Development of a standardized in vitro approach to evaluate microphysical, chemical, and toxicological properties of combustion-derived fine and ultrafine particles
Graphical abstract
Introduction
Air pollution is a serious worldwide issue due to its health impacts. As classified by the International Agency of Research on Cancer (IARC) as carcinogenic to humans (Group 1), air pollution led to 8.8 million early deaths in 2015 (Lelieveld et al., 2019). The strongest evidence for public health concerns mostly includes coarse (PM10, i.e. PM with a median aerodynamic diameter <10 µm) and fine particulates (FP, PM2.5, i.e. PM with a median aerodynamic diameter <2.5 µm). This has been demonstrated both after short- and long-term exposures for cardiovascular and respiratory diseases, as well as for all-cause mortality (Brook et al., 2010; Burnett et al., 2018; Pope et al., 1995).
There is a growing concern in the scientific community about the contribution of ultrafine particles (UFP, PM with a median aerodynamic diameter of 100 nm or less; PM0.1) to human health. Indeed, physical factors such as size and morphology participate in the toxicity of particles by letting them penetrate into the different regions of the respiratory system (Carvalho et al., 2011; Tyler et al., 2016). Depending on its size, PM is deposited in different parts of the respiratory system: PM10 in upper airways, PM2.5 in the lower airways including alveoli (Nemmar et al., 2013) and UFP, which can be translocated to interstitial sites in the respiratory tract and in extrapulmonary organs (Kreyling et al., 2009; Oberdörster et al., 2004). The large surface area of UFP enables them to deliver a greater quantity of chemical compounds to the respiratory system for a given mass of PM, playing an important role in their toxicological effects (Abdel-Shafy and Mansour, 2016; Chen and Lippmann, 2009; Tyler et al., 2016). Several studies have demonstrated the role played by PM in the oxidative stress and inflammation response (Donaldson et al., 2002; Leikauf et al., 2020; Li et al., 2003; Nemmar et al., 2013; Schraufnagel, 2020). However, despite their higher number and important reactivity, UFP have been less studied than coarse and FP (Schmid et al., 2009). Total PM emissions include both filterable PM, directly emitted from the source as solid or nonvolatile liquid particles, and condensable PM, initially in the vapor phase after combustion and immediately forming solid or liquid droplets. Both these condensable and filterable PM form UFP (Feng et al., 2018; Kwon et al., 2020). Another characteristic of the UFP are their very short atmospheric lifetimes: their concentrations decrease with increasing distance from the emission sources , hence efficient sampling strategies to collect sufficient PM mass are required (Kumar et al., 2021). An alternative is to conduct experiments with a source providing relevant emissions of UFP that can be easily produced in a reproducible way.
The mini-Combustion Aerosol Standard (mini-CAST) is a reference soot generator in which soot particles, produced from a co-flow propane diffusion flame, are quenched and quickly diluted to stabilize the soot particle stream and prevent water condensation. Many studies have been carried out to analyze the properties of soot particles, demonstrating that soot particle characteristics (such as Organic Carbon (OC) / Total Carbon (TC), morphology, number and size distribution) can be varied depending on the operating conditions, while maintaining a stable and repeatable source (Bescond et al., 2016; Jing, 1999; Moallemi et al., 2019; Moore et al., 2014). The mini-CAST has been used primarily as a calibration device for soot and to understand soot formation and has been adapted for liquid fuels. Recently, it has been used as a relevant tool in the production of aircraft soot analogues (Marhaba et al., 2019). Thus, the miniCAST soot generator should serve as an adequate device for studying particle toxicity with microphysical and chemical properties representative of real-world emissions.
The toxicity of inhaled airborne particles can be studied by different in vivo and in vitro techniques. Concerning the in vitro approaches, submerged assessments are commonly chosen, where cells are exposed to a particle suspension, but these are not representative of physiological conditions (Loret et al., 2016). Conversely, particle exposure of lung cells grown at the air-liquid interface (ALI) depicts a situation close to physiological conditions and allows estimation of the toxicity of combustion particles (BéruBé et al., 2010; Bhowmick and Gappa-Fahlenkamp, 2016; Paur et al., 2011). Here, cells were cultured on the semi-permeable membranes of a trans-well insert in a culture well where medium was supplied only from the basal pole. Then cells were exposed to an aerosol at the apical pole. Several devices have been developed in order to study this approach, such as the Cultex® System and the Vitrocell® System (Aufderheide and Mohr, 2004, 2000). Despite the advantages provided by these devices, controlled and repeatable cell exposures at relevant airborne concentrations are scarce. However, these devices, associated with the use of novel tissue engineering tools, are attractive tools to extend the knowledge on the cellular effects of UFP. Among these novel cellular tools, normal human bronchial epithelial (NHBE) cells can differentiate into a mucociliary phenotype similar to in vivo conditions. Additionally, when cultured at the air-liquid interface, NHBE cells are adapted to direct aerosol exposure, allowing preservation of the physical and chemical characteristics of the aerosol (Bhowmick and Gappa-Fahlenkamp, 2016; Zavala et al., 2020).
Several experimental in vitro studies at the ALI have demonstrated the role of oxidative stress and inflammation in FP- and UFP-induced respiratory toxicity (Crobeddu et al., 2017; Diabaté et al., 2008; Sotty et al., 2019). These effects may be due to the organic composition and polycyclic aromatic hydrocarbon (PAH) contents (Akhtar et al., 2014; Li et al., 2003), although other studies suggest that the relationship between biological response and chemical composition is much more complex (Schwarze et al., 2007). Indeed, in the study of Schwarze et al. (2007), the authors outlined the importance of size and composition, which may lead to different cellular responses. The issue of what characteristics make a particle more biologically reactive than others is pivotal, but needs further investigation and the development of suitable experimental tools.
This work aims to propose an integrated approach suitable for the physical, chemical, and toxicological characterization of FP and UFP. For that, a miniCAST soot generator was used under two different operating conditions (CAST1 and CAST3) generating particles made respectively of low and high OC/TC. First, we examined the microphysical properties of these particles as well as their deposition efficiency using an ALI device. Then, we characterized their chemical properties by a non-targeted approach using ultra-high resolution mass spectrometry and a direct insertion probe (DIP FTICR-MS), and a targeted approach using GC-MS. Finally, we conducted in vitro toxicity assays using the ALI-differentiated model of NHBE.
Section snippets
Experimental setup
The exposure system is comprised of several main components: soot generator miniCAST (5201c, Jing Ltd., Switzerland), the Vitrocell® exposure system (model VITROCELL® 6/4 CF, Vitrocell® Systems GmbH, Germany), and several devices for the physical characterization and control of aerosol generation, as described below. A schematic of the experimental setup is shown in Fig. 1.
Soot particles were produced with a miniCAST soot generator allowing the production of a stable and repeatable aerosol of
Particle characterization: size, mass, and morphology
The particle size distribution within the aerosol can be seen in Fig. 2 The geometric median (GM) mobility diameter, particle number, and mass concentration for each different operating condition are shown in Table 2. Particle emission depends on the operating conditions. CAST3 produced a smaller size distribution compared to CAST1. The mass concentration for CAST1 condition is higher (89.4 mg/m3) compared with CAST3 (57.9 mg/m3).
Concerning the particle morphology, the two operating conditions
Discussion
The present results illustrate a methodological approach which allows comparing, in an original and innovative way, the physical, chemical, and toxicological properties of different fine and ultrafine particles, using a mini-CAST generator. In this study, we used two different operating conditions to obtain model particles containing either high (CAST3) or low (CAST1) organic contents, and exposed differentiated NHBE cells “on-line” at the air-liquid interface to be closer to realistic
Conclusions
In conclusion, this study presents a new methodological approach to study freshly emitted FP and UFP on differentiated bronchial cells with respect to their physicochemical properties. It outlines the necessity of a thorough characterization to control dose delivery, which depends on the physical and chemical characteristics of particles. Although further testing and optimization are still required, this approach offers new perspectives to study FP and UFP having properties similar to those
Acknowledgments
This work was supported by ANSES (French Agency for Food, Environmental and Occupational Health and Safety; PUFBIO project, Grant number EST-2017-190) and co-supported by the Regional Council of Normandy and the European Union in the framework of the ERDF-ESF (CellSTEM project). Ana Teresa Juarez Facio received a PhD fellowship funded by ADEME (Agency for Ecological Transition). The authors would like to thank Cathy Logie, Violaine Martin de Lagarde and Marion Janona for their technical support.
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