Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Bundeswehr Institute of Pharmacology and Toxicology

Exposure of the respiratory tract to airborne particles is gaining more and more importance due to the ubiquitous application of these particles in the field of industry, pharmacy and in daily life. Remarkably, the toxic properties and the underlying pathomechanisms with regard to inhalable substances have been insufficiently investigated so far. Thus, the EU Chemicals Regulation demands toxicological data (including the identification of potential inhalation hazards) for all chemicals placed on the market until 2018 (REACH). This requires extensive, technically complex and expensive inhalation toxicology studies that are usually generated in animal experiments. However, the legislation demands the consideration of the "3Rs" principle. Thus, in vitro-based test systems for the assessment of pulmonary toxicity are required. One promising approach to assess acute pulmonary toxicity of airborne particles is the CULTEX(®) RFS methodology that allows exposure of human lung epithelial cells at the air-liquid interface mimicking the alveolar situation. A prevalidation study showed the general applicability of this method. However, the clean air exposure group, which served as unexposed controls, exhibited some variations with regard to cell viability compared to the incubator control group. The aim of this study was therefore the identification of the possible causes and the improvement of methodological aspects. Several parameters including the general workflow, adjustment of airflow parameters, and cleaning procedures were investigated and adapted. Finally, our results showed the successful optimization of the CULTEX(®) RFS methodology for clean air exposure of A549 cells. However, although viability data in incubator controls and clean air exposures were equal, a distinct difference in cell morphology was observed that required further optimization. Additional experiments identified that open-wall cell culture inserts with a 2-fold pore density were found to be superior compared to the standard inserts and thus the deciding factor for the improvement of cell morphology. The presented findings are an important step in providing the CULTEX(®) RFS methodology as a promising alternative method to current in vivo testing in inhalation toxicology. 

Optimization of the CULTEX(®) radial flow system for in vitro investigation of lung damaging agents.

Transient receptor potential family channels (TRPs) have been identified as relevant targets in many pharmacological as well as toxicological studies. TRP channels are ubiquitously expressed in different tissues and act among others as sensors for different external stimuli, such as mechanical stress or noxious impacts. Recent studies suggest that one member of this family, the transient receptor potential ankyrin 1 cation channel (TRPA1), is involved in pain, itch, and various diseases, suggesting TRPA1 as a potential therapeutic target. As a nociceptor, TRPA1 is mainly activated by noxious or electrophilic compounds, including alkylating substances. Previous studies already revealed an impact of 2-chloroethyl-ethyl sulfide on the ion channel TRPA1. In this study, we demonstrate that sulfur mustard (bis-(2-chloroethyl) sulfide, SM) activates the human TRPA1 (hTRPA1) in a dose-dependent manner measured by the increase in intracellular Ca concentration ([Ca]). Besides that, SM-induced toxicity was attenuated by antioxidants. However, very little is known about the underlying mechanisms. Here, we demonstrate that N-acetyl-L-cysteine (NAC) prevents SM-induced hTRPA1-activation. HEK293-A1-E cells, overexpressing hTRPA1, show a distinct increase in [Ca] immediately after SM exposure, whereas this increase is reduced in cells pretreated with NAC in a dose-dependent manner. Interestingly, glutathione, although being highly related to NAC, did not show an effect on hTRPA1 channel activity. Taken together, our results provide evidence that SM-dependent activation of hTRPA1 can be diminished by NAC treatment, suggesting a direct interaction of NAC and the hTRPA1 cation channel. Our previous studies already showed a correlation of hTRPA1-activation with cell damage after exposure to alkylating agents. Therefore, NAC might be a feasible approach mitigating hTRPA1-related dysregulations after exposure to SM.

N-Acetyl-L-cysteine inhibits sulfur mustard-induced and TRPA1-dependent calcium influx.

Inhalation of the chemical warfare agent sulfur mustard (SM) is associated with severe acute and long-term pulmonary dysfunctions and health effects. The still not completely elucidated molecular toxicology and a missing targeted therapy emphasize the need for further research. However, appropriate human data are extremely rare. In vivo animal experiments are often regarded as gold standard in toxicology but may exhibit significant differences compared to the human pulmonary anatomy and physiology. Thus, alternative in vitro exposure methods, adapted to the human in vivo situation by exposing cells at the air-liquid interface (ALI), are complimentary approaches at a cellular level. So far, it is unclear whether the enhanced experimental complexity of ALI exposure, that is potentially biologically more meaningful, is superior to submerged exposures which are typically performed. Aim of our study was the evaluation of an appropriate in vitro exposure system (CULTEX Radial Flow System (RFS) equipped with an eFlow membrane nebulizer) for the exposure of cultivated human lung cells (A549) with SM under ALI conditions. Cellular responses (i.e. cell viability) and formation of SM-specific DNA-adducts were investigated and compared between ALI and submerse SM exposures. Our results proved the safe applicability of our ALI exposure system setup. The aerosol generation and subsequent deposition at the ALI were stable and uniform. The technical CULTEX RFS setup is based on ALI exposure with excess of aerosol from that only some is deposited on the cell layer. As expected, a lower cytotoxicity and DNA-adduct formation were detected when identical SM concentrations were used compared to experiments under submerged conditions. A distinct advantage of SM-ALI compared to SM-submerse exposures could not be found in our experiments. Though, the CULTEX RFS was found suitable for SM-ALI exposures.

A novel exposure system generating nebulized aerosol of sulfur mustard in comparison to the standard submerse exposure.

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m for liquid and aerosol phase, respectively.

A wearable origami-like paper-based electrochemical biosensor for sulfur mustard detection.

The CULTEX® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 μg/cm). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.

Amelie Tsoutsoulopoulos

Bundeswehr Institute of Pharmacology and Toxicology

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface