4-hydroxy-2-nonenal-protein adducts and apoptosis in murine lung cells after acute ozone exposure

Ozone is a photochemically generated pollutant that can cause acute pulmonary inflammation and induce cellular injury and may contribute to the development or exacerbation of chronic lung diseases. Despite extensive investigation, the mechanisms of ozone and oxidant-induced cellular injury are still uncertain. Ozone has been reported to cause the formation of aldehydes in biological fluids that could explain many of the cellular effects caused by ozone. One of the most toxic aldehydes formed during oxidant-induced lipid peroxidation is 4-hydroxy-2-nonenal (HNE). HNE reacts primarily with Cys and secondarily with Lys and His amino acids, altering protein function and forming protein adducts that can be detected using specific adducts. In this study, we investigated whether ozone could cause the formation of HNE by assaying for HNE-protein adducts in cells isolated by lung lavage from C3H/HeJ mice exposed to 2.0 and 0.25 ppm ozone for 3 hr. Since oxidative stress and HNE have been shown to cause apoptosis we also examined the lung lavage cells for evidence of apoptosis following ozone exposure. Using a specific polyclonal antibody against HNE-amino acid adducts, two principle HNE-protein adducts were detected by Western analysis in cells obtained after ozone exposure at approximately 86-90 and 32 kDa. In addition to cell necrosis, apoptosis of lung cells was significant 3 hr after ozone exposure as detected using a Cell Death ELISA procedure and confirmed with DNA ladder and morphological analysis. The apoptotic cell injury peaked at 6 hr postexposure and decreased by 24 hr. Taken together, these results demonstrate that HNE is formed in vivo following ozone exposure and HNE appears to form specific protein adducts in lung cells. Furthermore, ozone can cause lung cell injury by an apoptotic mechanism in addition to a necrotic mechanism. Since HNE is toxic to cells and is formed as a result of ozone exposure, it may contribute to the lung cell injury following ozone exposure.