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Efficient protection by cationized catalase against H2O2 injury in primary cultured alveolar epithelial cells
Journal of Controlled Release 121 (2007) 74 – 80 www.elsevier.com/locate/jconrel
Efficient protection by cationized catalase against H2O2 injury in primary cultured alveolar epithelial cells Takayuki Nemoto, Shigeru Kawakami, Fumiyoshi Yamashita, Mitsuru Hashida ⁎ Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Received 15 November 2006; accepted 17 May 2007 Available online 24 May 2007
Abstract Increasing evidence suggests that hydrogen peroxide plays an important role in alveolar epithelial injury produced during many inflammatory lung diseases. In this study, the successful prevention of hydrogen peroxide (H2O2)-induced injury in primary cultured rabbit alveolar epithelial cells by cationized catalase is described. Cationized catalase was synthesized by direct chemical modification to enhance its association with alveolar epithelial cells. Cationized catalase exhibited a 22.3-fold higher cellular association at 2 h than native catalase, and incubation of cationized catalase with the cells produced a 2.19-fold intracellular catalase activity, which suggested that cationized catalase distributed both to the cell membrane and into the cell interior. Cationized catalase markedly suppressed H2O2-induced cell injury. In addition, electron spin resonance spectrometry analysis revealed that cationized catalase effectively eliminated H2O2 produced in the medium by glucose plus glucose oxidase. On the other hand, polyethylene glycol-modified catalase (PEG-catalase) did not have any protective effect against H2O2-induced cell injury although PEG-catalase exhibited a 2.49-fold higher cellular association at 2 h than native catalase. These results suggest that cationization of catalase is a promising strategy for the treatment of many of inflammatory lung diseases. © 2007 Published by Elsevier B.V. Keywords: Catalase; Cationization; Polyethylene glycol; Hydrogen peroxide; Alveolar epithelium
1. Introduction The lung epithelium is often directly exposed to oxidants in the ambient air, including ozone, nitrogen dioxide, diesel exhaust, and cigarette smoke. Exposure to hyperoxia [1] or paraquat [2] increases intracellular production of reactive oxygen species (ROS). As well as intracellular metabolic processes, alveolar macrophages and neutrophils are possible sources of ROS [3]. Increased ROS damages a variety of biomolecules such as DNA, lipids, and proteins, induces apoptosis, and causes inflammation via activation of transcriptional factors, leading to gene expression of pro-inflammatory mediators [4]. Thus, oxidative stress is the basis of many lung diseases. The alveolar epithelium is composed of alveolar type I and type II cells. In many types of inflammatory lung injury, alveolar type I cells exhibit more evidence of injury than alveolar type II
⁎ Corresponding author. Tel.: +81 75 753 4525; fax: +81 75 753 4575. E-mail address: [email protected] (M. Hashida). 0168-3659/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.jconrel.2007.05.020
cells [5–7]. One of the reasons for this is that alveolar type I cells are more likely to be injured because they contain less catalase activity than alveolar type II cells [8]. It has been shown that alveolar type II cells in intact rat lungs have many catalasecontaining organelles, whereas alveolar type I cells have relatively few. Furthermore, it has also been shown that there are no significant differences in the components of glutathionedependent systems between alveolar type I cells and type II cells. Therefore, efficient delivery of catalase to alveolar type I cells would be effective in the treatment of inflammatory lung injury. Catalase catalyzes the dismutation of H2O2 to H2O and is a potential antioxidant therapeutic agent. Much attention has been paid to inhalation of catalase as a strategy for the treatment of ROS-mediated lung injury. However, catalase exhibits a low cellular association due to its large molecular size and charge, which have limited its effective use. In order to overcome the limitation, liposomal encapsulation, conjugation with transferrin or polyethyleneglycol (PEG) had been investigated [9]. Although strategies for enhanced delivery of catalase to the sites of action have focused on the use of liposome encapsulation
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[10], the preparation of liposome-encapsulated catalase requires the succinoylation of catalase prior to use in order to prevent liposome aggregation [10,11]. Previously, Rojanasakul et al. [12] demonstrated that transferrin-conjugated catalase, taken up by alveolar cells via alveolar transferrin receptors, protects the cells from oxidative injury. However, the high level of native transferrin in the pulmonary lining fluid would compete with transferrin-conjugated catalase for the receptors [13]. Since polyethyleneglycol (PEG) can be adsorbed onto the cell membrane in the phospholipids headgroup region [14], PEGylation of catalase (PEG-catalase) enhances its cellular uptake and combats cell injury by H2O2 [15]. Cationized protein binds to the negatively charged proteoglycans of the cell surface via electrostatic interaction and is slowly internalized into the cells [16]. Considering that alveolar type I cells occupy 95 % of the alveolar epithelium [17], cationized catalase is expected to be efficiently taken up by alveolar type I cells. Isolated alveolar type II cells, when cultured, lose their phenotypic characteristics and differentiate into alveolar type I-like cells [18]. In the present study, the effectiveness of cationized catalase against H2O2-mediated oxidative stress was evaluated in primary cultured alveolar type II cells. The results obtained were compared with those of catalase and PEG-catalase. 2. Materials and methods 2.1. Materials Bovine liver catalase was obtained from Sigma (St. Louis, MO). Hexamethylenediamine was obtained from Tokyo Chemical Industry (Tokyo, Japan). A product of PEG (2,4-bis (O-methoxypolyethylene glycol)-6-chloro-s-triazin) was obtained from Seikagaku Corp. (Tokyo, Japan). 111Indium chloride was kindly supplied by Nihon Medi Physics (Takarazuka, Japan). Diethylenetriaminepentaacetic acid (DTPA) anhydride was purchased from Dojindo Laboratory (Kumamoto, Japan). Glucose oxidase and 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Hydrogen peroxide and Iron (II) Sulfate Heptahydrate (FeSO4·7H2O) were obtained from Wako Pure Chemical (Osaka, Japan). 5,5-Dimethyl-1pyrroline-N-oxide (DMPO) was obtained from Labotec (Tokyo, Japan). Porcine pancreatic elastase and pure Griffonia simplicifolia lectin (GS-I) were obtained from Worthington Biochemical (Freehold, NJ) and EY Laboratories (San Mateo, CO), respectively. Soybean trypsin inhibitor, human recombinant epidermal growth factor (EGF), hydrocortisone, Dulbecco's modified minimum Eagle's medium nutrient mixture F-12 Ham (DMEM/F12), and Eagle's minimum essential medium Joklik modified for suspension culture (SMEM) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Equitech-Bio (Kerrville, TX). ITS+Premix, type I rat tail collagen, and recombinant human fibronectin were obtained from Becton Dickinson Biosciences (Bedford, MA). Other cell culture reagents were obtained from Invitrogen (Grand Island, NY).
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2.2. Animals New Zealand white rabbits (1.2–1.5 kg) were purchased from Biotek (Saga, Japan). Animals were maintained under conventional housing conditions and all animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences of Kyoto University. 2.3. Primary culture of alveolar type II cells For isolation of alveolar type II cells, three in-house balanced salt solutions were prepared: one balanced salt solution (BSSA) was composed of 137 mM NaCl, 5.0 mM KCl, 0.7 mM Na2HPO4, 10 mM HEPES, 5.5 mM glucose at a pH adjusted to 7.4, another balanced salt solution (BSSB) was BSSA supplemented with 1.8 mM CaCl2 and 1.2 mM MgSO4, the other balanced salt solution (BSSC) was BSSA supplemented with 3 mM EDTA. Alveolar type II cells were isolated from the rabbit lung using the method of Shen et al. [19]. Rabbits were first injected with heparin (1000 U/kg) and then euthanized by rapid injection of sodium pentobarbital (1.5 ml/kg), via a marginal ear vein. The abdominal cavity was opened. While the lung was ventilated manually through a tracheal cannula with a 60 ml syringe, it was perfused with BSSC via the pulmonary vein. The lung was washed several times with BSSA and BSSC, alternately, and then once with SMEM containing 2 U/ml elastase. Following this, about 50 ml 2 U/ml elastase solution was instilled through the trachea and the lung was incubated in BSSB at 37 °C for 35 min. The lung was excised from the trachea and the bronchi, and transferred to SMEM containing 1.67 mg/ml trypsin inhibitor. The pieces of lung were minced, suspended in SMEM, and sequentially filtered through gauze, a 40 μm cell strainer, and a 15 μm nylon mesh. The cell suspension was centrifuged at 200 ×g for 8 min at 4 °C. The cell pellets were resuspended in SMEM, incubated with 16 μg/ml GS-I lectin at room temperature for 30 min to eliminate alveolar macrophages and red blood cells and subsequently filtered through a 15 μm nylon mesh. The filtered cell suspension was centrifuged and the cell pellet was resuspended in DMEM/F12 containing 10% FBS. Cell viability was estimated to be more than 95% by the trypan blue dye exclusion method. Purified type II cells were plated onto plastic plates (Becton Dickinson, Lincolin Park, NJ) at a density of 0.88 × 106 cells/ cm2. The plastic plates were pretreated for 4 hr with 0.25 ml/ cm 2 PBS (−) containing 30 μg/ml collagen, 10 μg/ml fibronectin, and 10 μg/ml BSA. From day 3 of culture onwards, the medium was changed to serum-free DMEM/F12 supplemented with ITS+, 10 ng/ml EGF and 1 μM hydrocortisone. The cultures were fed every other day with serum-free defined medium and maintained in a humidified 5% CO2 incubator at 37 °C. When the alveolar type II cells were plated on Transwells (Corning Costar, Cambridge, MA) pretreated with collagen and
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fibronectin, the transepithelial electric resistance exhibited more than 2 kΩ cm2 from day 4 to day 9. All experiments were carried out on day 5 of culture. 2.4. Synthesis and characterization of cationized catalase and PEG-catalase Cationized catalase was obtained by coupling catalase with hexamethylenediamine using carbodiimide as a catalyst. Twenty five mg catalase was added to 30 ml 2 M hexamethylenediamine and the pH was adjusted to 6.0 using HCl. Thirty minutes and one hour later, 25 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to each. The reaction mixture was agitated for 6 hr at a pH of 6.0, followed by extensive dialysis against distilled water. Then the eluent was concentrated by ultrafiltration, with replacement of the solvent with distilled water, and finally lyophilized. Polyetheleneglycol-conjugated catalase (PEG-catalase) was synthesized according to our previously report [20]. Briefly, activated PEG (250 mg) was added to 25 mg catalase in 5 ml 50 mM borate buffer (pH 9.2). The reaction mixture was agitated for 12 hr at 4 °C in the dark. Then the eluent was concentrated and unreacted activated PEG was removed by ultrafiltration, with replacement of the solvent with distilled water, and finally lyophilized. The activities of native catalase and catalase derivatives were determined by monitoring the ability to degrade H2O2 at 240 nm [21]. The enzymatic activity of native catalase, cationized catalase, and PEG-catalase was 12263.0, 4183.6, and 11485.6 U/mg protein, respectively. The number of amino groups was determined by trinitrobenzene sulfonic acid using glycine as a standard [22]. The Bradford method was used to measure the concentration of catalase using BSA as a standard [23]. The number of modified amino groups of cationized catalase and PEG-catalase was estimated to be 24.5 and 80.3 per catalase molecule, respectively.
off into 500 μl of saline. The cell suspension was homogenized by 3 cycles of freezing in liquid nitrogen and thawing in a water bath at 37 °C. After centrifugation at 15000 ×g for 12 min, the supernatant was used to measure catalase activity. Catalase activity was determined by monitoring the degradation of H2O2 at 240 nm [21]. 2.7. Cytotoxicity assays Cytotoxicity was evaluated by MTT assay. Alveolar type II cells were incubated with glucose oxidase (12.5–200 mU). DMEM-F12 medium used in this study includes 17.5 mM glucose. After given time periods at 37 °C, the cells were washed twice with PBS (−) and subjected to MTT assay. The protective effect of catalase, cationized catalase and PEGcatalase against H2O2 was also evaluated by MTT assay. In this experiment, 100–1000 U/ml catalase, cationized catalase and PEG-catalase were pre-incubated for 2 h with alveolar type II cells before the incubation with glucose oxidase. In addition, morphological changes were also observed using fluorescence microscopy (Biozero Bz-8000 Keyence Inc., Osaka, Japan). 2.8. Electron spin resonance spectrometry analysis U The OH produced by Fenton's reaction between Fe2+ and H2O2 was trapped by DMPO. Cell culture medium was added to the reaction mixture containing DTPA (400 μM), FeSO4 (100 μM), and DMPO (15 mM). The reaction mixture was transferred to a flat quartz cuvette and placed in the cavity of an U ESR spectrometer (JEOL, Tokyo, Japan). The OH produced by Fenton's reaction between Fe2+ and H2O2 was trapped by DMPO, and the amount of the DMPO-OH spin adduct formed was measured exactly 5 min after the addition of H2O2 by ESR U spectrometry. The quantity of OH generated is determined by
2.5. Cellular uptake of [111In]-labeled catalase and catalase derivatives Alveolar type II cells cultured on 24-well plastic plates were incubated with 1 ml serum-free culture medium containing 100,000 cpm of 111 In-labeled native catalase, cationized catalase and PEG-catalase at 37 °C. After incubation for given time periods, the cells were washed three times with PBS (−) and incubated overnight at 37 °C with 1 ml 0.3 N NaOH containing 0.1% Triton X-100. Aliquots of the lysate were taken for determination of 111In radioactivity and protein content. The radioactivity was counted in a well-type NaI scintillation counter (ARC-500, Aloka, Tokyo, Japan), and the protein content was determined by the Bradford method using BSA as a standard [23]. 2.6. Intracellular catalase activity Alveolar type II cells plated on a 6-well plate were incubated with 1000 U/ml native catalase, cationized catalase and PEGcatalase for 2 h. The cells were washed with PBS (−) and scraped
Fig. 1. Cellular association of catalase and catalase derivatives in primary cultured rabbit alveolar type II cells. Alveolar type II cells were incubated with 100,000 cpm of 111In-labeled native catalase, cationized catalase and PEGcatalase at 37 °C. After given time periods, the associated radioactivity and cellular protein were measured. % of dose means the ratio of associated radioactivity to the radioactivity of added each labeled 111In catalase. Results are expressed as means + S.D. (n = 3).
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amplitude, 1 G; response time, 0.1 s; amplitude, 7.9 × 100; and sweep time, 2 min. 2.9. Statistical analysis Statistical analysis was carried out by one-way ANOVA followed by the Student–Newmann–Keuls multiple comparison test, at a significance level of P b 0.05. 3. Results 3.1. Cellular association of catalase and catalase derivatives
Fig. 2. Intracellular catalase activity in primary cultured rabbit alveolar type II cells. Alveolar type II cells were incubated with catalase, cationized catalase and PEG-catalase (1000 U/ml) for 2 h, and then the intracellular catalase activity was measured. Results are expressed as means + S.D. (n = 3). ⁎P b 0.05 vs. control cells; N.S., not significant from control.
the intensity of the ESR signal of the DMPO-OH spin adduct. The signal intensity was evaluated using the relative peak of the second signal of the quartet of the DMPO-OH spin adduct and the intensity of Mn2+ as an internal standard. The ESR settings were as follows: magnetic field, 3350 ± 100 G; microwave power, 5 mW; modulation frequency, 100 KHz; modulation
Fig. 1 shows their cellular association in primary cultured rabbit alveolar type II cells. Their cellular association increased in a time-dependent manner for up to 2 h. The associated amount of native bovine liver catalase, cationized catalase, and PEG-catalase at 2 h was 0.51, 11.60, and 1.30% of dose per mg protein, respectively. Thus, cationized catalase showed a much higher association than native catalase and PEG-catalase. Intracellular catalase activity was also measured following 2 h incubation with 1000 U/ml of native catalase and catalase derivatives (Fig. 2). The level of endogenous catalase activity was 15.89 ± 1.82 U/mg protein. When the cells were incubated with native catalase, cationized catalase and PEG-catalase, the intracellular catalase activity was 16.23 ± 1.87, 34.84 ± 2.48, and 12.47 ± 1.27 U/mg protein, respectively. 3.2. Protective effect of catalase, catalase derivatives against the cytotoxic effect of hydrogen peroxide The viability of alveolar type II cells treated with glucose oxidase was evaluated by MTT assay (Fig. 3). As the concentration of glucose oxidase increased, the viability of the cells
Fig. 3. Cytotoxic effect of hydrogen peroxide in primary cultured rabbit alveolar type II cells. (A) Alveolar type II cells were incubated with a given concentration of glucose oxidase for 6 h. (B) Alveolar type II cells were incubated with glucose oxidase (50 mU/ml) for given time periods. Cellular viability was assessed by MTT assay. Results are expressed as means ± S.D. (n = 3). (C) Morphological change by hydrogen peroxide in primary cultured rabbit alveolar type II cells. Alveolar type II cells were observed using fluorescence microscopy after incubation with glucose oxidase (50 mU/ml) for 6 h. Control panel shows untreated cultures; + glucose oxidase panel demonstrate cultures treated with glucose oxidase (50 mU/ml) for 6 h.
Fig. 4. Protective effect of catalase and catalase derivatives against the cytotoxic effect of hydrogen peroxide in primary cultured rabbit alveolar type II cells. Alveolar type II cells were treated with catalase, cationized catalase and PEGcatalase (100–300–1000 U/ml) from 2 h before the addition of glucose oxidase (50 mU/ml). Cultures were further incubated with glucose oxidase for 6 h and cellular viability was assessed by MTTassay. Results are expressed as means + S.D. (n = 3). ⁎P b 0.05 vs. glucose oxidase (GO)-treated cells.
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Fig. 5. Effect of catalase and catalase derivatives on H2O2 formation by ESR analysis. Alveolar type II cells were treated with catalase, cationized catalase and PEG-catalase (100–300–1000 U/ml) from 2 h before the addition of glucose oxidase (50 mU/ml). Cultures were further incubated with glucose oxidase for 2 h and cell culture medium was added to the reaction mixture containing DTPA (400 μM), FeSO4 (100 μM), and DMPO (15 mM). The amount of DMPO-OH spin adduct generated was detected after exactly 5 min using ESR spectrometry.
decreased (Fig. 3A). When treated with 50 mU glucose oxidase, the cells were injured in a time-dependent manner (Fig. 3B). The injury of alveolar type II cells by glucose oxidase was also confirmed by morphological observation (Fig. 3C). The cells were pre-incubated with 100–1000 U/ml of native catalase, cationized catalase, and PEG-catalase for 2 h. Fig. 4 shows the protective effect of native catalase, cationized catalase and PEG-catalase against H2O2-induced cell injury. Cationized catalase showed its protective effect against H2O2induced cell injury in a dose-dependent manner, whereas native catalase and PEG-catalase had no effect. 3.3. Electron spin resonance spectrometry analysis To further confirm that H2O2 was actually eliminated by cationized catalase, ESR analysis was carried out. Fig. 5 shows the scavenging effect of native catalase and catalase derivatives on H2O2 formation produced by glucose plus glucose oxidase in the cell culture medium. HMD-catalase markedly reduced the DMPO-OH spectrum, whereas catalase and PEG-catalase had hardly any effect. 4. Discussion Oxidative stress is known to play a crucial role in many lung diseases. Accumulating evidence has emerged to support the crucial role of H2O2 in lung diseases. In comparison with appropriate control subjects, patients with ARDS have higher concentrations H2O2 in urine [24] or exhaled air [25]. Patients with COPD have higher levels of H2O2 in exhaled breath condensates than healthy smokers [26]. H2O2 reduces surfactant
metabolism [27] and the ATP level in alveolar type II cells [28,29], and inhibits alveolar epithelial wound repair by induction of apoptosis [30]. Thus, effective elimination of H2O2 would be an effective strategy for the treatment of many lung diseases. In inflammatory lung conditions, H2O2 is mainly produced from inflammatory cells such as alveolar macrophages and neutrophils [3]. Cationized catalase showed a much higher cellular association than native catalase (Fig. 1), and exhibited a significant protective effect against H2O2-induced cell injury (Fig. 4). Glucose oxidase produces mainly H2O2 outside the cells due to its large size, 160 kDa. In this study, therefore, cationized catalase present on the cellular membrane appears to mainly contribute to its protective effect against H2O2-induced cell injury. This statement is supported by our direct observation that cationized catalase significantly eliminated H2O2 produced by glucose oxidase (Fig. 5). These observations show that cationized catalase could be effective against the attack by H2O2 produced by lung inflammatory cells such as alveolar macrophages and neutrophils. A high concentration of oxygen is commonly used to treat premature babies, children, and adults with pulmonary failure. However, prolonged exposure to high oxygen tension is known to induce lung tissue damage. In hyperoxia, an excess of ROS is produced intracellularly, leading to cell death [31]. Cationized catalase showed a much higher cellular association than native catalase (Fig. 1). Incubation with cationized catalase enhanced intracellular catalase activity 2.19-fold (Fig. 2). These results suggest that a part of the cationized catalase is internalized by the cells. Thus, intracellular localization of cationized catalase would be effective against the attack by H2O2 produced into the alveolar epithelium during hyperoxia. In this study, the enzyme activity of cationized catalase and PEG-catalase was 34.1 % and 93.6 % of native catalase and the number of modified amino groups was 24.5 and 80.3 per catalase molecule, respectively. These were the consistent results of Ma et al. [32] about cationized catalase and Yabe et al. [20] about PEG-catalase. PEG-catalase maintained much higher activity than cationized catalase although the ratio of modified amino group in PEG-catalase was much higher than that in cationized catalase. Cationization of catalase with hexamethylenediamine can cause changes in conformation [32]. The change in the surface charge by cationization may be a cause of deactivation. Furthermore, the modification of the protoporphyrin IX with Fe3+ group and the change in the vicinity of Fe3+ group may be another cause of the deactivation. On the other hand, there is no information on change in conformation by PEGylation. The cellular uptake study reveals that PEG-catalase had a 2.49-fold higher cellular association at 2 h than native catalase (Fig. 1). This cellular uptake of PEG-catalase is much lower than that of cationized catalase. Cationized protein binds to the negatively charged proteoglycans of the cell surface via electrostatic interaction and is slowly internalized into the cells [16]. It is difficult to explain the difference in cellular uptake characteristics between PEG-catalase and cationized catalase because the cellular uptake mechanism of PEG-catalase remains unknown. The association of PEG with the cellular
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membrane may be much weaker than that of cationized protein, which binds to the cellular membrane via electrostatic interaction. Although PEG-catalase showed a 2.49-fold higher association at 2 h than native catalase (Fig. 1), PEG-catalase produced no enhancement in intracellular catalase activity at 2 h at all (Fig. 2). The associated amount in Fig. 1 indicates the total quantity of PEG-catalase both adsorbed onto the cellular membrane and internalized into the cells. On the other hand, catalase activity measured in Fig. 2 is intracellular. Therefore, PEGcatalase might be present on the cellular membrane at 2 h. Beckman et al. [33] demonstrated that PEG-catalase showed an initial rapid cellular association within the first 4 h in endothelial cells, followed by a slower, continuous uptake over the next 24 h. Walther et al. [15] also reported similar cellular uptake characteristics of PEG-catalase in alveolar type II cells. These observations support our suggestion that PEG-catalase might be present on the cellular membrane at 2 h. However, although cationized catalase enhances intracellular catalase activity 2.19fold after a 2 h incubation (Fig. 2), PEG-catalase did not show any protective effect against H2O2-induced cell injury. ESR analysis showed that pre-incubation of PEG-catalase with the cells does not lead to the elimination of resulting H2O2 into the medium (Fig. 5D). One possible explanation is that the 2.49-fold higher cellular association of PEG-catalase, compared with native catalase, is not sufficient to produce a protective effect. In conclusion, cationized catalase exhibited an efficient protective effect against H2O2-induced cell injury. It was suggested that cationized catalase bound to the cellular membrane and was internalized into the cells. This is also likely to happen after inhalation in vivo. Accordingly, cationized catalase is expected to be an effective treatment for various ROS-related lung diseases. Acknowledgments This work was supported in part by Grant-in-Aids for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] B.A. Freeman, J.D. Crapo, Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria, J. Biol. Chem. 256 (1981) 10986–10992. [2] T.E. Gram, Chemically reactive intermediates and pulmonary xenobiotic toxicity, Pharmacol. Rev. 49 (1997) 297–341. [3] A. Van der Vliet, C.E. Cross, Oxidants, nitrosants, and the lung, Am. J. Med. 109 (2000) 398–421. [4] I. Rahman, P.S. Gilmour, L.A. Jimenez, W. MacNee, Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation, Mol. Cell Biochem. 234–235 (2002) 239–248. [5] M. Bachofen, E.R. Weibel, Alterations of the gas exchange apparatus in adult respiratory insufficiency associated with septicemia, Am. Rev. Respir. Dis. 116 (1977) 589–615. [6] A. Burkhardt, Alveolitis and collapse in the pathogenesis of pulmonary fibrosis, Am. Rev. Respir. Dis. 140 (1989) 513–524. [7] G. Nash, F.D. Foley, P.C. Langlinais, Pulmonary interstitial edema and hyaline membranes in adult burn patients. Electron microscopic observations, Hum. Pathol. 5 (1974) 149–160.
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[33] J.S. Beckman, R.L. Minor Jr., C.W. White, J.E. Repine, G.M. Rosen, B.A. Freeman, Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance, J. Biol. Chem. 263 (1988) 6884–6892.
Efficient protection by cationized catalase against H2O2 injury in primary cultured alveolar epithelial cells Takayuki Nemoto, Shigeru Kawakami, Fumiyoshi Yamashita, Mitsuru Hashida ⁎ Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Received 15 November 2006; accepted 17 May 2007 Available online 24 May 2007
Abstract Increasing evidence suggests that hydrogen peroxide plays an important role in alveolar epithelial injury produced during many inflammatory lung diseases. In this study, the successful prevention of hydrogen peroxide (H2O2)-induced injury in primary cultured rabbit alveolar epithelial cells by cationized catalase is described. Cationized catalase was synthesized by direct chemical modification to enhance its association with alveolar epithelial cells. Cationized catalase exhibited a 22.3-fold higher cellular association at 2 h than native catalase, and incubation of cationized catalase with the cells produced a 2.19-fold intracellular catalase activity, which suggested that cationized catalase distributed both to the cell membrane and into the cell interior. Cationized catalase markedly suppressed H2O2-induced cell injury. In addition, electron spin resonance spectrometry analysis revealed that cationized catalase effectively eliminated H2O2 produced in the medium by glucose plus glucose oxidase. On the other hand, polyethylene glycol-modified catalase (PEG-catalase) did not have any protective effect against H2O2-induced cell injury although PEG-catalase exhibited a 2.49-fold higher cellular association at 2 h than native catalase. These results suggest that cationization of catalase is a promising strategy for the treatment of many of inflammatory lung diseases. © 2007 Published by Elsevier B.V. Keywords: Catalase; Cationization; Polyethylene glycol; Hydrogen peroxide; Alveolar epithelium
1. Introduction The lung epithelium is often directly exposed to oxidants in the ambient air, including ozone, nitrogen dioxide, diesel exhaust, and cigarette smoke. Exposure to hyperoxia [1] or paraquat [2] increases intracellular production of reactive oxygen species (ROS). As well as intracellular metabolic processes, alveolar macrophages and neutrophils are possible sources of ROS [3]. Increased ROS damages a variety of biomolecules such as DNA, lipids, and proteins, induces apoptosis, and causes inflammation via activation of transcriptional factors, leading to gene expression of pro-inflammatory mediators [4]. Thus, oxidative stress is the basis of many lung diseases. The alveolar epithelium is composed of alveolar type I and type II cells. In many types of inflammatory lung injury, alveolar type I cells exhibit more evidence of injury than alveolar type II
⁎ Corresponding author. Tel.: +81 75 753 4525; fax: +81 75 753 4575. E-mail address: [email protected] (M. Hashida). 0168-3659/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.jconrel.2007.05.020
cells [5–7]. One of the reasons for this is that alveolar type I cells are more likely to be injured because they contain less catalase activity than alveolar type II cells [8]. It has been shown that alveolar type II cells in intact rat lungs have many catalasecontaining organelles, whereas alveolar type I cells have relatively few. Furthermore, it has also been shown that there are no significant differences in the components of glutathionedependent systems between alveolar type I cells and type II cells. Therefore, efficient delivery of catalase to alveolar type I cells would be effective in the treatment of inflammatory lung injury. Catalase catalyzes the dismutation of H2O2 to H2O and is a potential antioxidant therapeutic agent. Much attention has been paid to inhalation of catalase as a strategy for the treatment of ROS-mediated lung injury. However, catalase exhibits a low cellular association due to its large molecular size and charge, which have limited its effective use. In order to overcome the limitation, liposomal encapsulation, conjugation with transferrin or polyethyleneglycol (PEG) had been investigated [9]. Although strategies for enhanced delivery of catalase to the sites of action have focused on the use of liposome encapsulation
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[10], the preparation of liposome-encapsulated catalase requires the succinoylation of catalase prior to use in order to prevent liposome aggregation [10,11]. Previously, Rojanasakul et al. [12] demonstrated that transferrin-conjugated catalase, taken up by alveolar cells via alveolar transferrin receptors, protects the cells from oxidative injury. However, the high level of native transferrin in the pulmonary lining fluid would compete with transferrin-conjugated catalase for the receptors [13]. Since polyethyleneglycol (PEG) can be adsorbed onto the cell membrane in the phospholipids headgroup region [14], PEGylation of catalase (PEG-catalase) enhances its cellular uptake and combats cell injury by H2O2 [15]. Cationized protein binds to the negatively charged proteoglycans of the cell surface via electrostatic interaction and is slowly internalized into the cells [16]. Considering that alveolar type I cells occupy 95 % of the alveolar epithelium [17], cationized catalase is expected to be efficiently taken up by alveolar type I cells. Isolated alveolar type II cells, when cultured, lose their phenotypic characteristics and differentiate into alveolar type I-like cells [18]. In the present study, the effectiveness of cationized catalase against H2O2-mediated oxidative stress was evaluated in primary cultured alveolar type II cells. The results obtained were compared with those of catalase and PEG-catalase. 2. Materials and methods 2.1. Materials Bovine liver catalase was obtained from Sigma (St. Louis, MO). Hexamethylenediamine was obtained from Tokyo Chemical Industry (Tokyo, Japan). A product of PEG (2,4-bis (O-methoxypolyethylene glycol)-6-chloro-s-triazin) was obtained from Seikagaku Corp. (Tokyo, Japan). 111Indium chloride was kindly supplied by Nihon Medi Physics (Takarazuka, Japan). Diethylenetriaminepentaacetic acid (DTPA) anhydride was purchased from Dojindo Laboratory (Kumamoto, Japan). Glucose oxidase and 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Hydrogen peroxide and Iron (II) Sulfate Heptahydrate (FeSO4·7H2O) were obtained from Wako Pure Chemical (Osaka, Japan). 5,5-Dimethyl-1pyrroline-N-oxide (DMPO) was obtained from Labotec (Tokyo, Japan). Porcine pancreatic elastase and pure Griffonia simplicifolia lectin (GS-I) were obtained from Worthington Biochemical (Freehold, NJ) and EY Laboratories (San Mateo, CO), respectively. Soybean trypsin inhibitor, human recombinant epidermal growth factor (EGF), hydrocortisone, Dulbecco's modified minimum Eagle's medium nutrient mixture F-12 Ham (DMEM/F12), and Eagle's minimum essential medium Joklik modified for suspension culture (SMEM) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Equitech-Bio (Kerrville, TX). ITS+Premix, type I rat tail collagen, and recombinant human fibronectin were obtained from Becton Dickinson Biosciences (Bedford, MA). Other cell culture reagents were obtained from Invitrogen (Grand Island, NY).
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2.2. Animals New Zealand white rabbits (1.2–1.5 kg) were purchased from Biotek (Saga, Japan). Animals were maintained under conventional housing conditions and all animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences of Kyoto University. 2.3. Primary culture of alveolar type II cells For isolation of alveolar type II cells, three in-house balanced salt solutions were prepared: one balanced salt solution (BSSA) was composed of 137 mM NaCl, 5.0 mM KCl, 0.7 mM Na2HPO4, 10 mM HEPES, 5.5 mM glucose at a pH adjusted to 7.4, another balanced salt solution (BSSB) was BSSA supplemented with 1.8 mM CaCl2 and 1.2 mM MgSO4, the other balanced salt solution (BSSC) was BSSA supplemented with 3 mM EDTA. Alveolar type II cells were isolated from the rabbit lung using the method of Shen et al. [19]. Rabbits were first injected with heparin (1000 U/kg) and then euthanized by rapid injection of sodium pentobarbital (1.5 ml/kg), via a marginal ear vein. The abdominal cavity was opened. While the lung was ventilated manually through a tracheal cannula with a 60 ml syringe, it was perfused with BSSC via the pulmonary vein. The lung was washed several times with BSSA and BSSC, alternately, and then once with SMEM containing 2 U/ml elastase. Following this, about 50 ml 2 U/ml elastase solution was instilled through the trachea and the lung was incubated in BSSB at 37 °C for 35 min. The lung was excised from the trachea and the bronchi, and transferred to SMEM containing 1.67 mg/ml trypsin inhibitor. The pieces of lung were minced, suspended in SMEM, and sequentially filtered through gauze, a 40 μm cell strainer, and a 15 μm nylon mesh. The cell suspension was centrifuged at 200 ×g for 8 min at 4 °C. The cell pellets were resuspended in SMEM, incubated with 16 μg/ml GS-I lectin at room temperature for 30 min to eliminate alveolar macrophages and red blood cells and subsequently filtered through a 15 μm nylon mesh. The filtered cell suspension was centrifuged and the cell pellet was resuspended in DMEM/F12 containing 10% FBS. Cell viability was estimated to be more than 95% by the trypan blue dye exclusion method. Purified type II cells were plated onto plastic plates (Becton Dickinson, Lincolin Park, NJ) at a density of 0.88 × 106 cells/ cm2. The plastic plates were pretreated for 4 hr with 0.25 ml/ cm 2 PBS (−) containing 30 μg/ml collagen, 10 μg/ml fibronectin, and 10 μg/ml BSA. From day 3 of culture onwards, the medium was changed to serum-free DMEM/F12 supplemented with ITS+, 10 ng/ml EGF and 1 μM hydrocortisone. The cultures were fed every other day with serum-free defined medium and maintained in a humidified 5% CO2 incubator at 37 °C. When the alveolar type II cells were plated on Transwells (Corning Costar, Cambridge, MA) pretreated with collagen and
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fibronectin, the transepithelial electric resistance exhibited more than 2 kΩ cm2 from day 4 to day 9. All experiments were carried out on day 5 of culture. 2.4. Synthesis and characterization of cationized catalase and PEG-catalase Cationized catalase was obtained by coupling catalase with hexamethylenediamine using carbodiimide as a catalyst. Twenty five mg catalase was added to 30 ml 2 M hexamethylenediamine and the pH was adjusted to 6.0 using HCl. Thirty minutes and one hour later, 25 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to each. The reaction mixture was agitated for 6 hr at a pH of 6.0, followed by extensive dialysis against distilled water. Then the eluent was concentrated by ultrafiltration, with replacement of the solvent with distilled water, and finally lyophilized. Polyetheleneglycol-conjugated catalase (PEG-catalase) was synthesized according to our previously report [20]. Briefly, activated PEG (250 mg) was added to 25 mg catalase in 5 ml 50 mM borate buffer (pH 9.2). The reaction mixture was agitated for 12 hr at 4 °C in the dark. Then the eluent was concentrated and unreacted activated PEG was removed by ultrafiltration, with replacement of the solvent with distilled water, and finally lyophilized. The activities of native catalase and catalase derivatives were determined by monitoring the ability to degrade H2O2 at 240 nm [21]. The enzymatic activity of native catalase, cationized catalase, and PEG-catalase was 12263.0, 4183.6, and 11485.6 U/mg protein, respectively. The number of amino groups was determined by trinitrobenzene sulfonic acid using glycine as a standard [22]. The Bradford method was used to measure the concentration of catalase using BSA as a standard [23]. The number of modified amino groups of cationized catalase and PEG-catalase was estimated to be 24.5 and 80.3 per catalase molecule, respectively.
off into 500 μl of saline. The cell suspension was homogenized by 3 cycles of freezing in liquid nitrogen and thawing in a water bath at 37 °C. After centrifugation at 15000 ×g for 12 min, the supernatant was used to measure catalase activity. Catalase activity was determined by monitoring the degradation of H2O2 at 240 nm [21]. 2.7. Cytotoxicity assays Cytotoxicity was evaluated by MTT assay. Alveolar type II cells were incubated with glucose oxidase (12.5–200 mU). DMEM-F12 medium used in this study includes 17.5 mM glucose. After given time periods at 37 °C, the cells were washed twice with PBS (−) and subjected to MTT assay. The protective effect of catalase, cationized catalase and PEGcatalase against H2O2 was also evaluated by MTT assay. In this experiment, 100–1000 U/ml catalase, cationized catalase and PEG-catalase were pre-incubated for 2 h with alveolar type II cells before the incubation with glucose oxidase. In addition, morphological changes were also observed using fluorescence microscopy (Biozero Bz-8000 Keyence Inc., Osaka, Japan). 2.8. Electron spin resonance spectrometry analysis U The OH produced by Fenton's reaction between Fe2+ and H2O2 was trapped by DMPO. Cell culture medium was added to the reaction mixture containing DTPA (400 μM), FeSO4 (100 μM), and DMPO (15 mM). The reaction mixture was transferred to a flat quartz cuvette and placed in the cavity of an U ESR spectrometer (JEOL, Tokyo, Japan). The OH produced by Fenton's reaction between Fe2+ and H2O2 was trapped by DMPO, and the amount of the DMPO-OH spin adduct formed was measured exactly 5 min after the addition of H2O2 by ESR U spectrometry. The quantity of OH generated is determined by
2.5. Cellular uptake of [111In]-labeled catalase and catalase derivatives Alveolar type II cells cultured on 24-well plastic plates were incubated with 1 ml serum-free culture medium containing 100,000 cpm of 111 In-labeled native catalase, cationized catalase and PEG-catalase at 37 °C. After incubation for given time periods, the cells were washed three times with PBS (−) and incubated overnight at 37 °C with 1 ml 0.3 N NaOH containing 0.1% Triton X-100. Aliquots of the lysate were taken for determination of 111In radioactivity and protein content. The radioactivity was counted in a well-type NaI scintillation counter (ARC-500, Aloka, Tokyo, Japan), and the protein content was determined by the Bradford method using BSA as a standard [23]. 2.6. Intracellular catalase activity Alveolar type II cells plated on a 6-well plate were incubated with 1000 U/ml native catalase, cationized catalase and PEGcatalase for 2 h. The cells were washed with PBS (−) and scraped
Fig. 1. Cellular association of catalase and catalase derivatives in primary cultured rabbit alveolar type II cells. Alveolar type II cells were incubated with 100,000 cpm of 111In-labeled native catalase, cationized catalase and PEGcatalase at 37 °C. After given time periods, the associated radioactivity and cellular protein were measured. % of dose means the ratio of associated radioactivity to the radioactivity of added each labeled 111In catalase. Results are expressed as means + S.D. (n = 3).
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amplitude, 1 G; response time, 0.1 s; amplitude, 7.9 × 100; and sweep time, 2 min. 2.9. Statistical analysis Statistical analysis was carried out by one-way ANOVA followed by the Student–Newmann–Keuls multiple comparison test, at a significance level of P b 0.05. 3. Results 3.1. Cellular association of catalase and catalase derivatives
Fig. 2. Intracellular catalase activity in primary cultured rabbit alveolar type II cells. Alveolar type II cells were incubated with catalase, cationized catalase and PEG-catalase (1000 U/ml) for 2 h, and then the intracellular catalase activity was measured. Results are expressed as means + S.D. (n = 3). ⁎P b 0.05 vs. control cells; N.S., not significant from control.
the intensity of the ESR signal of the DMPO-OH spin adduct. The signal intensity was evaluated using the relative peak of the second signal of the quartet of the DMPO-OH spin adduct and the intensity of Mn2+ as an internal standard. The ESR settings were as follows: magnetic field, 3350 ± 100 G; microwave power, 5 mW; modulation frequency, 100 KHz; modulation
Fig. 1 shows their cellular association in primary cultured rabbit alveolar type II cells. Their cellular association increased in a time-dependent manner for up to 2 h. The associated amount of native bovine liver catalase, cationized catalase, and PEG-catalase at 2 h was 0.51, 11.60, and 1.30% of dose per mg protein, respectively. Thus, cationized catalase showed a much higher association than native catalase and PEG-catalase. Intracellular catalase activity was also measured following 2 h incubation with 1000 U/ml of native catalase and catalase derivatives (Fig. 2). The level of endogenous catalase activity was 15.89 ± 1.82 U/mg protein. When the cells were incubated with native catalase, cationized catalase and PEG-catalase, the intracellular catalase activity was 16.23 ± 1.87, 34.84 ± 2.48, and 12.47 ± 1.27 U/mg protein, respectively. 3.2. Protective effect of catalase, catalase derivatives against the cytotoxic effect of hydrogen peroxide The viability of alveolar type II cells treated with glucose oxidase was evaluated by MTT assay (Fig. 3). As the concentration of glucose oxidase increased, the viability of the cells
Fig. 3. Cytotoxic effect of hydrogen peroxide in primary cultured rabbit alveolar type II cells. (A) Alveolar type II cells were incubated with a given concentration of glucose oxidase for 6 h. (B) Alveolar type II cells were incubated with glucose oxidase (50 mU/ml) for given time periods. Cellular viability was assessed by MTT assay. Results are expressed as means ± S.D. (n = 3). (C) Morphological change by hydrogen peroxide in primary cultured rabbit alveolar type II cells. Alveolar type II cells were observed using fluorescence microscopy after incubation with glucose oxidase (50 mU/ml) for 6 h. Control panel shows untreated cultures; + glucose oxidase panel demonstrate cultures treated with glucose oxidase (50 mU/ml) for 6 h.
Fig. 4. Protective effect of catalase and catalase derivatives against the cytotoxic effect of hydrogen peroxide in primary cultured rabbit alveolar type II cells. Alveolar type II cells were treated with catalase, cationized catalase and PEGcatalase (100–300–1000 U/ml) from 2 h before the addition of glucose oxidase (50 mU/ml). Cultures were further incubated with glucose oxidase for 6 h and cellular viability was assessed by MTTassay. Results are expressed as means + S.D. (n = 3). ⁎P b 0.05 vs. glucose oxidase (GO)-treated cells.
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Fig. 5. Effect of catalase and catalase derivatives on H2O2 formation by ESR analysis. Alveolar type II cells were treated with catalase, cationized catalase and PEG-catalase (100–300–1000 U/ml) from 2 h before the addition of glucose oxidase (50 mU/ml). Cultures were further incubated with glucose oxidase for 2 h and cell culture medium was added to the reaction mixture containing DTPA (400 μM), FeSO4 (100 μM), and DMPO (15 mM). The amount of DMPO-OH spin adduct generated was detected after exactly 5 min using ESR spectrometry.
decreased (Fig. 3A). When treated with 50 mU glucose oxidase, the cells were injured in a time-dependent manner (Fig. 3B). The injury of alveolar type II cells by glucose oxidase was also confirmed by morphological observation (Fig. 3C). The cells were pre-incubated with 100–1000 U/ml of native catalase, cationized catalase, and PEG-catalase for 2 h. Fig. 4 shows the protective effect of native catalase, cationized catalase and PEG-catalase against H2O2-induced cell injury. Cationized catalase showed its protective effect against H2O2induced cell injury in a dose-dependent manner, whereas native catalase and PEG-catalase had no effect. 3.3. Electron spin resonance spectrometry analysis To further confirm that H2O2 was actually eliminated by cationized catalase, ESR analysis was carried out. Fig. 5 shows the scavenging effect of native catalase and catalase derivatives on H2O2 formation produced by glucose plus glucose oxidase in the cell culture medium. HMD-catalase markedly reduced the DMPO-OH spectrum, whereas catalase and PEG-catalase had hardly any effect. 4. Discussion Oxidative stress is known to play a crucial role in many lung diseases. Accumulating evidence has emerged to support the crucial role of H2O2 in lung diseases. In comparison with appropriate control subjects, patients with ARDS have higher concentrations H2O2 in urine [24] or exhaled air [25]. Patients with COPD have higher levels of H2O2 in exhaled breath condensates than healthy smokers [26]. H2O2 reduces surfactant
metabolism [27] and the ATP level in alveolar type II cells [28,29], and inhibits alveolar epithelial wound repair by induction of apoptosis [30]. Thus, effective elimination of H2O2 would be an effective strategy for the treatment of many lung diseases. In inflammatory lung conditions, H2O2 is mainly produced from inflammatory cells such as alveolar macrophages and neutrophils [3]. Cationized catalase showed a much higher cellular association than native catalase (Fig. 1), and exhibited a significant protective effect against H2O2-induced cell injury (Fig. 4). Glucose oxidase produces mainly H2O2 outside the cells due to its large size, 160 kDa. In this study, therefore, cationized catalase present on the cellular membrane appears to mainly contribute to its protective effect against H2O2-induced cell injury. This statement is supported by our direct observation that cationized catalase significantly eliminated H2O2 produced by glucose oxidase (Fig. 5). These observations show that cationized catalase could be effective against the attack by H2O2 produced by lung inflammatory cells such as alveolar macrophages and neutrophils. A high concentration of oxygen is commonly used to treat premature babies, children, and adults with pulmonary failure. However, prolonged exposure to high oxygen tension is known to induce lung tissue damage. In hyperoxia, an excess of ROS is produced intracellularly, leading to cell death [31]. Cationized catalase showed a much higher cellular association than native catalase (Fig. 1). Incubation with cationized catalase enhanced intracellular catalase activity 2.19-fold (Fig. 2). These results suggest that a part of the cationized catalase is internalized by the cells. Thus, intracellular localization of cationized catalase would be effective against the attack by H2O2 produced into the alveolar epithelium during hyperoxia. In this study, the enzyme activity of cationized catalase and PEG-catalase was 34.1 % and 93.6 % of native catalase and the number of modified amino groups was 24.5 and 80.3 per catalase molecule, respectively. These were the consistent results of Ma et al. [32] about cationized catalase and Yabe et al. [20] about PEG-catalase. PEG-catalase maintained much higher activity than cationized catalase although the ratio of modified amino group in PEG-catalase was much higher than that in cationized catalase. Cationization of catalase with hexamethylenediamine can cause changes in conformation [32]. The change in the surface charge by cationization may be a cause of deactivation. Furthermore, the modification of the protoporphyrin IX with Fe3+ group and the change in the vicinity of Fe3+ group may be another cause of the deactivation. On the other hand, there is no information on change in conformation by PEGylation. The cellular uptake study reveals that PEG-catalase had a 2.49-fold higher cellular association at 2 h than native catalase (Fig. 1). This cellular uptake of PEG-catalase is much lower than that of cationized catalase. Cationized protein binds to the negatively charged proteoglycans of the cell surface via electrostatic interaction and is slowly internalized into the cells [16]. It is difficult to explain the difference in cellular uptake characteristics between PEG-catalase and cationized catalase because the cellular uptake mechanism of PEG-catalase remains unknown. The association of PEG with the cellular
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membrane may be much weaker than that of cationized protein, which binds to the cellular membrane via electrostatic interaction. Although PEG-catalase showed a 2.49-fold higher association at 2 h than native catalase (Fig. 1), PEG-catalase produced no enhancement in intracellular catalase activity at 2 h at all (Fig. 2). The associated amount in Fig. 1 indicates the total quantity of PEG-catalase both adsorbed onto the cellular membrane and internalized into the cells. On the other hand, catalase activity measured in Fig. 2 is intracellular. Therefore, PEGcatalase might be present on the cellular membrane at 2 h. Beckman et al. [33] demonstrated that PEG-catalase showed an initial rapid cellular association within the first 4 h in endothelial cells, followed by a slower, continuous uptake over the next 24 h. Walther et al. [15] also reported similar cellular uptake characteristics of PEG-catalase in alveolar type II cells. These observations support our suggestion that PEG-catalase might be present on the cellular membrane at 2 h. However, although cationized catalase enhances intracellular catalase activity 2.19fold after a 2 h incubation (Fig. 2), PEG-catalase did not show any protective effect against H2O2-induced cell injury. ESR analysis showed that pre-incubation of PEG-catalase with the cells does not lead to the elimination of resulting H2O2 into the medium (Fig. 5D). One possible explanation is that the 2.49-fold higher cellular association of PEG-catalase, compared with native catalase, is not sufficient to produce a protective effect. In conclusion, cationized catalase exhibited an efficient protective effect against H2O2-induced cell injury. It was suggested that cationized catalase bound to the cellular membrane and was internalized into the cells. This is also likely to happen after inhalation in vivo. Accordingly, cationized catalase is expected to be an effective treatment for various ROS-related lung diseases. Acknowledgments This work was supported in part by Grant-in-Aids for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] B.A. Freeman, J.D. Crapo, Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria, J. Biol. Chem. 256 (1981) 10986–10992. [2] T.E. Gram, Chemically reactive intermediates and pulmonary xenobiotic toxicity, Pharmacol. Rev. 49 (1997) 297–341. [3] A. Van der Vliet, C.E. Cross, Oxidants, nitrosants, and the lung, Am. J. Med. 109 (2000) 398–421. [4] I. Rahman, P.S. Gilmour, L.A. Jimenez, W. MacNee, Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation, Mol. Cell Biochem. 234–235 (2002) 239–248. [5] M. Bachofen, E.R. Weibel, Alterations of the gas exchange apparatus in adult respiratory insufficiency associated with septicemia, Am. Rev. Respir. Dis. 116 (1977) 589–615. [6] A. Burkhardt, Alveolitis and collapse in the pathogenesis of pulmonary fibrosis, Am. Rev. Respir. Dis. 140 (1989) 513–524. [7] G. Nash, F.D. Foley, P.C. Langlinais, Pulmonary interstitial edema and hyaline membranes in adult burn patients. Electron microscopic observations, Hum. Pathol. 5 (1974) 149–160.
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