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Key Event Title
Inhibition of lung surfactant function
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Lung surfactant function inhibition leading to decreased lung function||MolecularInitiatingEvent||Jorid Birkelund Sørli (send email)||Open for comment. Do not cite||Under Development|
|All life stages||High|
Key Event Description
Airborne substances that penetrate deep into the lungs and reach the alveoli will come into contact with the thin layer of lung surfactant prior to encountering the alveolar epithelial cells. In addition, blood components (such as albumin) that cross the alveolar-capillary membrane and reach the alveolar airspace can interact with the lung surfactant. The nature of this interaction between substances and lung surfactant depends on the origin (intrinsic versus extrinsic) of the substance, its molecular structure, size, and other physicochemical properties such as hydrophobicity, charge, etc. The interaction can be direct, with certain components of the lung surfactant film at the air-liquid interface i.e. by oxidation or cleaving of the phospholipids (Seeds, Grier et al. 2012, Stachowicz-Kusnierz, Cwiklik et al. 2018), or indirect, via competition with the adsorption of lung surfactant. In many cases, the interaction of substances with lung surfactant at the molecular level is responsible for lung surfactant function inhibition.
How It Is Measured or Detected
Measurements of lung surfactant function inhibition
The inhibition of lung surfactant function can be measured in vitro by evaluating the surface activity in dynamic assays that mimic the continuous compression and expansion of the surfactant films at the air-liquid interface in the alveoli during breathing. Values of minimum surface tension, i.e. the lowest value of surface tension reached upon compression of the surfactant film, is a good indicator of the proper functioning of the lung surfactant. Maximum surface tension, i.e. the highest value of surface tension reached upon expansion of the lung surfactant film, reflects the effective re-adsorption of the lung surfactant at the interface. This parameter was shown to be less sensitive than the minimum surface tension to identify inhibitors of lung surfactant function (Valle, Wu et al. 2015, Da Silva, Autilio et al. 2021). These tests can be performed in different setups.
Constrained drop surfactometer
In the constrained drop surfactometer (CDS) a droplet of lung surfactant is deposited on a sharp-edged pedestal, so that a surfactant film is formed at its air-water surface. The adsorbed lung surfactant film is cycled continuously, to mimic breathing (Zuo, Veldhuizen et al. 2008, Valle, Wu et al. 2015, Sørli, Da Silva et al. 2016, Yang, Wu et al. 2018). A camera continuously takes pictures of the droplet before and during exposure to aerosols of the test substance at the air-liquid interface. Alternatively, the lung surfactant and the test substance can be mixed prior to deposition on the pedestal (Sørli, Låg et al. 2020). Surface tension values are obtained by analysis of the drop shape in real-time (Yu, Yang et al. 2016). 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.
Captive bubble surfactometer
In the captive bubble surfactometer (CBS), the lung surfactant film is formed at the air-liquid interface of an air bubble suspended in liquid. The function can be studied by injecting the test substance in the proximity of the surfactant layer at the interface between the air bubble and the surrounding liquid, or by mixing the substance and the surfactant prior to injecting the lung surfactant into the chamber. The captive bubble surfactometer allows study of the rapid initial adsorption of the lung surfactant at the air-liquid interface, post-expansion adsorption, surface activity during dynamic and quasi-static cycles, and stability of the surfactant film to mechanical perturbations (Autilio and Perez-Gil 2019).
Pulsating bubble surfactometer
In the pulsating bubble surfactometer (PBS), an air bubble suspended on a capillary tube is formed in a chamber containing lung surfactant and is periodically compressed and expanded by a piston pulsator (Enhorning 2001, Autilio and Perez-Gil 2019). The method has been used to study the effects of nanoparticles (Schleh, Muhlfeld et al. 2009), bacterial lipopolysaccharides (Kolomaznik, Liskayova et al. 2018), glucocorticoids (Cimato, Facorro et al. 2018), or meconium (Stichtenoth, Jung et al. 2006) on lung surfactant. The pulsating bubble surfactometer was also used to investigate the surface activity of lung surfactant from patients with acute respiratory distress syndrome (Gregory, Longmore et al. 1991, Markart, Ruppert et al. 2007).
In the capillary surfactometer (CS), surfactant is deposited in a capillary tube of uneven diameter that simulate the cylindrical surfaces of the terminal conducting airways a constant airflow is led through the capillary. The percent of time with an open passage is used to assess the functionality of lung surfactant (Enhorning 2001, Larsen, Dallot et al. 2014, Sørli, Da Silva et al. 2016).
Surfactant adsorption test
The surfactant adsorption test is a fluorescence-based method that measures the extent and rate of adsorption of lung surfactant at the air-liquid interface. Lung surfactant is labelled with a fluorescent probe, and injected into the wells of a multi-well plate containing a light-absorbing agent (typically brilliant black). The plates are shaken and the fluorescence (of the lung surfactant sample reaching the surface of the wells) is measured. The fluorescence of the lung surfactant sample in the bulk (not adsorbed at the interface) is quenched by the light-adsorbing agent. This method is high-throughput compared to the biophysical assays described above and it allows to measure the effects of physiologically relevant factors, such as temperature, surfactant concentration, or presence of inhibitors in a high number of samples (Ravasio, Cruz et al. 2008). However, this assay does not measure other biophysical properties like pressure-area isotherms, compressibility etc.
Investigation of the interaction of a substance with lung surfactant
The interaction between a substance (exogenous airborne substances or biological components) and lung surfactant components can be investigated at the molecular level in vitro and estimated in silico. The methods rely on lung surfactant models, ranging from simple monolayers of dipalmitoylphosphatidylcholine (DPPC, the main surface-active component of lung surfactant), to the most complex native surfactant, obtained from broncho-alveolar lavage fluid or minced lung tissue. In most methods, a film of lung surfactant is formed at air-liquid interfaces and exposed to the substance of interest via aerosolisation or deposition. In some cases, the lung surfactant model is mixed directly with the test substance before spreading of the film.
Atomic force microscopy
The topography of surfactant structures formed at respiratory-like air-liquid interfaces upon exposure to test substances can be studied by atomic force microscopy on fixed samples.
Langmuir-Blodgett films are interfacial films of surfactant transferred from the air-liquid interface onto solid supports. They are used to gain information about the distribution of lipids and proteins within the surfactant film and the effect of the interaction with test substances (Cruz and Perez-Gil 2007). Surfactant films deposited at the air-liquid interface of a trough filled with liquid can be compressed by reducing the surface area of the trough. A sensor plate measures the variation in surface pressure over compression to yield surface pressure – area isotherms. It should be noted that in addition to the traditional Langmuir trough, the Langmuir-Blodgett technique has been adapted in the constrained drop surfactometer to study adsorbed surfactant films (Xu, Yang et al. 2020). The comparison of such isotherms in the presence or absence of the test substance gives insights in the interaction of a substance with lung surfactant at the molecular level. Shifts in the surface pressure-area isotherms are identified most easily using simple models such as DPPC monolayers, but can also be seen using the more complex lung surfactant. Structural changes can be identified during compression of the film when combined with epifluorescence or atomic force microscopy.
Cryogenic transmission electron microscopy
In aqueous dispersions, lung surfactant forms vesicles. Cryogenic transmission electron microscopy allows visualizing morphological and structural changes at the single membrane vesicle level. After incubation with the test substance, the surfactant model is applied onto a carbon grid and vitrified in liquid ethane cooled by liquid nitrogen. Changes in the size, circularity or lamerallity of the vesicles indicate disruption of the three-dimensional surfactant structures.
Differential scanning calorimetry
Differential scanning calorimetry allows the study of phase transitions occurring in lipid membranes (such as lung surfactant) over changes in temperature (Demetzos 2008). It can be used to characterize the thermotropic phase behaviours of phospholipids in the surfactant models in the absence or presence of interacting substances. Associated enthalpy, transition temperature, and cooperativity can be estimated from the thermograms. It is a very sensitive method when working with simple models such as pure DPPC bilayers. The method is much less sensitive when using complex lung surfactant models. This is because several transitions overlap in membranes made of complex mixtures, each occurring at different temperature so it is difficult to identify one specific variation.
Domain of Applicability
The applicability domain is restricted to the groups of organisms where the structure and the functioning of the pulmonary system, including the lung surfactant, are conserved and relevant. Lung surfactant is a vital component of the lungs found in all major vertebrate groups, but particularly, to sustain the delicate structure of the mammalian lung. The lung surfactant system has a single point of origin and was a prerequisite for the evolution of air breathing (Sullivan, Daniels et al. 1998). While the composition and function of lung surfactant are conserved in vertebrates, changes in composition among non-vertebrates are noted and likely reflect differences in the structure of the respiratory units (Veldhuizen, Nag et al. 1998). Decreased lung function has been observed after exposure to airborne toxicants in humans of all sexes and ages, and in common experimental animal species, such as mice, rats, and rabbits.
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