8-oxoguanine DNA glycosylase (Oggl) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Oggl is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Oggl isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Oggl functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOggl (mt-hOggl) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOggl mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOggl in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase and apoptosis suggesting that the protective effects of mt-hOgg 1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOggl co-precipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOggl silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOggl acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. We previously showed that iron-catalyzed ROS in part mediate asbestos-inducedAEC DNA damage and apoptosis. Mitochondria have a critical role in regulating apoptosis after exposure to agents causing DNA damage but their role in regulating asbestos-induced apoptosis is unknown. To determine whether asbestos causes AEC mitochondrial dysfunction, we exposed A549 cells to amosite asbestos and assessed mitochondrial membrane potential changes (delta(psi)m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. We show that amosite asbestos, but not an inert particulate, titanium dioxide, reduces delta(psi)m after a 4 h exposure period. Further, the delta(psi)m after 4 h was inversely proportional to the levels of apoptosis noted at 24 h as assessed by nuclear morphology as well as by DNA nucleosome formation. A role for iron-derived ROS was suggested by the finding that phytic acid, an iron chelator, blocked asbestos-induced reductions in A549 cell delta(psi)m and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell delta(psi)m after 4 h and diminished apoptosis. We conclude that asbestos alters AEC mitochondrial function in part by generating iron-derived ROS, which in turn can result in apoptosis. This suggests that the mitochondrial death pathway is important in regulating pulmonary toxicity from asbestos.

Airborne particulate matter (PM) increases morbidity and mortality resulting from cardiopulmonary diseases including lung cancer and chronic obstructive lung disease. PM induces alveolar epithelial cell (AEC) DNA damage and apoptosis in part by generating iron-derived reactive oxygen species (ROS) and activating the mitochondria-regulated death pathway. Since p53 is critically involved in the cellular DNA damage response that can result in mitochondrial dysfunction, we determined whether transcriptional activation of p53 is required for PM-induced A549 cell apoptosis. We found that pifithrin (30 μM for 24 h), an inhibitor of p53 transcriptional activation, blocked PM (50 μg/cm2) induced caspase 9 activation and apoptosis, as assessed by annexin V staining and DNA fragmentation (Table). Similar findings were noted in A549 cells overexpressing the E6 oncoprotein, which also blocks p53 transcription (data not shown). Finally, we found that PM caused the translocation of BAX, a pro-apoptotic Bcl-2 family member, from the cytosol to the mitochondrial membrane that was inhibited in cells treated with pifithrin. Thus, p53 mediates PM-induced AEC mitochondrial-dependent apoptosis. These data suggest an important role for p53 in the pathogenesis of PM-induced pulmonary toxicity in part by causing mitochondrial dependent apoptosis. Funded by Veterans Affairs Merit Review (D.W.K.); NIH-K08 (G.S.B.).Table 1.Pifithrin Blocks PM‐Induced Caspase 9 Activation and Apoptosis

Elevated levels of air pollution particles are associated with increased morbidity and mortality from acute and chronic cardiopulmonary injury. One mechanism underlying these effects involves oxidative damage to lung epithelial cells. We previously showed that Düsseldorf particulate matter (DPM) causes alveolar epithelial cell DNA damage and apoptosis by a mitochondria-regulated death pathway. In this work, we used several different types of well-characterized particulates to determine whether mitochondria-derived reactive oxygen species (ROS) are the primary cause of apoptosis. Washington particulate matter (WPM), residual oil fly ash (ROFA), and DPM each increased ROS production (ASSAY HERE) and apoptosis (DNA fragmentation) as compared to inert particulates such as desert dust (DD) and Mount St Helen volcanic dust (MSH) (Table 1). Notably, WPM, ROFA, and DPM did not induce ROS production or apoptosis in;gr0-A549 cells, which are incapable of mitochondrial ROS production. We also found that overexpression of MnSOD or CuZnSOD using adenoviral expression vectors blocks DPM-induced A549 cell ROS production and apoptosis as compared to null/sham adenoviral controls. We conclude that a diverse group of toxic airborne particulates, unlike inert particulates, induce mitochondria-derived ROS production and lung epithelial cell apoptosis. We propose that strategies aimed at reducing mitochondrial-derived ROS levels will protect the lung epithelium exposed to airborne particulate matter.TABLE 1Elevated Levels of ROS in Airborne Particulate Matter Increases A549 Cell Apoptosis Funded by Veterans Affairs Merit Review (D.W.K.); NIH-K08 (GSB).