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. The entire procedure was routinely done in <2 min. main pulmonary artery. Lungs were perfused free of blood Fluorocitrate and MnCl2 were included in the lysis buffer to with PBS for 15-30 s. The left lung was removed and limit the inactivation of aconitase by 02J-/O2 during extract homogenized at 40C for 15 s in 2.5 ml of buffer containing 50 preparation and storage (22). No loss of aconitase activity mM Tris-Cl (pH 7.4), 0.6 mM MnCl2, and 2 mM sodium was detected in cell lysates after 2 weeks ofstorage at -70°C. citrate using a Virtishear (VirTis) set at maximum power. Enzyme, Protein, and Lactate Assays. Freshly thawed cell Two 200-/l samples were rapidly frozen on dry ice/ethanol extracts were clarified by centrifugation for 20 s at 14,000 x and stored at -700C. Liver was excised, homogenized, and g, and the supernatants were promptly assayed for aconitase stored by a similar procedure. activity by following the linear absorbance change at 340 nm at 25°C in a 1.0-ml reaction mixture containing 50 mM Tris Cl (pH 7.4), 5 mM sodium citrate, 0.6 mM MnCl2, 0.2 mM RESULTS NADP+, 1-2 units of isocitrate dehydrogenase, and 10-100 Hyperoxia Inactivates Aconitase and Impairs Respiration mg of extract protein. Freshly thawed tissue homogenates and Growth of A549 Cells. Aconitase activity in A549 cells were briefly sonicated for 10 s, clarified by centrifugation, decreased by 83% and 91% of air control activities after 24 and assayed. One milliunit of aconitase activity was defined and 48 hr of exposure to hyperoxia, respectively (PO2 = 600 as the amount catalyzing the formation of 1 nmol ofisocitrate mmHg) (Fig. 1A). A 22% loss of aconitase activity was per min. The amount of the competitive inhibitor (±)- detected after only 3 hr, and aconitase activity was progres- fluorocitrate present from extract carryover did not affect the sively lost over 12 hr (Fig. 1B). Measurements of whole-cell assay. An initial lag in NADPH formation is seen in assays of respiration rates after 24 hr of exposure to hyperoxia (Fig. 2) low aconitase activity, which presumably reflects the delayed revealed impairments of basal and uncoupled respiration accumulation of cis-aconitate (36). Thus, linear rates during rates of 60%o and 71%, respectively, relative to those of the the latter half of a 60-min assay were used for the determi- 24-hr air-exposed controls. It should be noted that an in- nations of aconitase activity. Electrophoretic separation and crease in basal respiration rates was observed in air control activity staining of aconitase isozymes were done with minor cells during the 24-hr incubation. Nevertheless, hyperoxia modifications to the procedure of Koen and Goodman (37). decreased the respiratory capacity as measured with the a-Ketoglutarate dehydrogenase activity was determined uncoupler FCCP. We examined the potential role of aconi- spectrophotometrically by following the initial linear increase tase inactivation in the pathogenesis of 02 poisoning in in absorbance at 340 nm in a 1.0-ml assay containing 50 ,umol cultured A549 cells by comparing the effects of hyperoxia of Tris-Cl (pH 7.0), 0.2 ,umol of NAD+, 1.0 ,umol of a-keto- with those of fluoroacetate or fluorocitrate on cell respira- glutarate, 50 nmol of coenzyme A, 0.5 ,umol ofNaCN, and 10 tion. Fluoroacetate is metabolized by cells to form fluoro- nmol of cocarboxylase incubated at 25°C. Lactic acid was citrate, a potent and specific competitive and reversible determined in a 1.0-ml aliquot of cell culture medium after inhibitor of aconitase (K, = 10--10-8 M), and an extremely first deproteinizing the sample with 73 Al of 60%o (wt/wt) toxic poison (22, 29-34). Exposure of cells to 10 mM sodium perchloric acid by setting on ice for 15 min and then centri- fluoroacetate or to 0.1 mM sodium (+)-fluorocitrate for 24 hr fuging at 14,000 x g for 5 min. The supernatant was neutral- caused similar impairments of the mitochondrial respiratory ized with KOH, clarified again to remove potassium perchlo- capacity of cultured A549 cells (Fig. 2). rate, and stored at -70°C. Sample (100 ul) lactic acid was Exposure ofcultures ofhuman epithelial-like A549 cells to determined spectrophotometrically at 340 nm in a 1.0-mltotal hyperoxia (P02 = 600 mmHg) caused growth arrest after a reaction volume containing 10 units of lactate dehydroge- 24-hr exposure (Fig. 3A). Morphological changes (cell swell- nase, 80 Amol ofglycine buffer (pH 10.0), and 2 mg ofNADI ing) also were seen in growth-arrested cells, and death, as after a 60-min incubation at 250C with a lactic acid standard. grossly characterized by cell lysis and release of cells from Downloaded by guest on September 30, 2021 12250 Physiology: Gardner et al. Proc. Natl. Acad. Sci. USA 91 (1994) A B a-Ketoglutarate dehydrogenase was measured because it was shown to be more sensitive to inactivation 12Air- previously E 8 * Hyperoxia E during hyperoxic exposure of cultured CHO cells than either the mitochondrial NADH dehydrogenase or succinate dehy- E ~ ~ drogenase (14). Lactic acid was measured because its pro- duction has been shown to correlate with the impairment of oxidative phosphorylation and enhanced glycolysis elicited by hyperoxia in vitro and in vivo (9, 14, 41). After 24 hr of C~~~~~ exposure of A549 cell cultures to hyperoxia (Po2 = 600 mmHg), there were no significant differences either in a-ke- 0 ~00 toglutarate dehydrogenase activity (4.5 ± 0.1 vs. 4.1 ± 0.5 milliunits per mg of extract protein for air vs. hyperoxia; n = 0 24 48 0 3 612 2 ± SD) or in the rate of medium lactic acid accumulation (2.6 Hours Hours ± 0.1 vs. 2.8 ± 0.1 mM per 24 hr for air vs. hyperoxia; n = 2 ± SD). It is noteworthy that alterations in these two FIG. 1. Inactivation of aconitase by hyperoxia in cultured human markers were reported for cultured cells or tissues (9, 14, 41) A549 cells. Cells were grown to =70% confluency in 100-mm tissue culture dishes in 10 ml of F12K medium/10% fetal bovine serum/ exposed longer or to a higher Po2 than those that we have antibiotics at 370C under a humidified air/5% CO2. Cell cultures examined. Importantly, these results suggest that aconitase either were exposed to 95% 02/5% CO2 at a final Po2 of 600 mmHg inactivation precedes the loss of a-ketoglutarate dehydroge- in a humidified gas chamber or were maintained under humidified nase activity and lactic acid accumulation in the sequence of air/5% CO2 at a Po2 of 133 mmHg at 370C. Cells were harvested after hyperoxia-induced metabolic disturbances. 24 or 48 hr of exposure to air or hyperoxia (A) or after short exposures TNF-a (1 ng/ml) or interleukin la (1 ng/ml) pretreatment to hyperoxia (B). Cell extracts were prepared and assayed for ofA549 cells for 24 hr increased the level ofthe mitochondrial aconitase activity and protein as described. Results are the average manganese-containing superoxide dismutase (MnSOD) by of three independent exposures ± SEM. mU, milliunits. -10- and =16-fold, respectively (P.R.G. and C.W.W., un- the was seen after a 72- to 96-hr exposure. published work). We supposed that the elevated mitochon- dishes, By drial MnSOD in inhibiting aconitase with fluorocitrate, we could mimic the cytokine-differentiated A549 cells would protect the superoxide-sensitive mitochondrial aconitase growth-inhibiting effects of hyperoxia as measured by count- against hyperoxic damage because these agents can protect ing fluoroacetate-treated cells (Fig. 3B) or by the neutral red rats against the lethality of hyperoxia (40). Surprisingly, dye assay of fluorocitrate-treated cells (Fig. 3C). Fluoroci- neither cytokine pretreatment protected aconitase against trate or fluoroacetate also caused the characteristic cell inactivation during an 8-hr exposure of A549 cells to hyper- swelling observed with hyperoxia. Inhibition of cellular ac- oxia (Po2 = 600 mmHg). Aconitase activity was inactivated onitase with fluorocitrate did not cause visible cell death to 47% ± 1%, 54% ± 4%, and 51% ± 2% (average ± SEM; during the 72-hr exposure. n = 3) of its control level in untreated, TNF-a-treated or We measured two other markers of hyperoxic damage to interleukin la-treated A549 cells, respectively. cell metabolism-namely, the loss ofactivity of the citric acid Hyperoxia Inactivates Aconitase in Rat Lung. A 24-hr cycle enzyme a-ketoglutarate dehydrogenase and the accu- exposure of rats to a Po2 of 760 mmHg was previously found mulation of lactic acid in the culture medium (9, 14, 41). to decrease respiration capacity (10) and citric acid cycle activity and to cause the accumulation of citrate (9), the 80 aconitase substrate, in lung tissue extracted from treated rats. 70 We explored the effects of hyperoxia on lung aconitase Ca) activity in rats exposed to hyperoxia because these previ- 0 60 * Basal ously reported effects may be explained by the loss of total 50 0 Uncoupled lung aconitase activity. As shown by the data in Fig. 4, lung {- 40 aconitase activity decreased by 73% in rats maintained in a Po2 of 760 mmHg relative to air controls (Fig. 4A), whereas E 0 30 exposure of rats to a sublethal Po2 of 635 mmHg resulted in 20 a 61% loss of aconitase activity (Fig. 4B). C Electrophoretic separation and staining of aconitase activ- 10 ity (37) revealed a principal loss of the cathodal form or

0 mitochondrial isozyme in lung extracts prepared from rats exposed to hyperoxia (Fig. 5). We did not detect the anodal -0~~~~ cytoplasmic aconitase activity or iron-responsive element- I °ILO binding protein (42) in lung extracts by this method. No CM C c) c) t significant loss of total liver aconitase activity was measured ' CMJ I in the rats exposed to a Po2 of 635 mmHg for 24 hr. The total CM% liver aconitase activity was 19.9 ± 0.2 milliunits per mg of FIG. 2. Loss of respiratory capacity in cultured A549 cells extract protein and 20.0 ± 0.7 milliunits per mg of extract exposed to hyperoxia, fluoroacetate, or fluorocitrate. Cultures of protein (average ± SEM; n = 3) in air controls and hyperoxia- A549 cells were grown to 70% confluency in 10 ml of F12K treated rats, respectively. These results are consistent with medium/l0o fetal bovine serum/antibiotics in 100-mm dishes at the interpretation that aconitase inactivation is due to an 370C under a humidified air/5% CO2. Cultures were then exposed for elevated lung tissue Po2 because the liver would not be 24 hr to air/5% CO2 (Po2 = 133 mmHg), 95% 02/5% CO2 (Po2 = 600 expected to experience a homogenous elevation ofPo2 under mmHg), air/5% CO2 plus 10 mM sodium fluoroacetate (FA) or these conditions (2, 3). air/5% CO2 plus 0.1 mM sodium (+)-fluorocitrate (FC). Cells were harvested and respiration rates were measured as described. The protonophore FCCP was added to a final concentration of 4-6 AuM DISCUSSION to maximally uncouple mitochondrial respiration. Results are the average ± SD of independent trials (n = 2 for all excluding 24-hr air As pointed out earlier by Haugaard (13), there has been a control where n = 4). reluctance to attribute the toxicity of 02 tothe inactivation of Downloaded by guest on September 30, 2021 Physiology: Gardner et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12251 A B C

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FIG. 3. Inhibition of growth of A549 cells by hyperoxia, fluoroacetate, or fluorocitrate. Cultures of A549 cells were plated at 1.0-1.3 x 106 cells per 100-mm dish in 10 ml of F12K medium/10% fetal bovine serum/antibiotics. Cells were allowed to adhere and grow for 12 hr at 370C under humidified air/5% CO2 (P02 = 133 mmHg) and were then either placed under 95% 02/5% CO2 (P02 = 600 mmHg) or grown under normoxia. Cells were counted after 24- and 48-hr growth under normoxia (solid bars) or hyperoxia (hatched bars) (A). Sodium fluoroacetate was added at the indicated concentrations to cell cultures after 12 hr of growth, as described for hyperoxia, and cells were counted after 72 hr of exposure (B). A549 cells were plated at 3 x 104 cells per well in a 6-well plate and after 12 hr of growth, sodium (±)-fluorocitrate was added at the indicated concentrations. After 72 hr of fluorocitrate exposure, neutral red (NR) dye uptake and retention were measured (C). Results are the average ± SD of two or more trials for all experiments.

specific enzymes because most, if not all, enzymes examined 02 argue that, in vivo, lung energy levels indeed are com- are inactivated by only extreme 02 exposures. We have now promised by the loss of oxidative phosphorylation (41). demonstrated that the activity of aconitase decreases rapidly Our data do not address the mechanism of aconitase during a sublethal or lethal 02 exposure of cultured mamma- inactivation during hyperoxic exposure in either cultured lian cells or rat lung tissue. The importance of aconitase cells or in rat lung. However, we know that the production of activity to physiological function can be inferred from the 02- and H202 is increased during exposure to hyperoxia (5, poisonous effects of the potent aconitase inhibitor fluoroci- 27, 28) and that aconitase is extremely sensitive to inactiva- trate. In cultured cells, fluoroacetate/fluorocitrate impairs tion by 02- but much less susceptible to inactivation by 02 mitochondrial respiratory capacity (Fig. 2) and cell growth and H202 (16, 22, 23, 26). In fact, a 10-15% inactive fraction (Fig. 3). Furthermore, the demonstration of increased lung ofaconitase is present in bacteria (23) and in mammalian cells citrate levels after a 24-hr exposure of rats to 100% 02 (9) growing under normoxia (26) (P.R.G. and C.W.W., unpub- supports the hypothesis that the loss of aconitase activity lished work). This inactive aconitase fraction is proportion- limits citric acid cycle activity and mitochondrial respiratory ally increased when 02 production is augmented or when capacity in vivo. Although it has been shown that rat lung superoxide dismutase activity is lowered (23-26) (P.R.G. and ATP levels and energy charge are maintained after lungs are C.W.W., unpublished work). The failure ofTNF/interleukin exposed to 100% 02 for 48 hr followed by perfusion with 1-induced MnSOD to protect aconitase against hyperoxia in glucose for 80 min (43), in vivo substrate limitations, cell type A549 cells is not simple to interpret because these inflam- differences in metabolic emphasis, and specific work de- matory cytokines are thought to limit the intracellular avail- mands need to be considered when evaluating the importance ability of iron by increasing ferritin heavy-chain synthesis ofaconitase inactivation, citric acid cycle impairment (9), and (44). Decreased iron availability may impair the dynamic decreased respiratory capacity of mitochondria (10) in lung reinsertion of iron in the damaged aconitase, thus negating pathology. The compensatory increases in lung glycolysis any beneficial effects of elevated MnSOD and decreased °2 and lactate production after a 24-hr exposure of rats to 100% for aconitase activity. Recently, it has been reported that transgenic mice expressing elevated levels of the mitochon- A B drial MnSOD specifically in alveolar type II epithelial cells

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760 mm Hc. 635 mmrHc FIG. 4. Loss of total lung aconitase activity in rats exposed to hyperoxia. Rats were exposed for 24 hr to normoxia (21% 02) or hyperoxia (>99%o 02) at a standard atmospheric pressure of 760 FIG. 5. Gel separation and staining of rat lung aconitase activity. mmHg (A) or rats were exposed at an ambient Denver atmospheric Lung extracts prepared from rats exposed for 24 hr to normoxia or pressure of 635 mmHg (B). Rats were exposed and anesthetized; hyperoxia at an atmospheric pressure of 760 mmHg, as described in their lungs were then removed, homogenized, and assayed for the Fig. 4 legend, were electrophoresed (20 ,ug oflung extract protein aconitase activity and protein as described. Results are the average per lane) and stained for aconitase activity. The asterisk denotes of measurements from three rats + SEM. mitochondrial aconitase activity. Downloaded by guest on September 30, 2021 12252 Physiology: Gardner et al. Proc. Natl. Acad. Sci. USA 91 (1994) and in Clara cells were significantly protected from lung Lusiak, B. & Brown, 0. R. (1993) J. Biol. Chem. 268, 25547- edema and death during exposure to hyperoxia (45). Possibly 25552. the selective protection of lung function by elevated mito- 18. Gardner, P. R. & Fridovich, I. (1991) J. Biol. Chem. 266, chondrial MnSOD in these cell types is at least partially due 1478-1483. to the protection of the 02--sensitive mitochondrial aconi- 19. Kuo, C. F., Mashino, T. & Fridovich, I. (1987) J. Biol. Chem. tase. Additional 262, 4724-4727. factors affecting aconitase stability during 20. Liochev, S. I. & Fridovich, I. (1993) Arch. Biochem. Biophys. exposure to hyperoxia may include decreases in the avail- 301, 379-384. ability of ferrous ions or of glutathione/thiols required for 21. Smykrandall, E., Brown, 0. R., Wilke, A., Eisenstark, A. & repair of the aconitase iron-sulfur center (24, 46) or alter- Flint, D. H. (1993) Free Radicals Biol. Med. 14, 609-613. ations in the degradation and synthesis of the aconitase 22. Gardner, P. R. & Fridovich, I. (1991) J. Biol. Chem. 266, protein. 19328-19333. Further investigations of the mechanism of aconitase in- 23. Gardner, P. R. & Fridovich, I. (1992) J. Biol. Chem. 267, activation and ofits role in the loss ofcitric acid cycle activity 8757-8763. and the early damage to lung cell and tissue function may 24. Gardner, P. R. & Fridovich, I. (1993) Arch. Biochem. Biophys. provide important insights into the mechanisms of02 toxicity 301, 98-102. and may also facilitate the development ofpalliative therapies 25. Gardner, P. R. & White, C. W. (1994) Am. J. Resp. Crit. Care for this condition. Med. 149, (4: part 2), A455 (abstr.). 26. Gardner, P. R. & White, C. W. (1994) in The Oxygen Paradox, This work was supported by National Institutes of Health (NIH) ed. Davies, K. J. A. (Univ. of Padua Press, Padua, Italy), in Postdoctoral Training Grants T32 HL07670 and F32 HL08997, an press. 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