Mitochondrial Glutathione and Oxidative Stress: Implications for Pulmonary Oxygen Toxicity in Premature Infants

Molecular Genetics and Metabolism 71, 352–358 (2000) doi:10.1006/mgme.2000.3063, available online at http://www.idealibrary.com on

MINIREVIEW Mitochondrial Glutathione and Oxidative Stress: Implications for Pulmonary Oxygen Toxicity in Premature Infants Donough J. O’Donovan 1 and Caraciolo J. Fernandes Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030 Received August 1, 2000

fects are evident when intracellular GSH concentrations are increased. In conditions that increase mitochondrial production of ROS, such as exposure to high concentrations of oxygen, therapies based on enhancing mitochondrial GSH concentrations could be highly beneficial. © 2000 Academic Press Key Words: glutathione; antioxidant protection; oxidative stress; mitochondria; pulmonary oxygen toxicity; premature infants.

Administration of supplemental oxygen, despite being an important clinical therapy, can cause significant lung damage. Because they have underdeveloped lungs, prematurely born human infants frequently require supportive therapies that employ elevated oxygen concentrations, which put them at risk for developing pulmonary oxygen toxicity. This risk is made even greater by the immaturity of their cellular antioxidant defenses. Although the exact mechanisms of oxygen toxicity are still not fully defined, cellular damage is probably mediated by increased production of chemically reactive oxygen species (ROS) in the mitochondria. Cellular protection against ROS is provided by a variety of antioxidant molecules and enzymes, including the glutathione (GSH)-dependent antioxidant system. The GSH-dependent antioxidant enzyme system provides vital cellular protection against ROS, particularly hydrogen peroxide and certain organic hydroperoxides, under pathological and toxicological conditions, by using selenium-dependent and -independent peroxidases to reduce hydrogen peroxide or lipid peroxides to water or the respective alcohols, with the concurrent oxidation of GSH to glutathione disulfide (GSSG). In the mitochondria, limitations of GSH synthesis and transmembrane transport suggest that optimal functioning of the mitochondrial GSH system, and maintenance of adequate thiol– disulfide redox tone is essential to protect against the injurious effects of ROS. Manipulation of endogenous GSH concentrations can alter cellular responses to oxidant injury. Beneficial ef-

PULMONARY OXYGEN TOXICITY IN PREMATURE INFANTS Administration of supplemental oxygen is a common and necessary therapy in management of patients with respiratory insufficiency. However, high concentrations of oxygen are toxic and prolonged exposure to high concentrations of oxygen can cause lung injury and long-term pulmonary morbidity (1– 4). Prematurely born infants, who frequently require supplemental oxygen therapy, have an increased risk of developing pulmonary oxygen toxicity, probably attributable to inadequate host defenses, underdeveloped lungs, and immature antioxidant systems. In these infants, exposure to hyperoxia inhibits lung alveolarization, which can result in permanent deficiencies in alveolar formation and long-term pulmonary morbidity (5). Hence, oxygen-induced lung injury is a major factor in the pathogenesis of one of the most common and troublesome complications of prematurity, viz., bronchopulmonary dysplasia (BPD), often called chronic lung disease. BPD will complicate the hospital

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To whom correspondence should be addressed. Fax: (713) 798-5691. E-mail: [email protected]. 352 1096-7192/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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course of almost 15% of the 47,000 very low birth weight (VLBW, i.e., under 1500 g) infants born annually in the United States. In addition, as advances in neonatal care continue, survival rates for extremely immature infants will improve and the number infants at risk of developing BPD will also continue to rise. Hence, the toxic effects of hyperoxia on premature lungs represent a significant and costly health issue, and the development of therapies to prevent or decrease oxygen damage to immature lungs is an important clinical goal. At present use of the lowest effective oxygen concentrations, avoidance of certain drugs, and attention to nutritional and metabolic factors remain the best currently available strategies of avoiding or minimizing oxygen toxicity. More formal therapeutic approaches, such as might be afforded by lung-targeted therapies to augment cellular antioxidant defenses, could greatly diminish lung damage caused by pulmonary oxidant stresses and possibly prevent long-term pulmonary morbidity in premature infants. REACTIVE OXYGEN SPECIES AND OXYGEN TOXICITY Although the cellular mechanisms of oxygen toxicity are still not fully defined, oxidative damage of cellular components by increased generation of reactive oxygen species (ROS) is probably the mechanism by which high concentrations of oxygen damage cells (6 –11). In pulmonary oxygen toxicity, lung injury is probably initiated when the rates of generation of ROS are increased beyond the capacities of the antioxidant defenses, which is more likely when the functions of the antioxidant defense mechanisms are deficient, compromised, or developmentally unprepared, such as in prematurely born infants (5,12,13). The relationship between hyperoxia, ROS, and the induction of antioxidant enzymes has been demonstrated in bacterial models and strongly supported by data from cell cultures and animal experiments (14). In addition, the protective benefit of induction of antioxidant defense mechanisms in cell culture and animal studies has been well described (3,15). MITOCHONDRIA-MEDIATED CELL INJURY IN HYPEROXIA The provision of cellular energy by the mitochondria is essential for a multitude of cellular functions,

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including intermediary metabolism, ion regulation and other active transport processes, cell mobility, and cell proliferation. In most cells, the vast majority of energy is derived from oxidative phosphorylation, a process that involves the four-electron reduction of molecular oxygen to water and the production of ATP. Even under well-coupled conditions, as much as 2– 4% of the reducing equivalents escape the respiratory chain to liberate superoxide and hydrogen peroxide. Because ROS generation is a continuous physiological occurrence, mitochondria and extramitochondrial compartments possess efficient antioxidant systems: superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione (GSH), vitamins E and C, thiol peroxidases, and others. Under conditions in which mitochondrial ROS generation is increased, such as hyperoxia (6,7,16,17), or when the antioxidant systems are compromised, such as with prematurely born infants (12), deficient clearance by antioxidants of H 2O 2 may allow increased reaction with Fe 2⫹, resulting in the formation of the highly reactive hydroxyl radical (HO •) or related ferryl or perferryl species via Fenton-type chemistry, thus initiating cellular damage (18). Mitochondrially mediated cell injury by ROS has been implicated in cell dysfunction and identified as a critical event in both apoptotic and necrotic forms of cell death, and considerable data support this mechanism of cell injury in hyperoxia (19). THE GSH-DEPENDENT ANTIOXIDANT SYSTEM The tripeptide L-␥-glutamylcysteinylglycine, commonly called GSH, is found in relatively high concentrations (1–10 mM) in virtually all mammalian cells. GSH is found in separable intracellular compartments, with about 85% of total cellular content located in the cytoplasm and the remainder distributed between the mitochondria (10%) and other organelles. GSH serves a number of functions, which include providing a storage and transport form of cysteine, conjugation with xenobiotics and electrophilic intermediates, maintenance of sulfhydryl groups, and transfer of reducing equivalents. The transfer of reducing equivalents is a critical aspect of the interaction of GSH with H 2O 2 and other oxidants and products of peroxidation and the management of protein thiol– disulfide transformations and steady states (20 –30). Thus, the GSH redox cycle (Fig. 1) is a major endogenous antioxidant enzyme

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FIG. 1.

Glutathione redox cycle.

system and provides cellular protection against ROS, particularly hydrogen peroxide, by using selenium-dependent and -independent peroxidases to reduce H 2O 2 or lipid peroxides to water or the respective alcohols, with the concurrent oxidation of GSH to GSSG (21–23). IMPORTANCE OF GSH IN COMBATTING OXIDANT STRESS GSH in the most abundant intracellular thiol and the GSH-dependent antioxidant system plays a central role in cellular protection against oxidant injury. Because of its important biological proprieties, especially in detoxification, GSH function, synthesis, transport, and degradation have been studied extensively in many cell types. As a result, considerable evidence has accumulated supporting the role of intracellular GSH in protection against free radical processes under pathological and toxicological conditions (20 –30). Manipulation of endogenous GSH concentrations can alter cellular responses to oxidant injury. The data are compelling: increasing endogenous GSH concentrations by induction of de novo synthesis or by direct or indirect exogenous supplementation of GSH confers resistance to oxidant injury (31–33). For example, significant protection against paraquat-induced cell injury was achieved in rat alveolar type II cells by enhancing cellular GSH concentrations via exogenous supplementation (32). Similarly, Tsan et al. observed enhanced resistance to H 2O 2 in endothelial cells with increased GSH concentrations (31). The cytoprotective properties of intracellular GSH are further supported by studies on GSH depletion, which indicate an enhanced susceptibility to toxic injury in cells with diminished GSH concentrations (34 –39).

The flavoenzyme glutathione reductase (GR) catalyzes the reduction of GSSG back to GSH at the expense of NADPH oxidation. Normally, GSSG concentrations are less than 1% of the total GSH, the remainder being principally GSH. Under oxidative stress conditions, increases in GSSG concentrations occur but are usually transient, as reduction by GR is relatively effective, and some cell types actively export GSSG which helps maintain low cellular GSSG concentrations. Increased formation of GSSG has been proposed as a mechanism through which oxidants injure cells and GSSG, formed transiently, can exchange with protein sulfhydryls to produce protein– glutathione mixed disulfides. If the protein sulfhydryl is critical, cell structure, metabolism, or function may be altered. The cytoprotective significance of regenerating GSH from GSSG by GR is indicated by studies demonstrating that cells with attenuated GR activities are more susceptible to oxidative stresses (34 –36). Although it does not necessarily follow that increased GR activities would enhance antioxidant defense function significantly, we have observed greater resistance to oxidant stresses in cells with genetically enhanced GR activities, particularly if the mitochondrial GR activities are augmented (40 – 42). MITOCHONDRIA AND THE GLUTATHIONE-DEPENDENT ANTIOXIDANT SYSTEM Mitochondria constitute about 10% of the cell mass in many cell types and contain a comparable fraction of cellular GSH. During hyperoxia and other oxidant challenges, mitochondrial production of ROS increases and several observations support the need for effective glutathione redox cycling in this organelle. Mitochondria probably lack catalase (43) and depend on GPX to detoxify hydroperoxides. In addition, mitochondria are unable to synthesize GSH and, therefore, rely on the reduction of GSSG and on GSH uptake as a source of GSH (44). Olafsdottir et al. observed no increases in GSSG concentrations in the medium from isolated mitochondria exposed to tert-butyl hydroperoxide (t-BuOOH), indicating that mitochondria may not have an effective GSSG efflux system (45). Their data are consistent with the findings that the human multidrugresistant-associated protein, or ATP-dependent glutathione S-conjugate pump, which can export GSSG as a substrate, is located at the plasma membrane, but is not found associated with the mito-

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chondrial membrane (19,46). These studies and others suggest that a transport system for GSSG efflux is absent in the mitochondria and that all GSSG formed inside the mitochondria must be reduced in situ to maintain redox tone (47– 49). These limitations may place an additional burden on mitochondrial GR to maintain intramitochondrial GSH concentrations and GSH/GSSG ratios. To investigate the effects of mitochondria-specific GR enhancement in lung-type cells, we constructed a vector (LGR) that contained the coding sequence for human GR with a functional synthetic mitochondrial targeting “leader” sequence from human MnSOD (50,51). In these studies, H441 cells (transformed human lung cells with characteristics of Clara cells) transfected with adenovirus containing LGR showed highly selective enhancement of mitochondrial GR activities and were more resistant to t-BuOOH-induced oxidant stresses (41) (Fig. 2). In other studies adenovirus-mediated gene transfer of LGR protected H441 cells from oxygen-induced growth inhibition (42) (Fig. 3). Under hyperoxic conditions, H441 cells with enhanced mitochondrial GR activities had higher mitochondrial GSH concentrations, maintained higher mitochondrial GSH/GSSG ratios, and resisted oxygen-induced growth inhibition, suggesting that mitochondrial GSH homeostasis determined critical aspects of cellular resistance to hyperoxia. Depletion of mitochondrial GSH is associated with an increased susceptibility to oxidant injury (37–39). Meredith and Reed first proposed a cytoprotective role for mitochondrial GSH with the observation that the onset of cellular injury in isolated rat hepatocytes by ethacrynic acid is correlated with the depletion of mitochondrial GSH (38). Other reports followed and, more recently, Shan et al. firmly established the critical role of mitochondria GSH in cytoprotection with the observation that selective depletion of mitochondrial GSH concentrations by (R,S)-3-hydroxy-4-pentenoate significantly enhanced t-BuOOH-mediated cell injury in rat hepatocytes (53). Although the precise cytoprotective mechanisms are yet undetermined, mitochondrial GSH/ GSSG ratios may function in regulating the thiol/ disulfide status of mitochondrial protein thiol groups, such as the mitochondrial permeability transition (MPT) pore (54 –57). The MPT pore is a voltage-dependent channel that allows solutes of less than 1500 molecular weight to equilibrate across the mitochondrial inner membrane, and is responsible for the rapid efflux of solutes such as

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FIG. 2. (A) Mitochondria-specific enhancement of glutathione reductase activities in H441 cells transfected with adenovirus containing the LGR vector (41). CON, nontransfected control cells; DOS, cells transfected with adenovirus containing a control gene; LGR, cells transfected with adenovirus containing mitochondrially targeted glutathione reductase. MITO, mitochondrial compartment; CYTO, cytosolic compartment. *P ⬍ 0.01. (B) Resistance to oxidant injury in H441 cells with increased mitochondrial glutathione reductase activities. Lactate dehydrogenase (LDH) release was used to assess cellular injury in H441 cells exposed to tert-butyl hydroperoxide (t-BuOOH). Cells transfected with LGR released less LDH to the incubation medium when exposed to 400 ␮M t-BuOOH than did DOS or CON cells at all time points. *P ⬍ 0.01.

GSH and Ca 2⫹ (54). Oxidants can stimulate the release of Ca 2⫹ from the mitochondria, and interference in Ca 2⫹ homeostasis and increases in cytoplasmic-free Ca 2⫹ have been implicated in cell injury. Blockage by cyclosporin A of the MPT pore opening protects cells from-oxidant-mediated lethal injury and the redox status of the vicinal thiol groups in a mitochondrial protein appears to play a crucial role in determining the gating potential of the MPT pore (57,58).

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volves exposure to high concentrations of oxygen. Indeed, ROS have been implicated in the pathogenesis of BPD (4,11,27). In premature infants, antioxidant defense capacities could be compromised by low levels of GSH and other antioxidants that metabolize ROS, suggesting an increased risk of oxygen-induced lung injury in this developmentally unprepared population (27). Genetically enhancing the GSH-dependent antioxidant system may be particularly advantageous in these immature patients. CONCLUSION FIG. 3. Resistance to hyperoxia-induced growth inhibition is observed in H441 cells with increased mitochondrial glutathione reductase activities (LGR ⫽ 95%) (42). *P ⬍ 0.01. CON 21%, nontransfected cells in room air. DOS 21%, cells transfected with adenovirus containing a control gene in room air; LGR 21%, cells transfected with adenovirus containing a LGR in room air; CON 95%, nontransfected cells in 95% oxygen; DOS 95%, cells transfected with adenovirus containing a control gene in 95% oxygen; LGR 95%, cells transfected with adenovirus containing a LGR in 95% oxygen.

Further evidence supporting the cytoprotective role of the mitochondrial GSH system in cell injury is provided by the observation that agents preventing the loss of mitochondrial GSH also confer resistance to oxidant injury. The antioxidants diphenyl-p-phenylenediamine (DPPD) and vitamin E succinate reduce the loss of mitochondrial GSH during calcium ionophore treatment and diminish the loss of cell viability (59). GLUTATHIONE STATUS IN THE PREMATURE INFANT Premature infants, as a group, have lower and gestational age-related plasma concentrations of GSH and higher concentrations of GSSG than are measured in healthy adults (12). The mechanisms responsible for these differences and the implications of these differences are not known at present. However, the pivotal roles played by GSH in antioxidant defenses and in other physiological functions suggest that this deficiency may compound the burden of prematurely adapting to an extrauterine environment. Birth moves the fetus from an environment with low oxygen tension (intrauterine pO 2 ⫽ 20 –25 mm Hg) to one of much higher oxygen tension (room air pO 2 ⫽ 150 –160 mm Hg) (5). The challenges to the infant’s antioxidant defense mechanisms following birth are particularly critical in premature infants, whose medical management frequently in-

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