Evaluation of oxidative damage and Nrf2 activation by combined pollution exposure in lung epithelial cells

The lungs are one the main organs exposed to environmental pollutants, such as tropospheric ozone (O₃) and particulate matter (PM), which induce lung pathologies through similar mechanisms, resulting in altered redox homeostasis and inflammation. Although numerous studies have investigated the effects of these pollutants in the respiratory tract, there are only a few evidences that have evaluated the combined effects of outdoor stressors, despite the fact that humans are consistently exposed to more pollutants simultaneously. In this study, we wanted to investigate whether exposure to PM and O₃ could have an additive, noxious effect in lung epithelial cells by measuring oxidative damage and the activity of redox-sensitive nuclear factor erythroid 2-related factor 2 (Nrf2) which is a master regulator of cellular antioxidant defenses. First, we measured the cytotoxic effects of O₃ and PM individually and in combination. We observed that both pollutants alone increased LDH release 24 h post-exposure. Interestingly, we did observe via TEM that combined exposure to O₃ and PM resulted in increased cellular penetration of PM particles. Furthermore, we found that levels of 4-hydroxy-nonenal (4HNE), a marker of oxidative damage, significantly increased 24 h post-exposure, in response to the combined pollutants. In addition, we observed increased levels of Nrf2, in response to the combined pollutants vs. either pollutant, although this effect was not followed by the increase in Nrf2-responsive genes expression HO1, SOD1, GPX, or GR nor enzymatic activity. Despite these observations, our study suggests that O₃ exposure facilitate the cellular penetration of the particles leading to an increased oxidative damage, and additive defensive response.