In previous studies we found that zinc oxide nanoparticles (ZnONP) were highly fibrogenic and caused an eosinophil exudate into the BAL, a finding that is highly unusual and possibly unique following particle exposure . The current paper set out to determine the likely mechanism of the effects of ZnONP. This paper is notable by its lack of systematic inclusion of benchmark or control particles. The reason is that we have already published extensive findings on the ZnONP response compared to other metal oxides and control NP . The aim of the present paper was solely to investigate the detail of the ZnONP-induced response with a view to better understanding the mechanism whereby they cause such severe pathological effects in rat lungs following a single intratracheal instillation.
In this study, ZnONP were well dispersed with serum protein. Stability of NP depends on a balance between attractive and repulsive forces between particles . Incubation of NP with serum protein forms a protein corona which acts as steric stabilizer preventing agglomeration . When NP deposit in the lung, surfactant proteins and lipids are adsorbed onto the NP forming a lipoprotein corona. Therefore, intratracheal instillation of NP dispersed with serum protein partially mimics the interaction of NP with the lung surface environment. To minimize xenogeneic effects, the serum protein from the same strain animals as those used in the experiments was used as a dispersion medium.
All NP in this study showed negative charge by zeta potential measurement because of the negatively charged protein corona which is the actual charge that is encountered by cells . The recognition by phagocytes facilitates phagocytosis which ingests NP into phagosomes which are then acidified by phagosome/lysosomes fusion. Because ZnONP showed fast dissolution in acidic solution, they are not likely to persistent in the acid milieu of the phagolysosomes. Therefore the effect of surface charge is likely to be less important for high-solubility NP although insoluble positively charged NP are more toxic than neutral or negatively charged NP .
ZnONP induced pulmonary eosinophilia from 24 h to 4 wks after a single intratracheal instillation without any prior sensitisation process. There are published studies showing eosinophilic inflammation in animals following various sensitisation procedures; particles such as TiO2NP  and ambient particulate matter  have been implicated in enhancing development of the murine model of allergic asthma. In addition, instillation or intravenous injection of Sephadex beads (complex of cross-linked dextran polymers; diameter: 20 - 50 μm) also reported to cause eosinophilic inflammation in the lung . However, the eosinophilia caused by Sephadex beads may be produced by their extremely large size because ultrasonication of this particle produced only a transient neutrophilic inflammation . In addition, the Sephadex beads are polymer particles which are fundamentally different from ZnONP used in this study. NP-free BAL exudates collected 24 h after rats were instilled intratracheally with ZnONP at 150 cm2 did not produce any inflammatory reaction either at 1 wk or 4 wks. Considering that eosinophilia peaked 1 wk after instillation and pulmonary fibrosis was mature 4 wks after instillation, the lack of inflammation caused by NP-free BAL exudate instillation suggest that BAL fluid might be too diluted to produce the pathologies caused by ZnONP instillation.
In mice, ZnONP aspiration caused eosinophilia but eosinophils were not found in the alveoli (BAL fluid) but were present in the alveolar interstitium. The interstitial type of eosinophilia was consistent with the elevated levels of eotaxin and IL-13 in the BAL. Eosinophils in the interstitium are as likely to cause tissue injury as eosinophils in the bronchoalveolar space, if not more so .
We undertook a number of assays to investigate the mechanism of the complex and severe pathological syndrome seen following exposure to ZnONP. In the conventional rodent asthma model, recruited eosinophils are associated with airway remodelling including peribronchial fibrosis, smooth muscle hyperplasia, and mucus secretion with the involvement of eotaxin and IL-13 [24, 25]. In the ZnONP model we found that eotaxin and IL-13 were produced early in rats and mice exposed to ZnONP and these are key mediators of eosinophil recruitment [25, 26]. IL-13 is especially involved in the regulation of eosinophil infiltration, IgE synthesis, goblet cell hyperplasia, mucus hypersecretion, and sub-epithelial fibrosis in asthma [25, 27]. Therefore, IL-13 provoked by ZnONP might exert an important role on the wide spectrum of pathological effects seen here with ZnONP exposure.
Although the main inflammatory cells induced by ZnONP were eosinophils, PMN were also recruited during the acute phase. PMN have been regarded as the representative acute inflammatory cells playing a role in particle effects and their recruitment is highly correlated with the surface area dose of low-toxicity, low-solubility particles  and toxic particle such as crystalline silica . The pro-inflammatory cytokine recruiting PMN into ZnONP-exposed lungs was most likely IL-1β which is known to induce neutrophilic inflammation in the lung . However, PMN recruitment by ZnONP was confined to the 24 h time-point whilst significant IL-1β in BAL continued to the 1 week time-point at the highest dose.
Intratracheal instillation of ZnONP induced massive proliferation of airway epithelial cells and goblet cell hyperplasia. ZnONP were reported to be very cytotoxic to BEAS-2B cells in vitro by generating reactive oxygen species . In the present study, ZnONP dramatically increased the levels of LDH and total protein in the BAL at the acute phase indicating cell death and increased vascular permeability, respectively. Therefore, the proliferation of airway epithelial cells likely represents a regenerative response to the cytotoxicity induced by ZnONP. Proliferation of airway epithelial cells led to large-scale goblet cell hyperplasia. Goblet cell hyperplasia was most pronounced at 1 wk and had waned by 4 wks after instillation of ZnONP. Goblet cell hyperplasia plays an important role in protecting the airway from damage due to inhaled particles . The resulting increase in mucus flow traps inhaled particles and removes them from the airways by muco-ciliary clearance. In addition, goblet cells can be progenitors of ciliated cells to maintain mucus flow  and hyperplasia of goblet cells is reversible on cessation of administration . Hypersecretion of mucus by goblet cell hyperplasia is also a feature of airway injury including exposure to cigarette smoke, and sulphur dioxide, and in asthma where it can contribute to obstruction of airways . TGF-β is known to enhance goblet cell hyperplasia and mucus hyper-secretion in mice through an NF-κB-dependent mechanism . In this study, we found strong immunostaining for TGF-β at all time-points, but particularly at 1 and 4 wks after instillation when goblet cell hyperplasia was most pronounced. Consistent with the immunostaining results, total TGF-β concentration in the BAL was also increased at all time-points.
Massive pulmonary fibrosis with contraction and atelectasis was also induced by ZnONP instillation. Following ZnONP instillation collagen fibres, determined by PSR staining, were increased from 1 wk and were more marked at 4 wks. At 4 wks, TEM images showed that large swathes of collagen fibres were primarily located in the perivascular and alveolar interstitium co-localized with eosinophils. Myofibroblasts, the major source of extracellular matrix proteins and contractile forces during fibrogenesis , were also seen in the contracted and fibrotic lung lesions at 1 and 4 wks after treatment. TGF-β also is known to cause airway remodelling including peribronchial fibrosis and smooth muscle hyperplasia .
Instillation of ZnONP increased IgE levels in the serum but not in the BAL. Multi-walled carbon nanotubes , diesel exhaust particles , and ultrafine carbon black  have all been reported to increase serum IgE levels by an adjuvant-like mechanism in murine asthma models. Surprisingly, compared to previous studies, the increase in IgE levels by ZnONP were induced by just a single instillation without any sensitization process. The levels of IgA in the serum were decreased compared to vehicle control. Although there was no statistical significance, IgA levels in the BAL showed an increasing trend at 1 wk after instillation of ZnONP. Increases in mucosal secretory IgA were present in the BAL, and this might explain the decrease of IgA in serum if BAL IgA was derived from the vascular space. The increased IgA levels in the BAL might act be anti-inflammatory in inflamed lung . TGF-β is also known to induce IgA isotype expression by activating Smad3/4 complex translocation into the nucleus .
Our data on ZnONP durability in acid and neutral conditions suggests a mechanism for pathogenicity of ZnONP. ZnONP were stable at neutral pH or saline but very rapidly dissolved in the acidic artificial lysosomal fluid (pH 4.5). Thus ZnONP in the approximately neutral surfactant fluid or in the cytosol might be persistent. However, when ZnONP are internalized into the acid environment of the lysosome, they will be rapidly dissolved producing a high local concentration of Zn2+ ions. Zn2+ is one of the essential elements in cell homeostasis and remains in a bound form inside cells because free Zn2+ is very reactive and cytotoxic . The acute increase in free Zn2+ levels may damage lysosomes, allowing the contents to escape into the cytoplasm where they may damage other organelles leading to cell death [41, 42]. We suggest therefore that Zn2+ released from the phagolysosomes of dead or damaged cells is the source of the Zn2+ after ZnONP uptake in the lungs. The interaction of Zn2+ with cells generates oxidative stress and finally triggers cell death signalling cascades [41, 43]. When ZnONP were added to activated THP-1 cells (a differentiated macrophage cell line), lysosomes were destabilised by a mechanism which seems likely to involve dissolution of ZnONP under the acid condition of the lysosomes. Unlike ZnONP, TiO2NP showed no dissolution in acid or neutral conditions and when incubated with macrophages the fluorescence intensity of lysosomes was not reduced. The loss of lysosomal integrity induced by ZnONP was accompanied by cell death. A role for dissolved Zn2+ in the toxic mechanism of ZnONP is further supported by Muller et al.  who reported that ZnO nanowires were rapidly dissolved in the acidic pH of lysosomes causing structural changes in mitochondria and cell death including necrosis and apoptosis .
Instillation of dissolved Zn2+ from ZnONP treated under acid conditions in saline, produced eosinophilia 24 h after instillation. Moreover, the toxicity of soluble Zn2+ was much greater than similar mass dose of ZnONP based on mortality, number of eosinophils, and levels of LDH and total protein. The higher number of eosinophils by ZnONP than that of ZnONPalt might be due to the smaller primary particle size and their wider distribution inside of the lung. The chronic lung lesions caused by Zn2+ instillation were similar to those induced by ZnONP in terms of bronchocentric interstitial pulmonary fibrosis, goblet cell hyperplasia, and atelectasis.
There are many inhalation/instillation studies using zinc compounds although few papers on nanoparticles. No papers reported eosinophilia on exposure to zinc compounds except for our previous papers using ZnONP [10, 45] and one human case report with zinc oxide . The possible reason for this discrepancy is (1) species and strain differences, (2) location of eosinophils infiltration to the interstitium or airspaces, (3) dose, (4) polydispersity of particles, and (5) durability of particles. In particular the dispersed NP size might be important as we have shown by comparing well-dispersed and agglomerated NP which might specifically address the role of ZnONP agglomerate size. We noted that large poorly-dispersed agglomerates of ZnONP recruited much less number and percent eosinophils (1.3%) compared to well-dispersed ZnONP (37.7%). Based on this data we also conclude that micron-sized particles would not cause substantial eosinophilia in BAL following instillation. This finding is consistent with our previous study which showed no eosinophilic inflammation with ZnONPalt (137 nm) using same strain of rats and instillation technique used in here . In addition, other previous publications also showed no eosinophilic inflammation by nano-size ZnO (50 -70 nm) or micrometer-size ZnO (< 1000 nm) [9, 48]. These previous studies instilled ZnO without any dispersion which implies agglomeration into much larger particles whose compartmentation in the lung might differ from singlet NP or small agglomerates. Therefore, the eosinophilic inflammation by ZnONP may be elicited only by exposure to ZnONP well dispersed and at high doses.
ZnONP-induced eosinophilia, fibrosis, and goblet cell hyperplasia mediated by the soluble Zn2+ is to our knowledge a novel finding in rat lungs. Interestingly, one epidemiological study showed that the level of Zn2+ in ambient particulate matter was associated with asthma morbidity in USA . Therefore, ZnONP pose a unique and substantial hazard to the lungs and hygiene precautions and control of airborne exposure should be instituted in any situation with the potential for exposure in order to reduce the risks of the kinds of lung pathology described here.