Understanding PM2.5‐Induced Oxidative Stress In Alveolar Macrophages
Authors: Raval Kosha Jyotin , Kaur Mandeep , Waterston Anthony , Olivas Micah , Flores David , Hasson Alam , Dejean Laurent 
Published: June 2021

When oxidative stress exceeds normal levels, the antioxidant system of any eukaryotic organism can become overwhelmed. This may lead to various adverse health effects, including cardiovascular and pulmonary diseases. The risk for these conditions is heightened by increased exposure to particulate matter 2.5 (PM2.5), composed of transition metals and organic compounds 2.5μm or less in diameter. These particles reach the alveolar macrophages (AMs) in the respiratory tract, where they can induce an inflammatory response by producing excessive reactive oxygen species (ROS). During 2012 and 2013, samples of ambient PM2.5 samples were collected during periods of peak air pollution from Fresno and Claremont, CA. These samples were tested for their potential to induce a ROS response in the rat NR8383 AM cell line using the intracellular ROS‐sensitive fluorescent probe 2′‐7′‐Dichlorodihydrofluorescein diacetate. Interestingly, the specific cellular ROS response was dependent on the sample origin; strongly suggesting that the chemical composition of PM2.5 is a major determinant of its effect on AM ROS metabolism. The PM2.5 samples were characterized for their chemical content using chromatography and mass spectrometry based techniques. Two types of quinones (1,2‐Naphtaquinone, and 1,4‐Naphtaquinone) and one transition metal (Cu2+) were associated with samples that had a high capacity to create ROS in NR8383. Currently, each of the above components' potential to induce a cellular ROS response is being investigated using mock samples with concentration ranges similar to those of the PM2.5 samples. While establishing a cytotoxic link between PM2.5 and ROS, we also seek to understand the underlying biological mechanisms through which PM2.5 interacts with AM cells to produce excessive ROS. Previous studies have suggested the upregulation of heme oxygenase‐1 (HO‐1) during oxidative stress plays an essential role in intracellular ROS regulation. Our study consists of optimizing a gene knockdown protocol for HO‐1 in NR8383 to investigate the potential involvement of HO‐1 in a PM2.5‐induced ROS response. Having established an optimal protein concentration of 50μg to see distinct HO‐1 bands in western blots, a range of siRNA concentrations are being used in conjunction with our positive control, zymosan, to understand their cytotoxicity effects. Zymosan, extracted from the cell wall of saccharomyces cerevisiae, is a known inflammatory agent which stimulates ROS production in NR8383. Repeated trials of western blotting confirm that 4 hour zymosan treatments increase the HO‐1 protein levels in NR8383. The HO‐1 knockdown will be confirmed through quantitative reverse transcription polymerase chain reaction (qrt‐PCR) and western blotting. Cells with the HO‐1 knockdown will then be subjected to a fluorescence‐based microplate assay to monitor their PM2.5‐induced ROS response. We hypothesize enhanced ROS production in PM2.5 treated HO1‐knockdown cells due to the lack of the protective enzyme, HO‐1. Understanding the specific chemical components which have proved noxious to NR8383 and the biological mechanisms through which PM2.5 is able to induce ROS will help us understand exactly how pollution is pernicious and advocate for global reduction of PM2.5.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.