Acute Inhalation Toxicity After Inhalation of ZnO Nanoparticles: Lung Surfactant Function Inhibition In Vitro Correlates With Reduced Tidal Volume in Mice

Abstract

People can be exposed to zinc oxide (ZnO) by inhalation of consumer products or during industrial processes. Zinc oxide nanoparticle (NP) exposure can induce acute inhalation toxicity. The toxicological mechanisms underlying the acute effects on the lungs have long focused on the phagolysosomal dissolution of ZnO NPs in macrophages followed by the release of free Zn2+ ions. However, we postulate an alternative mechanism based on the direct interaction of ZnO NPs with the lung surfactant (LS) layer covering the inside of the alveoli. Therefore, we tested the effect of ZnO NPs and Zn2+ ions on the function of LS in vitro using the constrained drop surfactometer. We found that the ZnO NPs inhibited the LS function, whereas Zn2+ ions did not. To examine the role of lung macrophages in the acute toxicity of inhaled ZnO NPs, mice were treated with Clodrosome, a drug that depletes alveolar macrophages, or Encapsome, the empty carrier of the drug. After macrophage depletion, the mice were exposed to an aerosol of ZnO NPs in whole body plethysmographs recording breathing patterns continuously. Mice in both groups developed shallow breathing (reduced tidal volume) shortly after the onset of exposure to ZnO NPs. This suggests a macrophage-independent mechanism of induction. This study shows that acute inhalation toxicity is caused by ZnO NP interaction with LS, independently of NP dissolution in macrophages.

An adverse outcome pathway for lung surfactant function inhibition leading to decreased lung function

“…The main advantages of this method include the accessibility of the air–liquid interface for exposure to airborne substances, flexibility in controlling cycling rates, and ease of determination of the surface tension in real-time while cycling the surfactant film. The constrained drop surfactometer has been applied to the exposure to a broad range of substances, including nanoparticles ( Larsen et al, 2020 , Valle et al, 2015 , Fan et al, 2011 , Yang et al, 2018 , Wang et al, 2020 ), individual chemicals ( Da Silva et al, 2021 ), mixtures of chemicals ( Da Silva, et al, 2021 , Sørli et al, 2018 , Sørli et al, 2016 ), excipients for drug formulation ( Sørli et al, 2018 ), per- and poly-fluoroalkyl substances ( Sørli et al, 2020 ), or plasma ( Autilio et al, 2021 ).…”

“…Reduced tidal volume can be measured in humans by spirometry ( Miller et al, 2005 , Pellegrino et al, 2005 ) and in animals by plethysmography ( Sørli et al, 2018 , Sørli et al, 2018 , Larsen et al, 2020 ). Reduced tidal volume and total lung capacity has been observed in humans after to exposure to an impregnation spray products ( Lazor-Blanchet et al, 2004 , Khalid et al, 2009 ), and in exposed experimental animals ( Duch et al, 2014 , Sørli et al, 2018 , Sørli et al, 2018 , Nørgaard, et al, 2014 ).…”

“…The inhibition of function correlated with histological findings of lung collapse in the exposed animals ( Yang et al, 2018 ). In addition, the key event relationship between lung surfactant function inhibition in vitro leading to rapid decrease in tidal volume in vivo has also been demonstrated for zinc oxide nanoparticles ( Larsen et al, 2020 ), bile salt enhancers for drug formulation ( Sørli et al, 2018 ), and impregnation products ( Tashiro et al, 1998 , Duch et al, 2014 , Sørli et al, 2018 , Nørgaard, et al, 2010 , Nørgaard, et al, 2014 ). In some cases, when the animals were given clean air to breathe, the decrease in tidal volume was not reversed, indicating that the damage was too severe to be restored by the lungs in the absence of exposure ( Duch et al, 2014 , Larsen, et al, 2014 , Nørgaard, et al, 2010 ).…”

“…The biological plausibility and empirical evidence of the inhibition of lung surfactant function by inhaled substances or by blood components reaching the alveolar airspaces, are high. Studies have been conducted for a number of substances including airborne nanoparticles ( Chen et al, 2017 , Yang et al, 2018 , Larsen et al, 2020 ), airborne chemicals ( Da Silva et al, 2021 ), and biological components such as albumin, cholesterol meconium, and serum ( Zuo et al, 2006 , Lopez-Rodriguez et al, 2011 , Lopez-Rodriguez et al, 2012 , Lopez-Rodriguez et al, 2013 , Zhang et al, 2012 , Autilio et al, 2021 , Gómez-Gil et al, 2009 , Lugones et al, 2018 , Gunasekara et al, 2005 ). For impregnation spray products, the inhibition of lung surfactant function has been reported across multiple methods in vitro , such as the lung surfactant bioassay ( Sørli et al, 2018 ), the capillary surfactometer ( Sørli et al, 2016 ), the pulsating bubble surfactometer ( Tashiro et al, 1998 ), and the Langmuir trough ( Duch et al, 2014 , Larsen, et al, 2014 ).…”

Toxicological effects of zinc oxide nanoparticle exposure: an in vitro comparison between dry aerosol air-liquid interface and submerged exposure systems

“…This release can in turn cause lysosomal destabilization and result in leakage to the cytoplasm and damage to other organelles (Cho et al 2011). In addition to inducing cytotoxic effects on cells, ZnO NPs can inhibit the lung surfactant function independently of the dissolution of the particles into zinc ions, causing acute toxic effects (Larsen et al 2020).…”