Toxicology study of emissions from a burn pit simulator

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Carbonaceous (i.e., carbon-containing) particulate matter (PM) emitted by the combustion of waste in military burn pits is associated with post-deployment sudden illnesses, such as the Gulf War Illness (GWI). GWI is a syndrome where otherwise healthy soldiers return with debilitating conditions ranging from acute respiratory inflammations to chronic impaired endocrine function. The nature by which it develops is unknown. To shed light over the effect of military burn pit emissions contributing to GWI, we propose to evaluate the combustion envi- ronments within, using a simulator we built, and understand how these are conducive to toxicant formation. To identify underlying mechanisms, our immediate goal is to modify and calibrate the simulator. Then, we will syn- thesize carbonaceous PM representative of burn pit emissions and expose it to mammalian cell lines. The simulator operates on the bench scale and, to the best of our knowledge, is the only one of its kind within the VA system capable of reliably reproducing carbonaceous PM (and gases) emitted by military burn pits. The simulator’s design allows modulation of the feed (fuel, oxygen, and nitrogen) in the respective inlet streams. The ability to modulate these three independent variables offers tight control over the structure and temperature of the flame that precedes PM synthesis. As a result, the physical and chemical properties of the PM can be predicted. We will generate a statistically designed full-factorial experiments for mixtures. In this approach, we will characterize the PM for size, shape, charge, and surface area-to-volume ratio as well as polycyclic aromatic hydrocarbon (PAH) content, and plot these responses as a function of the independent variables. The response surface methodology (RSM) is a statistical tool, used commonly in engineering, which will allow us to explore the relationship between the three independent factors (that regulate the flame structure and temperature) and the properties of the PM using the least number of experiments. While several PAH molecules that exist in the gas-phase are toxic, the known carcinogens are larger hydrophobic molecules adsorbed on or deposited within the PM. To separate the toxicological effect of PM from the gaseous species, we will collect PM on filters. We will sterilize and disperse the PM samples in cell culture media. To ensure the dispersions are stable, we will record their sizes and zeta potentials using dynamic light scattering (DLS) measurements, and sedimen- tation rates. Cell viability will be monitored by electric cell-substrate impedance sensing (ECIS). ECIS measures the total electrical impedance across two gold electrodes at the bottom of the tissue culture plates, so cell viability is determined by the area of the gold electrodes covered by cell attachment. Biocompatibility will be monitored by apoptosis (cell death) and reactive oxygen species (ROS) generation using chemiluminescence measure- ments. ROS measurements are an indication of oxidative stress to the cells; an environment adverse for healthy cell function. Finally, to determine the chemical pathways initiated by the PM at the molecular level, we will perform RNA sequencing. Before the cells show signs of death or detachment from the electrode, we will extract the total RNA. The RNA will then be analyzed in the Genome Technology Access Center (GTAC) at Washington University. Through GTAC we will determine which genes are under- or over-expressed in each cell line com- pared to control cell lines. We anticipate this data will illustrate the effects of carbonaceous PM on the cells. By initially excluding other types of PM encountered by military personnel (e.g., metallic, halogenated, heterocyclic, or mineral) we will be able to pinpoint PAH-initiated pathways that we believe are significantly re- sponsible for GWI. This will serve as a foundation to direct efforts on how studies (using real material) are to be undertaken. After identifying how real carbonaceous PM causes disease, we will explore therapeutic options.