(416d) Surface Chemistry Toxicity Parameters Associated with Combustion Produced PM2.5 by in Vitro Assays

Introduction: The International Agency for Research on Cancer (IARC) has labeled diesel exhaust as carcinogenic within class 1. While the mechanism(s) by which soot causes the adverse health effects are not known, a great deal of these harmful effects relate to its ability to cause oxidative stress. Thus, oxidative potential, expressed through reactive oxidative species concentration, can be used as a good estimate for its reactivity and toxicity. Accordingly, physical structure and surface chemistry become surrogate measures of its oxidative potential as together they determine the redox properties and polar/acidic character. Based on this premise, we are testing the role and impact of soot structure and surface chemistry upon interaction with bronchial and alveolar epithelial cells. The proposed project’s goal is the identification of toxicity, oxidative, and pro-inflammatory factors in combustion produced soot arising from using alternative fuels, by studying the particles directly instead of studying compounds adsorbed on (and removed from) the soot particles. Model soots with tailored surface chemistry, and specific particle physical structures are being tested for toxicity, oxidative stress markers, effects of lung inflammation and and signaling pathways using cell culture bioassays, as well as protein carbonylation and DNA damage marker assessment.

Methods: An epithelial cell line (BEAS-2B) was incubated with lab-generated carbon (nascent, nitric acid-treated, and ozone-treated carbon) for 6 hr. and 24 hr. incubation times, at different concentrations (0-100 μg/mL). After incubation, we measured cell viability using the MultiTox-Fluor Multiplex Assay. In addition, we extracted RNA and protein from cells to evaluate expression of inflammatory cytokines (IL-1b, IL-6, IL-8) and genes related to the inflammatory response and oxidative stress (TLR4, CCL2, MMP1, and NRF2).

Results: Treatment of carbon particles with nitric acid and ozone resulted in differential oxygen and carboxylic acid content in the PM_2.5 surface. In BEAS-2B cells, an inverse relationship of cell viability and PM_2.5 treatment concentration was observed at both time points assessed. Of the three PM_2.5 preparations, BEAS-2B cells exposed to nitric acid-treated carbon resulted in the largest decrease (89% and 96%) of cell viability after 6 hr. and 24 hr., respectively, indicating that both carboxylic acid and oxygen content of the particle surface strongly contribute to PM_2.5 toxicity. Additional results showed an increase in gene expression of the inflammatory cytokines IL-1b, and IL-6, and the oxidative stress markers TLR4 and NRF2 at higher concentrations of PM_2.5 exposure (25-100 μg/mL).

Conclusion: In summary, exposure of different types of particulate matter to lung epithelial cells increases inflammatory responses, with different effects associated with soot components such as particle surface chemistry. Future experiments will determine how functional groups in the PM_2.5 surface chemistry affect the activation of the observed inflammatory response, and decrease in cell viability of lung epithelial cells.