Pramod Narula

Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells

Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO + hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia + NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO + hyperoxia. These results indicate that combined NO + hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO+hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia+ NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO+hyperoxia. These results indicate that combined NO+hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.

Inhibition of Nuclear Factor-κB (NF-κB) Activation by Nitric Oxide May Be Responsible for Synergistic Cytotoxicity from Combined Nitric Oxide (NO) and Hyperoxia (HYP)

Inhibition of Nuclear Factor-κB (NF-κB) Activation by Nitric Oxide May Be Responsible for Synergistic Cytotoxicity from Combined Nitric Oxide (NO) and Hyperoxia (HYP)

Lung Apoptosis In Response To Therapy With Nitric Oxide (No) And Hyperoxia(HYP) • 1733

Previous in vivo studies indicate that prolonged exposure to NO+HYP causes significant lung injury in newborn piglets. Peroxynitrite-mediated oxidant damage has been implicated in the pathogenesis of this injury. However, little is known about the in vivo mechanisms of cell injury or the mode of subsequent cell death. Our previous in vitro studies in human alveolar epithelial (A549) cells demonstrated that NO+HYP directly induced acute and synergistic cytotoxicity, but the mode of cell death was found to be nonapoptotic. To determine if NO+HYP results in apoptosis in vivo, we studied 1-3 day old piglets, mechanically ventilated for 48h with 100% O2, 100 PPM NO + 21% O2, 5 PPM NO + 100% O2, 100 PPM NO + 90% O2 or unventilated control animals(n=2/group). Animals were sacrificed, lungs removed and instillation-fixed in buffered formalin and embedded in paraffin. TUNEL (TdT dUTP Nick End Labeling) assays were performed on sections of lung tissue (right lower lobe) and the number of apoptotic (TUNEL+) nuclei/field were quantified. Unventilated animals had 3.5±0.5 (mean ± SE) apoptotic nuclei/field while animals ventilated with 100 PPM NO + 21% O2 had 7.0±2, 100% O2 had 27±3, 5 PPM NO + 100% O2 had 31±4, and 100 PPM NO+ 90% O2 had 68±9 (p< 0.05, ANOVA). Preliminary data suggest that combined therapy with NO+HYP results in increased apoptosis in the lung which may correlate with previously documented pulmonary injury.

Hyperoxia (HYP) Resistant HeLa 80 Cells Are Sensitive To Combined Cytotoxicity From Nitric Oxide (NO) and Hyperoxia |[bull]| 187

Nitric oxide (NO) is a potent vasodilator being used clinically to treat pulmonary hypertension in infants and children. We have previously demonstrated that NO and HYP have synergistic cytotoxic effects on both cultured human alveolar epithelial cells (A549) and human microvascular endothelial lung cells. To better understand the basis of this cytotoxicity, we studied the effects of combined NO and HYP on HeLa 80 cells, a substrain of HeLa cells capable of growing normally in 80% oxygen. Cultured HeLa 80 cells were grown under 4 separate experimental conditions:1) room air (RA), 2) 80% oxygen, 3) NO donor (0.5mM NONOate - ethanolamine, 2,2-hydroxynitrosohydrazono bis-) and RA, 4) combined 0.5mM NONOate and 80% oxygen. Live cells were counted daily (using Trypan blue) in each of the above groups for six consecutive days, and culture media and gasses were replenished each day. Cells continued to grow normally in RA or 80% oxygen. Cells grew more slowly with the NO donor and RA. In sharp contrast, cells treated with the combination of the NO donor and 80% oxygen began to die on day 3, with approximately 75% of the total cells having died by day 6. To study the mode of cell death, cells were grown on cover slips and divided into the 4 experimental conditions as described above. The cells were then stained with DAPI (4,6-diamidine-2-phenylindole -dihydrochloride), a DNA binding dye and viewed under a fluorescent microscope. Significant numbers of cells were found to be apoptotic when treated with the NO donor + 80% oxygen. These data indicate that HeLa 80 cells, which are normally resistant to the effects of HYP, die rapidly when exposed to combined NO and HYP. This suggests that NO and HYP are synergistically cytotoxic and the most significant mode of cell death is apoptosis. This is different from typical hyperoxia-sensitive cells, which do not undergo apoptosis in combined NO + HYP.

High levels of triglycerides in pleural fluid in parapneumonic effusion and empyema may indicate severe disease. 1942

High levels of triglycerides in pleural fluid in parapneumonic effusion and empyema may indicate severe disease. 1942

Early use of low dose intrapleural urokinase in pediatric parapneumonic pleural effusion and empyema. 1941

Early use of low dose intrapleural urokinase in pediatric parapneumonic pleural effusion and empyema. 1941

PROTEIN NITRATION IN THE LUNG IN RESPONSE TO NITRIC OXIDE (NO) AND HYPEROXIA(HYP). † 1579

Previous in vivo studies have indicated that prolonged exposure to NO + HYP(but not NO alone) causes significant lung injury in neonatal piglets. Peroxynitrate (ONOO), the product of NO and superoxide, can cause protein nitration and oxidant tissue injury and is thought to be a causative agent in the injury process. Nitrotyrosine (NT), a stable byproduct of ONOO, has been used as a specific indicator of ONOO formation. To determine whether NT formation occurs in vitro, cultured human alveolar epithelial cells (A549) were grown in room air (RA), 95% oxygen (HYP), 2mM SNAP (an NO donor) or HYP + SNAP. Western blot analysis, using a polyclonal antibody against NT, revealed NT-containing proteins at 25 and 35 kD from cells exposed to SNAP or SNAP + HYP. In vivo studies were then performed with 1-3 d old piglets, mechanically ventilated with RA (n=2), 100% oxygen (n=3), 100 PPM NO (n=4) or 100 PPM NO and 90% oxygen (n=4) for 48 h. Animals were sacrificed, the lungs removed and Western blot analysis performed for NT. Results demonstrated one major 35 kD protein in lung tissue from animals exposed to NO or NO + 90% oxygen, but not in animals exposed to RA or 100% oxygen. Control blots with preabsorbed antibody showed no specific staining. These results indicate that while NT may be a specific marker of ONOO formation, the presence of NT alone may not be a specific indicator of lung tissue injury. In addition, these data suggest that sufficient amounts of superoxide may be present both in tissue culture and in vivo to yield ONOO in the absence of an additional superoxide donor. Further studies are necessary to more clearly elucidate the role of protein nitration in lung injury.

PULMONARY ENDOTHELIAL CELLS ARE MORE RESISTANT THAN PULMONARY EPITHELIAL CELLS TO SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE (NO) AND HYPEROXIA (HYP).† 1553

PULMONARY ENDOTHELIAL CELLS ARE MORE RESISTANT THAN PULMONARY EPITHELIAL CELLS TO SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE (NO) AND HYPEROXIA (HYP).† 1553

SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE AND HYPEROXIA. |[dagger]| 2042

Nitric oxide (NO) has been discovered to be a potent vasodilator. Endogenous NO is produced by many cell types and is important in regulating basal vasomotor tone. Exogenous NO has recently been used very successfully to treat pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen therapy. Unfortunately, there are few reports on the combined toxicity of NO and hyperoxia, either of which can be toxic alone. Together, the potential for the formation of peroxynitrite(ONOO˙) - an extremely reactive and toxic free radical - is increased. To directly test this potential toxicity, cultured human alveolar epithelial(A549) cells were grown in room air (control), hyperoxia (95% O2), 2 mM SNAP (S-nitroso-N-acetyl penicillamine - an NO donor) and hyperoxia + SNAP. Live cells were counted in each of the groups above daily for the next six days, and media and gasses were refreshed each day. Cells began to die in hyperoxia after 4 to 5 d, but cell counts were relatively unchanged in NO. However, cells exposed to both NO and hyperoxia began to die on day 2, and died rapidly thereafter. This cytotoxic effect was clearly synergistic, the death rate far exceeding any additive effect. Some cells were grown on cover slips for later immunocytochemistry for nitrotyrosine, which is a stable byproduct of peroxynitrite (which is too reactive to detect directly). Immunoreactive nitrotyrosine was most abundant only in cells exposed to NO + hyperoxia. Western blots indicate that a variety of proteins become nitrated under various conditions. However, a unique band appeared on blots only after culture in NO + hyperoxia. These experiments indicate that NO and hyperoxia have a synergistic cytotoxic effect on alveolar cells in culture, which is probably mediated by production of peroxynitrite.(Funded by grants from the NIH-NHLBI, ALA and Winthrop-University Hospital)