Augmentation of Superoxide Dismutase and Catalase Activity in Alveolar Type II Cells Frans J. Walther, Alma B. Wade, David Warburton, and Henry J. Forman Department of Pediatrics, King-Drew Medical Center, UCLA School of Medicine, and the Cell Biology Group, Children's Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, California
This study tested whether adducts formed by covalent linkage of superoxide dismutase (SOD) or catalase to polyethylene glycol (PEG) could augment SOD and catalase activity in alveolar type II cells and document enhanced resistance to oxidant damage. Alveolar type II cells were isolated from adult, pathogenfree rats. Antioxidant enzymes were added to the medium of cell cultures in various concentrations for periods up to 48 h. Incubation with 500 to 3,000 U of PEG-SOD or 10,000 to 40,000 U of PEGcatalase/1Q6 cells produced a dose-response-related increase in intracellular enzyme activity in comparison with controls (untreated or treated with SOD or catalase, inactivated PEG-SOD or PEG-catalase, or PEG alone). Uptake was maximal during the first 4 h. Using fluorescent label (fluorescein isothiocyanate) bound to PEG-catalase, we found intracellular localization of the labeled enzyme. Exposure to H20 2 led to reduced cytotoxicity in cells pretreated with PEG-catalase than in controls. We conclude that supplementation with PEG-SOD or PEG-catalase enhanced the activity of these enzymes in alveolar type II cells and increased their resistance to oxidant stress.
The alveolar epithelium is exposed to oxygen and free oxygen radicals derived from intracellular metabolic processes and inflammatory cells (1, 2). Hyperoxia increases the intracellular production of free oxygen radicals and causes an increase in alveolar epithelial permeability (3), morphologic damage of the alveolar type II cells (4), and inhibition of the repair processes following epithelial injury (5). Free oxygen radicals released by activated inflammatory cells that migrate into the alveoli also contribute to lung injury (6). During the final 10 to 15% of gestation, both fetal lung antioxidant enzyme levels and lung surfactant content increase rapidly (7). Therefore, some premature infants are born both with surfactant insufficiency and with antioxidant enzyme insufficiency (8). An imbalance between the production and the scavenging of free oxygen radicals may be important in the pathogenesis of various lung diseases such as bronchopulmonary dysplasia of premature infants (9). Manipulation of intracellular antioxidant enzyme activity may therefore eventually lead to therapeutic strategies. Successful attempts to modify pulmonary oxygen toxicity in adult animals have involved the augmentation of the intra(Received in original form June 19, 1990 and in revised form September 26, 1990) Address correspondence to: Frans J. Walther, M.D., Ph.D., Department of Pediatrics, King-Drew Medical Center, 12021 S. Wilmington Avenue, Los Angeles, CA 90059. Abbreviations: Oulbecco's modified Eagle's medium, DMEM; fetal bovine serum, FBS; fluorescein isothiocyanate, FITC; hydrogen peroxide, H202; lactate dehydrogenase, LDH; polyethylene glycol, PEG; superoxide dismutase, SOD. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 364-368, 1991
cellular antioxidant capacity by endotoxin, preexposure to hyperoxia, hormonal stimulation, or the administration of exogenous superoxide dismutase (SOD) and catalase, which are responsible for the metabolism of superoxide anions and hydrogen peroxide (H20 2 ) , respectively. The therapeutic potential of SOD and catalase is limited by their short circulatory half-life of 10 to 20 min (10, 11), sensitivity to Proteases, and inability to penetrate cell membranes. Covalent attachment of SOD and catalase to the inert linear polymer, monomethyoxy-polyethylene glycol (PEG), or encapsulation in liposomes avoids these limitations. PEG conjugation increases their circulatory half-life from 6 min to 30 to 40 h in the rat (12), reduces their antigenicity and sensitivity to proteases, and improves their stability in aqueous solutions (10, 13, 14). Beckman and associates (11) showed that cultured endothelial cells take up PEG-SOD and PEG-catalase and that the increased cellular enzyme activity provides protection from the damaging effectsof reactive oxygen species. Alveolar type II cells produce pulmonary surfactant and are the progenitor cells for the type I cells that line most of the alveolar surface and facilitate alveolar-arterial gas exchange. We have studied whether addition of SOD and catalase conjugated to PEG to primary cultures of alveolar type II cells could increase intracellular activity of these enzymes and reduce their vulnerability to oxidant stress.
Materials and Methods Materials Native bovine copper/zinc-SOD and catalase and PEG-SOD (10) and PEG-catalase (10) were obtained from Sigma Chemical Co. (St. Louis, MO), elastase from Worthington
Walther, Wade, Warburton et al.: Antioxidant Enzyme Activity in Alveolar Type IT Cells
365
Biochemical Corp. (Freehold, NJ), fetal bovine serum (FBS) from Hyclone Laboratories (Logan, UT), tissue culture media from GIBCO (Grand Island, NY), plasticware from Falcon (Oxnard, CA), and pathogen-free rats from Charles Rivers Labs (Portage, MO. All other enzymes and biochemicals, of the highest grade available, were obtained from Sigma.
Fluorescent Labeling of Antioxidant Enzymes Antioxidant enzymes were labeled by adding 0.3 mg of fluorescein isothiocyanate (FITC) to 1.0ml of 10 mg/ml catalase or PEG-catalase in 50 mM sodium carbonate (21). Following stirring for 1 hat 4° C at a pH of 9.5, unbound FITC was removed by gel filtration chromatography on Sephadex G-25 eluted with PBS.
Primary Culture of Alveolar Type II Cells Alveolar type IT cells were harvested from male, 250- to 300-g, pathogen-free rats following the lung lavage, elastase digestion, and rat IgG differential adherence protocol of Dobbs and colleagues (15). This methodology yields 2 x 107 to 3 X 107 alveolar type IT cells/rat with> 90% viability by trypan blue staining and> 90 % purity by phosphene 3R fluorescent staining (16). Alveolar type IT cells were placed in culture in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS at a density of 3 x 106 to 4 x 106 cells/2 ml and incubated for 20 h at 37° C in a 5% CO 2:air incubator. Nonadherent cells were then removed by washing 3 times with phosphatebuffered saline (PBS), test solutions dissolved in DMEM were added, and the plates were returned to the incubator for the duration of the uptake study. Cells were washed 3 times with PBS, once with 1% PEG, and once with trypsin (0.05% wt/vol and 0.2% EDTA in DMEM), and then lysed with 0.1% Triton X-100. The suspension was centrifuged at 4° Cat 1,000 x g for 10 min. The supernatant was used for SOD, catalase, DNA, and protein assays.
Protein and DNA Content The amount of protein in the samples was determined by a modification of the Lowry method (22), and the DNA content by the fluorimetric method of Erwin and coworkers (23).
SOD and Catalase Assays SOD activity was assayed by inhibition of the reduction of cytochrome C in the xanthine oxidase reaction (17) using a modification described by Forman and Fisher (18). Endogenous reduct ants in the samples, which could interfere with the assay by producing nonsuperoxide-dependent cytochrome C reduction, were eliminated by mixing the sample and 5 X 10-5 M cytochrome C in 0.6 ml of 0.5 M potassium phosphate buffer, pH 7.8, and allowing the reduction and reoxidation by endogenous cytochrome oxidase of the cytochrome C to occur. The reaction was followed at 550 nm in a DU-7 spectrophotometer (Beckman Instruments, Fullerton, CA). After initial incubation, 10-5 M cyanide to inhibit cytochrome oxidase was added, followed by xanthine, xanthine oxidase, and H20 to a final volume of 3 ml. One unit of SOD equaled 50% inhibition of cytochrome C reduction. Catalase activity was measured by the rate of reduction of H 20 2 substrate, followed spectrophotometrically at 240 nm (19). Inactivation of PEG-SOD and PEG-catalase PEG-SOD was inactivated by mixing 10 mg PEG-SOD/ml with 100 mM H20 2 at room temperature for 6 h, then was dialyzed 3 times for 8 h in PBS at 4° C, filtered (0.22 j-tm), and stored at 4°C (20). PEG-catalase was inactivated by heating at 90° C for 15 min, then passed through a 27-gauge needle and stored at 4° C. Inactivated PEG-SOD and PEGcatalase lacked SOD or catalase activity when assayed and did not precipitate following inactivation.
Cytotoxicity Following the initial 20 h of incubation and 24 h of treatment with antioxidant enzymes, alveolar type IT cells pretreated with PEG-SOD or SOD were exposed to 1 mM xanthine in DMEM plus 50 mU of xanthine oxidase/ml medium for 3 h and cells pretreated with PEG-catalase or catalase to 500 ItM H2 0 2 in DMEM for 3 h. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release into the media as a percentage of total cellular LDH activity (LDL-20 kit; Sigma) before and after exposure to oxidant stress. The sensitivity of the LDH assay is 1 mU/ml. Control incubations of 1 x 106 alveolar type IT cells release 1 to 3 mU of LDH over 3 h. Statistical Methods All experiments were done in triplicate. All studies were replicated 6 times. The results are expressed as mean ± SEM of the 6 means/study. Comparisons between antioxidant enzyme activities at various experimental conditions were done using Student's t test for unpaired samples.
Results Mean ± SD plating efficiency of alveolar type IT cells measured by cell counting after incubation for 20 h was 30 ± 3%, so that the experiments reported below were performed with 1 x 106 cells (in 2 ml DMEM). Protein content was 99 ± 14 Itg protein/lfr' alveolar type IT cells, and DNA content 7.41 ± 0.54ItgDNA/106 cells. The activity of SOD was 2,900 U/mg protein, of catalase 37,200 U/mg protein, of PEG-SOD 3,020 U/mg protein, and of PEG-catalase 37,200 U/mg protein. In freshly isolated alveolar type IT cells, mean ± SEM SOD activity was 213 ± 11 U/mg DNA and catalase activity 2,710 ± 180 U/mg DNA. All experiments were started 20 h after establishment in culture, when SOD activity was 185 ± 11 U/mg DNA and catalase activity 945 ± 51 U/mg DNA. At 44 h after establishment in culture, the endpoint for 24-h uptake studies and starting point for exposure to oxidant stress, SOD activity was 177 ± 11 U/mg DNA and catalase activity 791 ± 60 U/mg DNA. Incubation of cultured alveolar type IT cells with PEG-SOD for 24 h produced a dose-dependent increase of intracellular SOD activity (Figure 1). Uptake reached a maximum of 0.12% of the added enzyme at 750 to 1,500 U PEG-SOD/ 106 cells and decreased to 0.07% at 3,000 U of PEG-SOD/ 106 cells. SOD activity in cells treated with similar amounts
366
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 41991
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Figure 1. Dose-dependent changes in SOD activity in U/mg DNA in 1()6 alveolar type II cells incubated for 24 h with 500 to 3,000 U of PEG-SOD or SOD. Triangle indicates P < 0.005 and stars indicate P < 0.001 versus control.
of SOD or inactivated PEG-SOD did not increase by more than 11%. At 3,000 V of PEG-SOD/IQ6 cells, mean SOD activity had increased by 173%. Incubation with PEG-catalase gave results similar to those with PEG-SOD, with a mean increase in catalase activity of 130% at 40,000 V of PEG-catalase/l06 cells (Figure 2). Uptake reached a maximum of 0.03% of the added enzyme at 20,000 V of PEGcatalase/lQ6 cells. Catalase activity in cells treated with catalase or inactivated PEG-catalase did not increase by more than 11%. The increments in both SOD and catalase activity after PEG-SOD and PEG-catalase could not be removed with extensive washing of treated cells with either PEG alone or trypsin. Addition of 1% PEG to alveolar type II cells did not significantly influence intracellular SOD or catalase activity. Using 106 cells treated with 750 or 1,500 V of PEG-SOD for periods up to 24 h (Figure 3), we found that cellular uptake of PEG-SOD was maximal by 4 h (0.011 %/U/h of added PEG-SOD) and lowest by 24 h (0.002%/V/h). Addition of 20,000 and 40,000 V of PEG-catalase/lQ6 cells for periods up to 48 h (Figure 4) led to maximal uptake velocity by 4 h: 0.0019%/V/h in the presence of 20,000 V and 0.0012%IVlh in the presence of 40,000 V of PEG-catalase. By 48 h, uptake had fallen to 0.0002 %/V /h for 20,000 V and to 0.00001%/ V/h for 40,000 V of PEG-catalase/lQ6 cells.
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This study tested whether adducts formed by covalent linkage of superoxide dismutase (SOD) or catalase to polyethylene glycol (PEG) could augment SOD and catalase activity in alveolar type II cells and document enhanced resistance to oxidant damage. Alveolar type II cells were isolated from adult, pathogenfree rats. Antioxidant enzymes were added to the medium of cell cultures in various concentrations for periods up to 48 h. Incubation with 500 to 3,000 U of PEG-SOD or 10,000 to 40,000 U of PEGcatalase/1Q6 cells produced a dose-response-related increase in intracellular enzyme activity in comparison with controls (untreated or treated with SOD or catalase, inactivated PEG-SOD or PEG-catalase, or PEG alone). Uptake was maximal during the first 4 h. Using fluorescent label (fluorescein isothiocyanate) bound to PEG-catalase, we found intracellular localization of the labeled enzyme. Exposure to H20 2 led to reduced cytotoxicity in cells pretreated with PEG-catalase than in controls. We conclude that supplementation with PEG-SOD or PEG-catalase enhanced the activity of these enzymes in alveolar type II cells and increased their resistance to oxidant stress.
The alveolar epithelium is exposed to oxygen and free oxygen radicals derived from intracellular metabolic processes and inflammatory cells (1, 2). Hyperoxia increases the intracellular production of free oxygen radicals and causes an increase in alveolar epithelial permeability (3), morphologic damage of the alveolar type II cells (4), and inhibition of the repair processes following epithelial injury (5). Free oxygen radicals released by activated inflammatory cells that migrate into the alveoli also contribute to lung injury (6). During the final 10 to 15% of gestation, both fetal lung antioxidant enzyme levels and lung surfactant content increase rapidly (7). Therefore, some premature infants are born both with surfactant insufficiency and with antioxidant enzyme insufficiency (8). An imbalance between the production and the scavenging of free oxygen radicals may be important in the pathogenesis of various lung diseases such as bronchopulmonary dysplasia of premature infants (9). Manipulation of intracellular antioxidant enzyme activity may therefore eventually lead to therapeutic strategies. Successful attempts to modify pulmonary oxygen toxicity in adult animals have involved the augmentation of the intra(Received in original form June 19, 1990 and in revised form September 26, 1990) Address correspondence to: Frans J. Walther, M.D., Ph.D., Department of Pediatrics, King-Drew Medical Center, 12021 S. Wilmington Avenue, Los Angeles, CA 90059. Abbreviations: Oulbecco's modified Eagle's medium, DMEM; fetal bovine serum, FBS; fluorescein isothiocyanate, FITC; hydrogen peroxide, H202; lactate dehydrogenase, LDH; polyethylene glycol, PEG; superoxide dismutase, SOD. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 364-368, 1991
cellular antioxidant capacity by endotoxin, preexposure to hyperoxia, hormonal stimulation, or the administration of exogenous superoxide dismutase (SOD) and catalase, which are responsible for the metabolism of superoxide anions and hydrogen peroxide (H20 2 ) , respectively. The therapeutic potential of SOD and catalase is limited by their short circulatory half-life of 10 to 20 min (10, 11), sensitivity to Proteases, and inability to penetrate cell membranes. Covalent attachment of SOD and catalase to the inert linear polymer, monomethyoxy-polyethylene glycol (PEG), or encapsulation in liposomes avoids these limitations. PEG conjugation increases their circulatory half-life from 6 min to 30 to 40 h in the rat (12), reduces their antigenicity and sensitivity to proteases, and improves their stability in aqueous solutions (10, 13, 14). Beckman and associates (11) showed that cultured endothelial cells take up PEG-SOD and PEG-catalase and that the increased cellular enzyme activity provides protection from the damaging effectsof reactive oxygen species. Alveolar type II cells produce pulmonary surfactant and are the progenitor cells for the type I cells that line most of the alveolar surface and facilitate alveolar-arterial gas exchange. We have studied whether addition of SOD and catalase conjugated to PEG to primary cultures of alveolar type II cells could increase intracellular activity of these enzymes and reduce their vulnerability to oxidant stress.
Materials and Methods Materials Native bovine copper/zinc-SOD and catalase and PEG-SOD (10) and PEG-catalase (10) were obtained from Sigma Chemical Co. (St. Louis, MO), elastase from Worthington
Walther, Wade, Warburton et al.: Antioxidant Enzyme Activity in Alveolar Type IT Cells
365
Biochemical Corp. (Freehold, NJ), fetal bovine serum (FBS) from Hyclone Laboratories (Logan, UT), tissue culture media from GIBCO (Grand Island, NY), plasticware from Falcon (Oxnard, CA), and pathogen-free rats from Charles Rivers Labs (Portage, MO. All other enzymes and biochemicals, of the highest grade available, were obtained from Sigma.
Fluorescent Labeling of Antioxidant Enzymes Antioxidant enzymes were labeled by adding 0.3 mg of fluorescein isothiocyanate (FITC) to 1.0ml of 10 mg/ml catalase or PEG-catalase in 50 mM sodium carbonate (21). Following stirring for 1 hat 4° C at a pH of 9.5, unbound FITC was removed by gel filtration chromatography on Sephadex G-25 eluted with PBS.
Primary Culture of Alveolar Type II Cells Alveolar type IT cells were harvested from male, 250- to 300-g, pathogen-free rats following the lung lavage, elastase digestion, and rat IgG differential adherence protocol of Dobbs and colleagues (15). This methodology yields 2 x 107 to 3 X 107 alveolar type IT cells/rat with> 90% viability by trypan blue staining and> 90 % purity by phosphene 3R fluorescent staining (16). Alveolar type IT cells were placed in culture in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS at a density of 3 x 106 to 4 x 106 cells/2 ml and incubated for 20 h at 37° C in a 5% CO 2:air incubator. Nonadherent cells were then removed by washing 3 times with phosphatebuffered saline (PBS), test solutions dissolved in DMEM were added, and the plates were returned to the incubator for the duration of the uptake study. Cells were washed 3 times with PBS, once with 1% PEG, and once with trypsin (0.05% wt/vol and 0.2% EDTA in DMEM), and then lysed with 0.1% Triton X-100. The suspension was centrifuged at 4° Cat 1,000 x g for 10 min. The supernatant was used for SOD, catalase, DNA, and protein assays.
Protein and DNA Content The amount of protein in the samples was determined by a modification of the Lowry method (22), and the DNA content by the fluorimetric method of Erwin and coworkers (23).
SOD and Catalase Assays SOD activity was assayed by inhibition of the reduction of cytochrome C in the xanthine oxidase reaction (17) using a modification described by Forman and Fisher (18). Endogenous reduct ants in the samples, which could interfere with the assay by producing nonsuperoxide-dependent cytochrome C reduction, were eliminated by mixing the sample and 5 X 10-5 M cytochrome C in 0.6 ml of 0.5 M potassium phosphate buffer, pH 7.8, and allowing the reduction and reoxidation by endogenous cytochrome oxidase of the cytochrome C to occur. The reaction was followed at 550 nm in a DU-7 spectrophotometer (Beckman Instruments, Fullerton, CA). After initial incubation, 10-5 M cyanide to inhibit cytochrome oxidase was added, followed by xanthine, xanthine oxidase, and H20 to a final volume of 3 ml. One unit of SOD equaled 50% inhibition of cytochrome C reduction. Catalase activity was measured by the rate of reduction of H 20 2 substrate, followed spectrophotometrically at 240 nm (19). Inactivation of PEG-SOD and PEG-catalase PEG-SOD was inactivated by mixing 10 mg PEG-SOD/ml with 100 mM H20 2 at room temperature for 6 h, then was dialyzed 3 times for 8 h in PBS at 4° C, filtered (0.22 j-tm), and stored at 4°C (20). PEG-catalase was inactivated by heating at 90° C for 15 min, then passed through a 27-gauge needle and stored at 4° C. Inactivated PEG-SOD and PEGcatalase lacked SOD or catalase activity when assayed and did not precipitate following inactivation.
Cytotoxicity Following the initial 20 h of incubation and 24 h of treatment with antioxidant enzymes, alveolar type IT cells pretreated with PEG-SOD or SOD were exposed to 1 mM xanthine in DMEM plus 50 mU of xanthine oxidase/ml medium for 3 h and cells pretreated with PEG-catalase or catalase to 500 ItM H2 0 2 in DMEM for 3 h. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release into the media as a percentage of total cellular LDH activity (LDL-20 kit; Sigma) before and after exposure to oxidant stress. The sensitivity of the LDH assay is 1 mU/ml. Control incubations of 1 x 106 alveolar type IT cells release 1 to 3 mU of LDH over 3 h. Statistical Methods All experiments were done in triplicate. All studies were replicated 6 times. The results are expressed as mean ± SEM of the 6 means/study. Comparisons between antioxidant enzyme activities at various experimental conditions were done using Student's t test for unpaired samples.
Results Mean ± SD plating efficiency of alveolar type IT cells measured by cell counting after incubation for 20 h was 30 ± 3%, so that the experiments reported below were performed with 1 x 106 cells (in 2 ml DMEM). Protein content was 99 ± 14 Itg protein/lfr' alveolar type IT cells, and DNA content 7.41 ± 0.54ItgDNA/106 cells. The activity of SOD was 2,900 U/mg protein, of catalase 37,200 U/mg protein, of PEG-SOD 3,020 U/mg protein, and of PEG-catalase 37,200 U/mg protein. In freshly isolated alveolar type IT cells, mean ± SEM SOD activity was 213 ± 11 U/mg DNA and catalase activity 2,710 ± 180 U/mg DNA. All experiments were started 20 h after establishment in culture, when SOD activity was 185 ± 11 U/mg DNA and catalase activity 945 ± 51 U/mg DNA. At 44 h after establishment in culture, the endpoint for 24-h uptake studies and starting point for exposure to oxidant stress, SOD activity was 177 ± 11 U/mg DNA and catalase activity 791 ± 60 U/mg DNA. Incubation of cultured alveolar type IT cells with PEG-SOD for 24 h produced a dose-dependent increase of intracellular SOD activity (Figure 1). Uptake reached a maximum of 0.12% of the added enzyme at 750 to 1,500 U PEG-SOD/ 106 cells and decreased to 0.07% at 3,000 U of PEG-SOD/ 106 cells. SOD activity in cells treated with similar amounts
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 41991
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Figure 1. Dose-dependent changes in SOD activity in U/mg DNA in 1()6 alveolar type II cells incubated for 24 h with 500 to 3,000 U of PEG-SOD or SOD. Triangle indicates P < 0.005 and stars indicate P < 0.001 versus control.
of SOD or inactivated PEG-SOD did not increase by more than 11%. At 3,000 V of PEG-SOD/IQ6 cells, mean SOD activity had increased by 173%. Incubation with PEG-catalase gave results similar to those with PEG-SOD, with a mean increase in catalase activity of 130% at 40,000 V of PEG-catalase/l06 cells (Figure 2). Uptake reached a maximum of 0.03% of the added enzyme at 20,000 V of PEGcatalase/lQ6 cells. Catalase activity in cells treated with catalase or inactivated PEG-catalase did not increase by more than 11%. The increments in both SOD and catalase activity after PEG-SOD and PEG-catalase could not be removed with extensive washing of treated cells with either PEG alone or trypsin. Addition of 1% PEG to alveolar type II cells did not significantly influence intracellular SOD or catalase activity. Using 106 cells treated with 750 or 1,500 V of PEG-SOD for periods up to 24 h (Figure 3), we found that cellular uptake of PEG-SOD was maximal by 4 h (0.011 %/U/h of added PEG-SOD) and lowest by 24 h (0.002%/V/h). Addition of 20,000 and 40,000 V of PEG-catalase/lQ6 cells for periods up to 48 h (Figure 4) led to maximal uptake velocity by 4 h: 0.0019%/V/h in the presence of 20,000 V and 0.0012%IVlh in the presence of 40,000 V of PEG-catalase. By 48 h, uptake had fallen to 0.0002 %/V /h for 20,000 V and to 0.00001%/ V/h for 40,000 V of PEG-catalase/lQ6 cells.
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