Proc. Nati. Acad. Sci. USA Vol. 91, pp. 12248-12252, December 1994 Physiology Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs PAUL R. GARDNER*, DEE-DEE H. NGUYEN, AND CARL W. WHITE Department of Pediatrics, Division of Pulmonary Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206 Communicated by Irwin Fridovich, August 29, 1994 ABSTRACT The effect of hyperoxia on activity of the su- damage in lung tissue (9, 10) and in cell culture models (14). peroxide-sensitive citric acid cycle enzyme aconitase was mea- In rats exposed to a Po2 of 760 mmHg for 24 hr, lung sured in cultured human epithelial-like A549 cells and in rat mitochondrial respiratory capacity and citric acid cycle ac- lungs. Rapid and progressive loss of >80% of the aconitase tivity are impaired (9, 10). Potential enzymatic sites for the activity in A549 cells was seen during a 24-hr exposure to a Po2 poisoning action of hyperoxia have been described. The of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial mitochondrial enzymes NADH dehydrogenase (14), succi- respiratory capacity correlated with loss of aconitase activity in nate dehydrogenase (14, 15), and a-ketoglutarate dehydro- A549 cells exposed tohyperoxia, and thiseffectcould be mimicked genase (14) have various sensitivities to hyperoxic exposure. by fluoroacetate (or fluorocitrate), a metabolic poison of aconi- However, evidence is lacking for a loss of dehydrogenase tase. Exposure ofrats to an atmospheric Po2 of760 mmHg or 635 activities during the early impairment of rat lung oxidative mmHg for 24 hr caused respective 73% and 61% decreases in metabolism by normobaric hyperoxia. total lung aconitase activity. We propose that early inactivation of The citric acid cycle enzyme aconitase is a member of a aconitase and inhibition ofthe energy-producing and biosynthetic growing family of 02--sensitive [4Fe-4S]-containing (de)- reactions ofthe citric acid cycle contribute to the sequelae oflung hydratases that have been implicated to be important sites of damage and edema seen during exposure to hyperoxia. 02 -/02toxicity (16-26). The activity ofaconitase is sensitive to inactivation by 02- (16, 22) and is modulated by changes Oxygenation of tissues must be carefully controlled to avoid in 02- levels in bacteria and mammalian cells (23, 24, 26). the deleterious effects of hypoxia or hyperoxia. Countering Thus, the ability of elevated levels of 02 to exacerbate the imbalance of hypoxia with oxygen therapy, as is fre- mitochondrial production of 02 (27, 28) and to impair citric quently done, necessarily means exposing the lung, and acid cycle activity and respiratory capacity of lung cell possibly other tissues as well, to a greater than normal Po2 mitochondria (9, 10) led us to investigate the sensitivity ofthe (1-4). Therapeutic exposures of individuals to elevated Po2 mitochondrial aconitase in cultured human epithelial-like range from slightly above normoxia (>160 mmHg 02; 1 lung cells (A549 cells) and in the lungs of rats exposed to mmHg = 133 Pa) for extended periods to brief exposures to hyperoxia. We now report that aconitase is a sensitive target hyperbaric 02 (usually <2280 mmHg 02), as in treatment for ofhyperoxic damage in vitro and in vivo and demonstrate that carbon monoxide poisoning. Under both situations, hyper- inhibition of aconitase with fluoroacetate (or fluorocitrate) oxygenation damages O2-sensitive biomolecules and elicits (29-34) can mimick the inhibitory effects of hyperoxia on increases in the level of reactive oxygen intermediates, A549 cell growth and respiratory capacity.t including superoxide radical (O2-), hydrogen peroxide (H202), alkyl peroxides, hydroxyl radical, and alkoxyl radi- cals. These reactive oxygen intermediates can, and do, MATERIALS AND METHODS overwhelm the natural antioxidant defenses and repair sys- tems (5, 6). Dioxygen and reactive oxygen intermediates Cells and Reagents. The human epithelial-like lung carci- ultimately cause reversible and irreversible pathologies. noma cell line A549 (CCL 185) was obtained from the Lung function is highly susceptible to hyperoxic damage American Type Culture Collection (Rockville, MD). Carbo- (1, 4, 7-13). Morphological and biochemical alterations in the nyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), fe- lung ultimately cause morbidity resulting from decreased tal calf serum, nitro blue tetrazolium, neutral red, barium blood oxygenation (4). Edema of the interstitial space and (±)-fluorocitrate, sodium fluoroacetate, lactic acid, lactate increased permeability of pulmonary microvasculature are dehydrogenase, and porcine heart isocitrate dehydrogenase early signs of pathology in the lungs of rats exposed to lethal were from Sigma. Sodium (+)-fluorocitrate was prepared by levels of dioxygen (Po2 of 760 mmHg). This initial damage is titrating barium fluorocitrate with a slight excess of sulfuric followed by and amplified by the activation and infiltration of acid, centrifuging to remove BaSO4, and then neutralizing the platelets, macrophages, and neutrophils and precedes the supernatant with NaOH. Recombinant human tumor necro- death of the animal or individual (3, 12). After sublethal sis factor a (TNF-a) (6 x 107 units per mg) was supplied by exposures of rats to hyperoxia (Po2 of 456-650 mmHg), Genentech, Inc. (San Francisco, CA), and human recombi- damage is marked by fibrosis, alveolar type II cell prolifer- nant interleukin la was provided by P. LoMedico of Hoff- ation (11, 12), and mitochondrial structural deformities (7, 8, man-La Roche. F12K growth medium, trypsin-EDTA, and 11, 12). The contribution of individual reactive oxygen inter- penicillin-streptomycin were obtained from GIBCO-BRL. mediates and dioxygen to the morphological and biochemical Heparin was obtained from Abbott. alterations in the lungs, however, remains only vaguely defined (5, 6, 12). Abbreviations: MnSOD, manganese-containing superoxide dismu- Mitochondrial respiration and energy production have tase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; been identified as sensitive and critical sites of hyperoxic TNF, tumor necrosis factor. *To whom reprint requests should be addressed at: National Jewish Center for Immunology and Respiratory Medicine, D301, 1400 The publication costs of this article were defrayed in part by page charge Jackson Street, Denver, CO 80206. payment. This article must therefore be hereby marked "advertisement" tA preliminary report of part of this work has been presented in in accordance with 18 U.S.C. §1734 solely to indicate this fact. abstract form (25). 12248 Downloaded by guest on September 30, 2021 Physiology: Gardner et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12249 Cell Growth and Extract Preparation. Cells were grown in Protein concentration was assayed by the method of Brad- 100-mm Falcon tissue culture dishes in 10 ml ofF12K growth ford (38) using Coomassie brilliant blue staining reagent medium containing 10% fetal calf serum, penicillin (100 units (Bio-Rad) and bovine serum albumin, fraction V (Calbio- per ml), and streptomycin (100 pg per ml) incubated at 370C chem) as the standard. under a humidified atmosphere of air containing 5% Co2. Respiration Measurements. A549 cell cultures were washed Exposures to hyperoxia at ambient Denver atmospheric with Dulbecco's PBS and gently trypsinized. Cells were pressure (635 mmHg) were in a humidified air-tight plastic washed with medium and pelleted at 1000 x g for 10 min. incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) Oxygen consumption by 3-7 x 106 cells per ml (cells har- gassed with 95% 02/5% CO2 and incubated at 37TC. Equil- vested from two 100-mm dishes) was measured by using a ibration of 02 was initially achieved by gentle agitation ofthe Gilson oxygraph with a Clark electrode. Assays were done in chambers and dishes. A549 cultures were routinely passaged a total volume of 2.0 ml of the conditioned cell growth by trypsinization and were subcultured in a 1:5 split ratio. medium incubated at 370C, and respiration rates were mea- Cell growth was assessed by trypsinization and counting with sured with or without the uncoupler FCCP (4-6 ,uM). A a hemacytometer, or viability (and growth) were assessed by medium 02 saturation value of 167 pM, which was estimated the neutral red dye-retention assay (35). To prepare cell from the 02 saturation value at sea level (39) and corrected extracts, the growth medium was aspirated from the adherent for a Denver atmospheric pressure of 635 mmHg, was used A549 cell cultures, and the cells (=5 x 106 per dish) were for rate calculations. chilled and washed with 5 ml of ice-cold Dulbecco's phos- Exposure of Rats to Oxygen and Preparation of Lung phate-buffered saline (PBS) (1.1 mM KH2PO4/8.1 mM Extracts. Two-mo-old male Sprague-Dawley rats weighing Na2HPO4/138 mM NaCl/2.7 mM KCI/0.5 mM MgCl2/0.9 -300-315 g (Harlan Laboratories, Indianapolis, IN) were mM CaCl2). The wash was removed by aspiration, and the exposed to air or hyperoxia (>99%6 02, 10 liters/min; 635 or cells were scraped into S ml ofice-cold PBS. Cells were then 760 mmHg) for 24 hr in Plexiglas chambers. All other centrifuged at 1500 x g with rapid braking after 20 s. The conditions of exposure were as described (40). After expo- supernatant was aspirated, and the cell pellet was disrupted sure, rats were injected with sodium pentobarbital and given in 100 01 ofbuffer containing 50 mM Tris Cl (pH 7.4), 0.6mM air or >99%6 02 (635 mmHg). Tracheostomies were done, and MnCl2, 20 ,uM (+)-fluorocitrate by applying 10 1-s bursts with rats were ventilated with room air or pure oxygen with a a microtip sonic oscillator (Fisher Scientific). The lysate was respirator (Harvard Apparatus model 6700). After midsternal then transferred to a chilled 1.5-ml Eppendorf tube and thoracotomy, heparin (150 units) was injected into the right immediately placed in a dry ice/ethanol bath and stored at ventricular outflow tract, and a cannula was placed in the -70°C.