidants such as blueberry and curcumin have also shown a signification upregulation of Hsp70 leading to increased cell recovery and survival 51,52 .
On the other hand, phosphorylation of eIf2α as a target of drug development route has been less studied. Salubrinal, an inhibitor of GADD34 has been recently shown to upregulate phosphorylation of eIF2α 53 . Although its use for any toxicity related therapy has not been investigated. Epithelial to mesenchymal transition has long gained a spotlight for cancer progression 54 . Although this signalling cascade has not been evaluated as a stress revival strategy in normal cells.
Our research discovers three novel routes, that are related and can together confer 20 times of dose tolerance to nanoparticle exposure. Cellular mechanisms such as upregulation of Hsp70, increased phosphorylation of eIf2α and induction of epithelial to mesenchymal transition, can enable human alveolar type II cells to migrate away from a zone of stress in the alveolar lining. They can tolerate a higher level of toxic treatment. These strategies either by themselves or in combination with other novel approaches have huge potential to be developed into therapeutic regimens for nanotoxicity especially in the case of pulmonary distress. A world of raising pollution and aerosolized nano material need the discovery and development of such approaches to combat lethal toxicities to human health.

Methods
All the general laboratory chemicals were purchased from Sigma-Aldrich (USA) and Himedia (India), antibodies from Cell Signalling (USA), Biolegend (USA) and Abcam (USA). Human broncho-alveolar carcinoma-derived (A549) cells were obtained from National Centre for Cell Sciences, Pune. Characterized nanoparticles were purchased from Sigma, Aldrich (USA). Culture plastic ware were procured from Corning Life Science. All experiments were carried out in triplicates.
Cell Culture. A549 cells were cultured in vitro and maintained in DMEM supplemented with 10% FBS (37 °C with 5% CO 2 ). Experiments were conducted at 80% confluence unless otherwise mentioned.
Charging of Nanoparticles. ZnO Table 1. Doses were chosen based on our viability assays 35 along a range commonly observed at  Mitochondrial activity Assay. Cell viability was assayed using Resazurin as per Santimano et al. 35 . Cells were seeded at a low seeding density of 2.5 × 10 4 cells per well in a 24 well plate. This was done to ensure resolution along the range of doses tested. Post 24 hours of seeding, fresh media was added maintaining 10% serum to better dispense nanoparticles away from aggregation. Nanoparticles were added after thorough vortex from the least concentrated stock to maintain a minimum aggregation at charging, this method was developed by us for proper dispersion of NPs at the time of charging to mimic the dispersion typical of aerosolized NPs. The design is detailed in Table 1. Resazurin was added in a working concentration of 440 µM. After 4 hours of incubation at 37 °C, positive difference (absolute value) in absorbance at wavelength of 580 nm and 615 nm of each well culture against control (+) was monitored and the percentage resazurin reduction was calculated and reported as a measure of toxicity. The limiting value corresponding to 0% reduction was obtained by measuring OD 580 -OD 615 of negative control. Percentage Resazurin reduction was used to calculate dose at 50% death (LD 50 ).
Cell Morphology Documentation. Cells were charged with nanoparticles and incubated for time periods of 24 and 48 hours respectively. The monolayer was washed with sterile phosphate buffered saline and fixed with 3.7% formaldehyde for 2 minutes. This was followed by permeabilization with 100% ice cold methanol and staining with Hoechst (33342, thermos fischer) for 15 minutes. Fluorescent pictures of nuclei were captured by an inverted microscope to aid visualization of nucleus. A DAPI filer was used that allowed for illumination of light around 340-380 nm and emission around 465 nm.
Studying mRNA level expression by RT PCR. Total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. RNA extracted was reverse transcribed and cDNA synthesized using the Bioline cDNA synthesis kit. cDNA was amplified by PCR. Resolution was done using 1.2% agarose gel electrophoresis (TBE buffer) with ethidium bromide staining, photographed under ultraviolet light (BioRad) and analysed by densitometry. The quantity of each transcript was normalized to that of GAPDH, which served as the internal control.
Studying the protein level expression by Western blot analysis. After cell lysate (40 µg) was resolved by 12% SDS-PAGE the western blot was carried out as per Sarkar et al. 56 . In brief, the PVDF membranes were washed with tris-buffered saline followed by blocking with 5% non-fat dried milk or 3% BSA as was suited. The membranes were incubated at 4 °C overnight with primary target specific antibodies.The membranes were then incubated with secondary antibodies coupled to horseradish peroxidase for optimised time periods at room temperature. The membranes were washed in combinations of TBS and TBST at room temperature. Immunoreactivities were detected by ECL reagents (Amersham GE Healthcare). Expression of target proteins was normalized to β-Actin.