Cheng, W; Currier, J; Zucker, R; Bromberg, P; Silbajoris, R; Hofer, TP; Samet, JM
RATIONALE. Exposure to ambient particulate matter (PM) has been associated with adverse health effects, including inflammatory responses. Diesel exhaust particles (DEP) are a ubiquitous contributor to the fine and ultrafine PM burden in ambient air. Toxicological studies of DEP have shown oxidant-dependent inflammatory effects that are associated with its organic content. However, the link between specific organic species in DEP, oxidant stress and inflammatory gene expression has not been established. Here, we have investigated the oxidative and inflammatory effects of 1,2-Naphthoquinone (1,2-NQ), a redox-active organic component of DEP that has been found previously to activate pro-inflammatory signaling in human lung cells. We employed live cell imaging using genetically encoded fluorescent indicators to follow real time changes in cytosolic redox status and mitochondrial H2O2 production.
METHODS. BEAS-2B cells were exposed to 1–100 uM 1,2-NQ and interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HOX-1) transcriptional activities were monitored with the use of lentiviral promoter reporter constructs. Redox status and intracellular concentrations of H2O2 were monitored in live cells by Nikon C1Si confocal imaging using the genetically-encoded GFP-based indicators roGFP2 and HyPer, respectively.
RESULTS. The expression of IL-8, COX-2, and HOX-1 was markedly induced by exposure to 1,2-NQ in a dose-dependent manner. Concomitantly, imaging analysis of BEAS cells expressing roGFP2 showed a rapid elevation in cytoplasmic redox potential with exposure to 10 uM 1,2-NQ. Over the same time course, treatment with 1,2-NQ induced a dose-dependent elevation in mitochondrial H2O2 production, as measured in cells transfected with a mitochondrial targeted version of HyPer. The H2O2 effect was observed to lag behind the change in redox status in BEAS cells exposed to 1,2-NQ. The temporal relationship between the oxidative effect and the production of mitochondrial H2O2 suggests that reactive oxygen species generation in the mitochondria occurs as a consequence and not a cause of the oxidative damage induced by 1,2-NQ.
CONCLUSIONS. This is the first mechanistic study examining oxidative and inflammatory effects of exposure to an organic component of PM. These findings demonstrate the utility of integrating live cell imaging approaches to mechanistic studies of the toxicity of environmental air contaminants in human lung cells. This abstract of a proposed presentation does not necessarily reflect EPA policy.
