Development of a standardized in vitro approach to evaluate microphysical,chemical,and toxicological properties of combustion-derived fine and ultrafine particles

Ultrafine particles represent a growing concern in the public health community but their precise role in many illnesses is still unknown.This lack of knowledge is related to the ex-perimental difficulty in linking their biological effects to their multiple properties,which are important determinants of toxicity.Our aim is to propose an interdisciplinary approach to study fine (FP) and ultrafine (UFP) particles,generated in a controlled manner using a miniCAST (Combustion Aerosol Standard) soot generator used with two different operating conditions (CAST1 and CAST3).The chemical characterization was performed by an untar-geted analysis using ultra-high resolution mass spectrometry.In conjunction with this ap-proach,subsequent analysis by gas chromatography-mass spectrometry (GC-MS) was per-formed to identify polycyclic aromatic hydrocarbons (PAH).CAST1 enabled the generation of FP with a predominance of small PAH molecules,and CAST3 enabled the generation of UFP,which presented higher numbers of carbon atoms corresponding to larger PAH molecules.Healthy normal human bronchial epithelial (NHBE) cells differentiated at the air-liquid inter-face (ALI) were directly exposed to these freshly emitted FP and UFP.Expression of MUCSAC,FOXJ1,OCLN and ZOI as well as microscopic observation confirmed the ciliated pseudos-tratified epithelial phenotype.Study of the mass deposition efficiency revealed a difference between the two operating conditions,probably due to the morphological differences be-tween the two categories of particles.We demonstrated that only NHBE cells exposed to CAST3 particles induced upregulation in the gene expression of IL-8 and NQO1.This ap-proach offers new perspectives to study FP and UFP with stable and controlled properties.