Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

Abstract

The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of >80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a Po2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure ofrats to an atmospheric Po2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition ofthe energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia. (1,4,(7)(8)(9)(10)(11)(12)(13). Morphological and biochemical alterations in the lung ultimately cause morbidity resulting from decreased blood oxygenation (4). Edema of the interstitial space and increased permeability of pulmonary microvasculature are early signs of pathology in the lungs of rats exposed to lethal levels of dioxygen (Po2 of 760 mmHg). This initial damage is followed by and amplified by the activation and infiltration of platelets, macrophages, and neutrophils and precedes the death of the animal or individual (3, 12). After sublethal exposures of rats to hyperoxia (Po2 of 456-650 mmHg), damage is marked by fibrosis, alveolar type II cell proliferation (11, 12), and mitochondrial structural deformities (7,8,11,12). The contribution of individual reactive oxygen intermediates and dioxygen to the morphological and biochemical alterations in the lungs, however, remains only vaguely defined (5, 6, 12).Mitochondrial respiration and energy production have been identified as sensitive and critical sites of hyperoxic damage in lung tissue (9, 10) and in cell culture models (14). In rats exposed to a Po2 of 760 mmHg for 24 hr, lung mitochondrial respiratory capacity and citric acid cycle activity are impaired (9, 10). Potential enzymatic sites for the poisoning action of hyperoxia have been described. The mitochondrial enzymes NADH dehydrogenase (14), succinate dehydrogenase (14, 15), and a-ketoglutarate dehydrogenase (14) have various sensitivities to hyperoxic exposure. However, evidence is lacking for a loss of dehydrogenase activities during the early impairment of rat lung oxidative metabolism by normobaric hyperoxia.The citric acid cycle enzyme aconitase is a member of a growing family of 02--sensitive [4Fe-4S]-containing (de)-hydratases that have been implicated to be important sites of 02 -/02 toxicity (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The activity ofaconitase is sensitive to inactivation by 02- (16,22) and is modulated by changes in 02-levels in bacteria and mammalian cells (23, 24, 26).Thus, the ability of elevated levels of 02 to exacerbate mitochondrial productio...

Middle age aggravates myocardial ischemia through surprising upholding of complex II activity, oxidative stress, and reduced coronary perfusion

“…In the present study, we evaluated the instant mitochondrial ROS impregnation at two moments of the perfusion: just before ischemia and at the end of reperfusion. It was performed by measuring the aconitase to fumarase ratio in cardiac homogenates (Gardner et al 1994). Transition from normoxia to end of reperfusion triggered a significant decrease in the aconitase to fumarase ratio in the middle-aged hearts, whereas it did not alter this parameter in the hearts of young adult animals.…”

“…Aconitase and fumarase activities were determined according to Gardner et al (1994), with the modification that the extraction medium was supplemented with tri-sodium citrate to stabilize aconitase activity ex vivo. In order to evaluate myocardial mitochondrial density, activity of the citrate synthase was divided by the protein amount of the homogenate.…”

Alterations in fatty acid metabolism and sirtuin signaling characterize early type-2 diabetic hearts of fructose-fed rats

“…Aconitase activity was measured in mitochondria that were isolated in the presence of citrate and MnCl 2 , to limit aconitase inactivation during sample preparation and storage (Gardner et al. 1994; Bulteau et al. 2003).…”

Autoxidation of Ubiquinol-6 Is Independent of Superoxide Dismutase

“…However, SOD is also unable to prevent Q 6 H 2 autoxidation when incubated over time in the presence of 100% oxygen ( Figure 4 and Table 2). Under such hyperoxia conditions, superoxide and various peroxides are known to be generated (Gardner et al, 1994), and liposome peroxidation was initiated and chain-carried in 100% O 2 as determined by assays of LOOH (Table 2). Thus, the addition of SOD to this liposome system does not slow Q 6 H 2 oxidation, even under conditions which promote the formation of superoxide.…”

“…To ensure that superoxide is generated in the reaction system, liposomes containing Q 6 H 2 , Q 6 , or PBS were incubated, with either SOD or catalase, in 100% O 2 at 37°C for 4 h, and the ability of Q 6 H 2 to protect cPN against peroxidation was assessed at 0, 1, 2, and 4 h. Hyperoxia conditions are known to induce formation of reactive oxygen species, including superoxide (Gardner et al, 1994). LOOH were measured at each time point before the cPN protection assay to determine whether lipid peroxidation was initiated and chain-carried during O 2 incubation, and again at 90 min to measure the extent of peroxidation incurred during the cPN assay.…”