Vijayalakshmi Panduri

Asbestos-induced alveolar epithelial cell apoptosis: Role of mitochondrial dysfunction caused by iron-derived free radicals

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-induced AEC 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 (ΔΨm) 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 ΔΨm after a 4 h exposure period. Further, the ΔΨm 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 ΔΨm and attenuated apoptosis. Finally, overexpression of Bcl-xl, an anti-apoptotic protein that localizes to the mitochondria, prevented asbestos-induced decreases in A549 cell ΔΨm 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.

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) 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 alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 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.

13 A MITOCHONDRIA-TARGETED DNA REPAIR ENZYME, HOGG-1, PREVENTS OXIDANT-INDUCED A549 CELL APOPTOSIS BY PRESERVING MITOCHONDRIAL ACONITASE.

Oxidant-induced alveolar epithelial cell (AEC) apoptosis is implicated in mediating lung injury, fibrosis, and malignant transformation by mechanisms that are not fully established. We previously reported that oxidative stress (eg, amosite asbestos or H2O2) induces mitochondria-regulated AEC apoptosis and that overexpression of a mitochondria-targeted DNA repair enzyme, human 8-oxoguanine-DNA glycosylase 1 (mt-hOgg-1), is protective. In this study, we explore the mechanism underlying the protective effects of mt-hOgg-1 (kind gift of Dr. M. Gillespie). We find that the protective effects are not due to redox activity of mt-hOgg-1 since, compared with empty vector controls, mt-hOgg-1 overexpressing A549 cells did not alter asbestos-induced ROS production as assessed by either DCF fluorescence or an adenovirally expressed redox sensor (ro-GFP) targeted to the mitochondria. As expected, mt-hOgg-1 enhanced DNA repair assessed by an 8-oxoG incision assay (1.7 ± 0.2-fold vs control; p < .05). Both asbestos and H2O2 reduced mitochondria (mt)-aconitase activity and protein expression, but, notably, these effects were completely blocked in mt-hOgg-1 over-expressing A549 cells. Immunoprecipitation studies show that hOgg-1, similar to frataxin, coprecipitates with mt-aconitase. We also found that hOgg-1 mutants that completely lack 8-oxoguanine DNA repair activity (V317, Long AB; kind gift of Dr. V. Bohr) blocked oxidant-induced caspase 9 activation and DNA fragmentation comparable to wild-type hOgg-1. We conclude that mt-hOgg-1 has a dual function as a DNA repair protein as well as a mt-aconitase chaperone. These data suggest a novel role of Mt-hOgg-1 preservation of AEC mitochondria aconitase in the pathogenesis of oxidant-induced lung toxicity. Funding: VA Merit Award (DK), NIH-HL67835-01 (GRSB).

40 OVEREXPRESSION OF ANTIOXIDANT ENZYMES IN LUNG EPITHELIAL CELLS PREVENTS DAMAGE FROM AIRBORNE PARTICULATE MATTER-INDUCED OXIDANT INJURY.

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 1 Elevated Levels of ROS in Airborne Particulate Matter Increases A549 Cell Apoptosis Funded by Veterans Affairs Merit Review (D.W.K.); NIH-K08 (GSB).

40 p53 MEDIATES PARTICULATE MATTER-INDUCED A549 CELL MITOCHONDRIA-DEPENDENT APOPTOSIS

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