Cytotoxicity analysis of biomass combustion particles in human pulmonary alveolar epithelial cells on an air–liquid interface/dynamic culture platform

Abstract

Background Exposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air–liquid interface (ALI) and dynamic culture (DC) systems. Methods HPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI–DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI–DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting. Results Scanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI–DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI–DC platform, indicating that the ALI–DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs. Conclusion A novel ALI–DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis.

Zero-Carbon and Carbon-Neutral Fuels: A Review of Combustion Products and Cytotoxicity

“…In recent years, the air-liquid interface (ALI) in vitro cell-exposure technology has been a way to expose cells to all or part of the In vitro, cell models have evolved from simple cell-free biochemical assays to monoculture systems under submerged conditions to the latest and advanced multicellular cultures grown at air-liquid interfaces. Monoculture cell lines commonly used in respiratory toxicity studies include human nasal epithelial cells (HNECs), human bronchial epithelial cell lines (HBECs) (e.g., BEAS-2B, HBEC3KT, 16-HBE, NL-20), human alveolar basal epithelial cells (e.g., A549, NCI-H441, HPAEpiC), human lung adenocarcinoma cell line (CALU-3), and mouse lung fibroblast cell line (L929) [191,[251][252][253][254][255]. Cell models commonly used in other human toxicity studies are the human epidermal carcinoma cell line (A431), normal human keratinocytes (NHKs), the human hepatocellular carcinoma cell line (HepG2), mouse macrophages (RAW264.7), the human lymphoblastoid cell line (MCL-5), the human monocytic cell line (U937, THP-1) and monocyte-derived macrophages (MDM), vascular endothelial cells (VECs), human pulmonary artery endothelial cells (HPAECs), human embryonic kidney cells (HEK 293T), and the pheochromocytoma line (PC12) [208,211,254,[256][257][258][259][260][261].…”