Mitigation of Pulmonary Oxygen Toxicity in Rats by Intratracheal Instillation of Polyethylene Glycol-Conjugated Antioxidant Enzymes

003 1 -3998/93/3304-0332$03.00/0 PEDIATRIC RESEARCH Copyright Q 1993 International Pediatric Research Foundation. Inc. Vol. 33. No. 4. 1993 Pnnn,d in l S . 4 . 1 Mitigation of Pulmonary Oxygen Toxicity in Rats by Intratracheal Instillation of Polyethylene Glycol-Conjugated Antioxidant Enzymes FRANS J. WALTHER. FELIX L. NUNEZ, REMEDIOS DAVID-CU, AND KATHLEEN E. HILL Division qf Neonatologj~.Department of Pediatrics. Los Ange1e.y Coztntj. Kin,g/Drenl ,Medical Cenrrr and C'CL4 School qfMedicine, Los Angeles, Caljfornia 90059 ABSTRAm. Hyperoxic lung injury may be mitigated by increasing alveolar epithelial antioxidant activity. We examined whether intratracheal instillation of superoxide dismutase (SOD) and catalase, conjugated to polyethylene glycol (PEG) to permit cellular access, reduces hyperoxic lung injury. Adult rats, pretreated intratracheally with 1 500 U PEG-SOD and 10 000 U PEG-catalase or with inactivated PEG-SOD/catalase, 1% PEG, or saline (treated controls), were exposed to hyperoxia (fraction of inspired oxygen > 0.95) for 48 h and compared with untreated air controls. Alveolar wash protein values in the treated control groups were significantly higher than in the PEG-SOD/catalase and air control groups, which had comparable values. Lung homogenate and alveolar type I1 cell SOD and catalase activities were higher after PECSOD/catalase treatment and lower after the control treatments when compared with untreated air controls. Lung homogenate dipalmitoyl phosphatidylcholine decreased and alveolar wash dipalmitoyl phosphatidylcholine increased after hyperoxia, but these changes were less after PEG-SOD/catalase treatment. Rats pretreated intratracheally with PEG-SOD/catalase survived significantly longer in hyperoxia than saline controls. These data indicate the potential of intratracheal antioxidant treatment to reduce pulmonary oxygen toxicity. (Pediatr Res 33: 332335, 1993) Abbreviations DPPC, dipalmitoyl phosphatidylcholine PEG, polyethylene glycol SOD, superoxide dismutase Successful attempts to modify pulmonary oxygen toxicity in adult animals have involved the augmentation of the intracellular antioxidant capacity by endotoxin (l), preexposure to hyperoxia (2, 3) or hypoxia (4), and the administration of exogenous antioxidant enzymes. SOD, catalase, and glutathione peroxidase cooperate in the detoxification of free oxygen radicals. SOD is responsible for the metabolism of superoxide anions; catalase and glutathione peroxidase catalyze the reduction of Hz02 to H20, with glutathione peroxidase also playing a role in the detoxification of intracellular hydroperoxides. Native SOD and catalase injected i.v. (5) or SOD administered intratracheally to Received July 31. 1992; accepted December 9. i992. Correspondence and reprint requests: Frans J. Walther, M.D., Ph.D., Department of Pediatrics, King/Drew Medical Center. 12021 S. Wilmington Ave.. Los Angeles. CA 90059. Supported in pan by a grant from Minority Access to Research Careers ( 5 ~ 3 4 GM07971-10) and Grant HL 40666 from the National Institutes of Health. rats (6) does not provide protection against hyperoxia, because their large molecular size does not permit penetration across cell membranes and they have a short half-life. When the molecular weight of SOD and catalase is increased through covalent attachment of the inert linear polymer, monomethoxy-PEG, they can undergo endocytosis and are taken up by cultured endothelial cells (7) and primary cultures of alveolar type I1 cells (8), thereby increasing cellular antioxidant enzyme activity and providing protection from the damaging effects of reactive oxygen species. Intravenous administration of PEGSOD and PEG-catalase to rats (9) and preterm lambs (10) provides partial protection against hyperoxic lung injury. Like PEG-conjugated antioxidants. liposome-encapsulated antioxidant enzymes enter cells in active form, either by fusion with plasma membranes or by endocytosis, and are protective against oxygen toxicity after i.v. or intraperitoneal administration (5, 1 1 ) or addition to alveolar type I1 cells (12. 13). Liposomal antioxidant enzymes instilled into the airway of adult rats prolong survival after exposure to hyperoxia (14). We investigated whether intratracheal instillation of PEG-SOD and PEG-catalase increases the activity of these antioxidant enzymes in lung tissue, especially in alveolar type I1 cells, and reduces pulmonary oxygen toxicity in adult rats. MATERIALS AND METHODS Chemicals. Native bovine Cu, Zn-SOD and catalase and PEGSOD and PEG-catalase ( I 5) were obtained from Sigma Chemical Company (St. Louis. MO), elastase was obtained from Worthington Biochemical Corp. (Freehold. NJ). fetal bovine serum from Hyclone Laboratories (Logan, UT), tissue culture media from GIBCO (Grand Island, NY). and plasticware from Falcon (Oxnard, CA). Antioxidant enzyme solutions were shown to be free of endotoxin contamination by the limulus amebocyte lysate assay ( 16, 17). All other enzymes and biochemicals, of the highest grade available, were obtained from Sigma. Inacrivafion of PEG-SOD and PEG-catalase. For control experiments, PEG-SOD was inactivated by mixing 10 mg PEGSOD/mL with 100 mM H202 at room temperature for 6 h. then dialyzed three times for 8 h in potassium phosphate (10 mM) buffered (pH 7.4) saline (0.15 M NaCI) at 4% assayed for activity, filtered (0.22 pM),and stored at 4°C (18). PEG-catalase (10 mg protein/mL) was inactivated by heating at 90°C for 15 min, assayed for activity, passed through a 27-gauge needle, and stored at 4°C. Inactivated PEG-SOD and PEG-catalase lacked SOD and catalase activity when assayed and did not precipitate after inactivation. P;e:;eu":mcn! efrc!s. Pathogen-free. adn!? rr?a!e prague-Dawley rats weighing 250-300 g were obtained from Charles Rivers (portage, MI). ~ 1 experiments 1 were performed humanely and with the approval of the ~ n i m a Care l and use committee. Rats were injected intratracheally with 0.3 mL of test solution 1 1 333 PULMONARY OXYGEN TOXICITY AND PEG-ANTIOXIDANTS after receiving 30 mg pentobarbital sodium by intraperitoneal injection and having their trachea exposed by an incision in the neck. Test solutions consisted of 0.2 mL of rat surfactant (12.5 ctrnol DPPC/mL) plus 0.1 mL containing 1 500 U of PEG-SOD + 10 000 U PEG-catalase (PEG-SOD/catalase group), equal amounts of inactivated PEG-SOD + PEG-catalase (inactive PEG-SOD/catalase group). 3% PEG ( 1 % PEG group). or normal saline. The rats were then exposed to hyperoxia for 48 h or until they died. Untreated. air-exposed rats were used as baseline controls. Groups of six to eight rats were used for collection of alveolar washes and lung homogenates and groups of four rats were used for type I1 cell isolates after 48 h of hyperoxia. Six untreated. air-exposed rats were used for collection of alveolar washes and lung homogenate and four were used for type 11 cell isolates. Eight pairs of rats (PEG-SOD/catalase and saline g (...truncated)