1 Particle and Fibre Toxicology 2011 Vol: 8(1):27. DOI: 10.1186/1743-8977-8-27

Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes

Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the pathological changes induced by ZnONP and their possible mechanism of action. Two doses of ZnONP (50 and 150 cm2/rat) were intratracheally instilled into the lungs of rats with assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses. Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 μg/rat) were also instilled to rats. Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane destabilization by NP. ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased myofibroblast accumulation and transforming growth factor-β positivity. Serum IgE levels were up-regulated by ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of macrophages with ZnONP in vitro. We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main cause of ZnONP-induced diverse progressive severe lung injuries.

Mentions
Figures
Figure 1: Durability of ZnONP in artificial lysosomal fluid (ALF, pH 4.5) and artificial interstitial fluid (Gamble's solution, pH 7.4). ZnONP were incubated at 37°C for 24 h with gentle shaking. (A) Gross picture taken by digital camera. The photographs were taken before centrifugation. (B) Percentage of mass compared to initial mass (50 mg). Values are mean ± S.D. n = 3. Figure 2: Bronchoalveolar lavage (BAL) analysis 24 h, 1 wk, and 4 wks after instillation of ZnONP to rats. (A - G), cytological analysis of BAL after instillation of ZnONP at 50 and 150 cm2/rat. (A), number of total cells; (B), number of PMN; (C), number of eosinophils. Representative BAL cell images of vehicle control (D) and ZnONP instillation (150 cm2) at 24 h (E), 1 wk (F), and 4 wks (G). Black arrows indicate alveolar macrophages; blue arrows indicate PMN; red arrows indicate eosinophils; green arrows indicate giant cells. (H and I), levels of total protein (H) and LDH (I) in the BAL from rats treated with ZnONP. Levels of LDH in BAL expressed as fold-change compared to vehicle control. (J - M), expression of inflammatory mediators in the BAL from rats instilled with ZnONP. (J), IL-1β; (K), eotaxin; (L), IL-13; (M), TGF-β. Values are mean ± S.D. n = 6 for 24 h and 4 wks groups and n = 4 for 1 wk groups. Significance versus vehicle control (VEH): * p < 0.05, ** p < 0.01, # p < 0.001. Figure 3: IgE and IgA levels after instillation of ZnONP at 150 cm2/rat. (A) IgE levels in serum; (B) IgA levels in serum; (C) IgA levels in BAL fluid. Values are mean ± S.D. n = 4 - 8 for each treatment group. Significance versus vehicle control: * p < 0.05, ** p < 0.01, # p < 0.001. Figure 4: Representative gross lesion and histology of lungs after instillation of ZnONP at 150 cm2/rat. (A), lungs were contracted and collapsed by ZnONP treatment and this caused puckering of the lung surface. (B - E), each figure is composed of a low power view (left, 25×), high power view of bronchiolar epithelium (middle, 400×), and alveoli (right, 400×). (B) vehicle control at 24 h; (C) ZnONP at 24 h; (D) ZnONP at 1 wk; (E) ZnONP at 4 wks. Red arrows indicate eosinophils and arrowheads indicate foamy macrophages. Black arrows indicate goblet cells. Figure 5: Immunohistochemistry for Ki-67 in the lung tissues treated with ZnONP at 150 cm2/rat. Each figure is composed of a low power view (left, 25×), high power view of bronchiolar epithelium (middle, 400×), and alveoli (right, 400×). (A) vehicle control at 24 h; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks. Note that ZnONP increased the proliferating Ki-67 positive cells (arrow) in the bronchiolar epithelium and alveolar epithelium. Figure 6: PAS staining for goblet cells in the lung tissues treated with ZnONP at 150 cm2/rat. Each panel is composed of low power view (left, 100×) and high power view of bronchiolar epithelium (right, 400×). (A) Vehicle control; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks; Percentage of PAS-positive cells in the bronchus (> 1 mm in diameter) (E) and bronchiole (< 1 mm in diameter) (F). Goblet cells (arrow) peaked at 1 wk and still present 4 wks after ZnONP instillation. Each data point represents an independent bronchus or bronchiole in the lung tissues. Significance versus vehicle control: * p < 0.05, ** p < 0.01, # p < 0.001. Figure 7: Picrosirius red staining of the lung tissues treated with ZnONP at 150 cm2/rat. (A) vehicle control; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks. Note that collagen deposition (arrow) was increased 1 and 4 wks after instillation of ZnONP. Bar scale: 50μm. Figure 8: Immunohistochemistry for α-SMA, and TGF-β in the lung tissues following treatment with ZnONP at 150 cm2/rat. (A - D), α-SMA in the lungs. (A) vehicle control at 24 h; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks. Note that α-SMA positive cells (dark brown staining) were seen in smooth muscle layer of airways and vessels of normal lung (A and B). In addition, they were seen profusely in the fibrotic lesions (arrow) at 1 (C) and 4 wks (D) after ZnONP instillation. (E - H), TGF-β in the lungs. (E) vehicle control at 24 h; (F) ZnONP at 24 h; (G) ZnONP at 1 wk; (H) ZnONP at 4 wks. Note that 'I' denotes the area of TGF-β positive fibrosis and the whole image of (H) was dominated by TGF-β positivity. Figure 9: Transmission electron microscopy images of lungs at 4 wks after instillation of ZnONP at 150 cm2/rat. (A), vehicle control; (B - D), ZnONP treatment group. Bar scale: B and C = 2 μm; A and D = 5 μm. Collagen bundles (arrowhead) were found in the perivascular region (B) and alveolar interstitium (C) where they co-localized with eosinophils (black arrow) and PMN. Foamy macrophages (white arrow) were localized in the alveolar spaces. Figure 10: Pulmonary toxicity of ZnONPalt at 24 h after instillation into lungs of rats. (A-C), BAL cell analysis at 24 h after instillation of ZnONPalt to rats. (A) Number of total cells; (B) number of PMN; (C) number of eosinophils. Levels of LDH (D) and total protein (E) in the BAL fluid at 24 h after instillation of ZnONPalt to rats. Values are mean ± S.D. n = 4 for each treatment group. Significance versus vehicle control (VEH): * p < 0.05, ** p < 0.01, # p < 0.001. Figure 11: Pulmonary toxicity of dissolved Zn2+ at 24 h after instillation into lungs of rats. Dissolved Zn2+ in saline was instilled at 92.5 μg/rat. (A - C), cytological analysis of BAL cells. (A), number of total cells; (B), number of PMN; (C), number of eosinophils. Levels of LDH (D) and total protein (E) in the BAL at 24 h after instillation of ZnONP. Values are mean ± S.D. n = 4 for each treatment group. Significance versus vehicle control (VEH): * p < 0.05, ** p < 0.01, # p < 0.001. Zn2+ (92.5 μg/rat) was compared with ZnONP at 50 cm2/rat: $ p < 0.05. Figure 12: Representative transmission electron microscopy images of lungs 24 h after instillation of ZnONP at 15 cm2 to C57BL/6 mice. (A), vehicle control; (B - D), ZnONP treatment group. Bar scale: A = 10 μm; B = 5 μm; C and D = 2 μm. Eosinophils (arrow) were infiltrated in the alveolar interstitium. Macrophage (arrowhead) was severely vacuolated. Figure 13: Lysosomal destabilization by ZnONP in THP-1 cells. Differentiated THP-1 cells by PMA (10 ng/ml) were stained with acridine orange for 15 min and cells were treated with NP for 24 h. (A), vehicle control; (B) TiO2NP at 10 cm2/ml (36.4 μg/ml); (C) ZnONP at 10 cm2/ml (20.6 μg/ml). Acridine orange dye aggregates inside of lysosome which showing red fluorescence. ZnONP showed less signals compared to vehicle control or TiO2NP. Figure 14: Diagram of lung injury caused by ZnONP instillation. ZnONP induced (1) goblet cell hyperplasia, (2) thickening of bronchiolar smooth muscle layer, (3) eosinophilia, (4) fibrosis with increased numbers of myofibroblasts (yellow) and collagen (thin blue fibres), (5) perivascular edema, and (6) type II cell hyperplasia.
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    • . . . 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 46 . . .
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    • . . . 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 47 . . .
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    • . . . 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 . . .
  49. JM Hirshon; M Shardell; S Alles; JL Powell; K Squibb; J Ondov; CJ Blaisdell Elevated ambient air zinc increases pediatric asthma morbidity Environ Health Perspect 116, 826-831 (2008) .
    • . . . Interestingly, one epidemiological study showed that the level of Zn2+ in ambient particulate matter was associated with asthma morbidity in USA 49 . . .
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