Inflammatory Gene Expression and Oxidative Stress in Human Lung Epithelial Cells Exposed to Combustion‐Produced PM_2.5

Abstract

Objective Air pollution is a lasting global health hazard which has a serious toxicological effects on human health. Long and short term exposure to air pollutants has been associated with respiratory and cardiovascular diseases, skin diseases, and long‐term chronic diseases such as cancer and asthma. Although air pollution is produced by a variety of sources, one of the major air pollutants is fine particulate matter ranging mostly from 2.5 to 10 μm (PM2.5 to PM10). Carbon black, produced by incomplete combustion of heavy petroleum products (soot), comprises a significant portion of PM. Growing evidence suggests that many of the adverse health effects of PM are associated with oxidative stress and inflammatory responses. This work focuses on the identification of oxidative and pro‐inflammatory factors induced by exposure of human lung epithelial cells to combustion‐produced soot in various surface chemistry forms. Methods A lung epithelial cell line (BEAS‐2B) was exposed to lab‐generated R‐250 carbon black (nascent, nitric acid‐treated, and ozone‐treated carbon) for 6hr and 24hr incubation times, at different concentrations (0–100 ug/mL). Cell viability was measured using the MultiTox‐Fluor Multiplex Assay. RNA and protein were extracted from cells to evaluate expression of inflammatory cytokines (IL‐1b, IL‐6) and genes related to the inflammatory response and oxidative stress (SOD2, NFE2L2, HO‐1, and CCL2) by real time polymerase chain reaction (PCR). The expression of protein oxidation products (carbonylated proteins) was measured using ELISA Protein Carbonyl Kit. Cell apoptosis was measured by western blot detection of cleaved Caspase‐3. Results Cell viability was decreased with increasing PM2.5 treatment concentrations at both time points. Nitric acid‐treated carbon resulted in the largest decrease in cell viability. Nitric acid and ozone‐treated particles resulted in increased protein carbonylation when compared to the nascent particles, indicating oxidative stress. Protein carbonylation significantly increased from 6hr to 24hr PM2.5 exposure, particularly for the nitric acid‐treated particles. Gene expression of the inflammatory cytokines IL‐1b, and IL‐6 was upregulated with increasing concentrations of PM2.5 exposure (1.56–50 ug/mL), particularly for nitric acid‐treated particles. An increase in gene expression was observed for oxidative stress markers CCL2, SOD2, NFE2L2 and HO‐1 higher concentrations of PM2.5 exposure (1.56–12.5 ug/mL). Conclusion Exposure of PM2.5 to human lung epithelial cells decrease cell viability and increases inflammatory responses and oxidative stress. PM2.5 surface functional groups strongly influence the extent of toxicity, and future experiments will focus on how functional groups affect cell viability and the observed inflammatory responses of human lung epithelial cells. Support or Funding Information This work was supported by startup funds from The University of North Carolina at Chapel Hill and Penn State College of Medicine (PS), and the Penn State University Human Healt...