Effect of Native and Modified Forms of Superoxide Dismutase and Catalase on Experimental Silicosis in Rats ALEXANDER V. MAKSIMENKO, LJUDMILA M. BEZRUKAVNIKOVAP EKATERINA L. GRIGORIEVA, VLADIMIR V. YAGLOVP AND VLADIMIR P. TORCHILIN Institute of Experimental Cardiology CardiologyResearch Center Academy of Medical Sciences of Russia 3rd Cherepkovskaya str. 15A Moscow, Russia a Institute of Trade Hygiene and Occupational Diseases Academy of Medical Sciences of Russia Moscow, Russia
INTRODUCTION
Silicosis is a rather widespread occupational disease which is induced by industrial quartz-containing aerosols affecting the lungs. According to published data and our data, modified forms of enzymes with depolymerizing and antiradical action (hyaluronidase, collagenase, superoxide dismutase) are capable of inhibiting the progress of fibrosis.' The efficacy of superoxide dismutase (a scavenger for superoxide radicals) is likely to be increased with its simultaneous administration with catalase, which converts toxic hydrogen peroxide to harmless water and oxygen.2 Preparations of native and modified catalase (CAT) were used to study the possibility of enhancing the effects. Native CAT and experimentally obtained aldehyde dextran-modified catalase (CAT-AD) and catalase-aldehyde dextran-superoxide dismutase covalent conjugate (CAT-AD-SOD) were used for the investigation. The aim of the present study was to perform chemical modification and to carry out a comparative study on the properties of native and modified forms of SOD and CAT, as well as their covalent conjugate, and to test them on the model of experimental silicosis in rats.
EXPERIMENTAL WORK
Currier Dextran [m.m. 70 kilodaltons (kDa)] modified by partial periodate oxidation was used as a carrier for SOD modification. This aldehyde dextran contained 20 aldehyde groups per 100 glycoside units of polymer chain. 118
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Preparation of SOD-AD
Reaction mixture containing 10 pM SOD from rat liver and 0.3 mM AD was incubated for 18 hours (0.05 M K,Na-phosphate buffer) at room temperature (pH 8.5). Then N a B b reduction was performed and the conjugate obtained was isolated by ultrafiltration (Amicon, XM-50) and subsequently lyophilized. Enzyme activity of SOD was defined by a spectrophotometry method (560 nm) following inhibition of nitrotetrasolium blue reduction during generation of superoxide radical in a xanthine-xanthine oxidase system at pH 7.8. Carrier and Cross-linkingAgent
Aldehyde dextran obtained as the result of partial periodate oxidation of dextran (mm. 150 kDa) was used for CAT modification and covalent cross-linking of SOD and CAT. The dextran mentioned exceeded dextran of another molecular mass in the following: catalase amount coupled to carrier, its residual catalytic activity, simplicity of the isolation of modification products. All these factors indicated the choice of this dextran (m.m. 150 kDa) as an optimal carrier or cross-linking agent (for SOD coupling) for chemical modification of CAT. The aldehyde dextran obtained contained 20-22 aldehyde groups per 100 glycoside units of polymer chain. Preparation of CAT-AD
Catalase (10 pM) from bovine liver was incubated with AD (0.02-30 pM) in 0.05 M buffer saline (pH 8.5) for 18 hours at 4°C. Then the mixture was carefully reduced by NaBH4 and the product obtained was isolated by ultracentrifugation (Amicon, XM-100) and rinsed with 0.05 M of KHzP04 (pH 4.0). Such acid washing provides dissociation of enzyme not coupled to subunits, and it can be easily separated from the CAT-AD covalent complex by ultracentrifugation. The product isolated was lyophilized. Catalase enzyme activity was determined by spectrophotometry following disappearance of hydrogen peroxide at 240 nm. Preparation of CAT-AD-SOD
AD (18 mg) was dissolved in 30 ml of 0.05 M buffer saline (pH 8.5) and supplemented with 7.2 mg of CAT (243 pl), and then the mixture was incubated with careful agitation for 3 hours at room temperature. After that 1.92 mg of SOD was added to the reaction mixture and incubation was carried out for 18 hours at 4°C. The incubation mixture was reduced with N a B h , the conjugate was isolated by ultrafiltration, and then lyophilized (Amicon, XM-300). Model of Erperimental Silicosis in Rats
Investigation was done on Wistar male rats. Intensive progression of silicosis (morphology similar to that observed in humans) was noted upon administration of
120
ANNALS NEW YORK ACADEMY OF SCIENCES
quartz dust into the lungs. Experimental silicosis was induced by a single intratracheal administration of 20 mg of quartz dust (dispersity less than 5 km). Respirable fractions of dust were isolated by sedimentation in water. Quartz dust accumulation in the lungs induced development of fibrosis, which results in an increase of lung mass and an elevation of connective tissue proteins (determined by oxyproline content). Therefore, fibrosis progression was monitored by mass of wet lungs and lymph nodes, mass of dry lungs and lymph nodes, as well as oxyproline level in dry tissue. Rats were killed at different steps of the experimental procedure and the indices mentioned above were determined calculating lung and lymph node mass coefficients (i.e., mass of the latter per 100 g of body weight). Stable fibrosis was observed during a three-week administration of quartz dust into rat lungs.
Experimental Therapy
Inhalation was applied to animals three weeks after quartz dust administration. SOD in native and modified (SOD-AD) form was administered in a dose of 200 U per animal. Inhalation continued for two months (three times a week), each duration being 1hour. Native and modified (CAT-AD) catalase preparations were inhaled into rats in a similar way. The dose of native or modified CAT was 200 U per animal, and the dose of the CAT-SOD mixture of native enzymes was 200 U of each enzyme per animal. CAT-AD-SOD bienzyme conjugate and the mixture of modified enzymes SOD-AD and CAT-AD were injected into rats intraperitoneally to study the antifibrosis effect of these preparations. CAT-AD-SOD was administered in a dose of 400 U SOD and 750 U CAT, and modified enzyme mixture was administered in a dose of 400 U of each enzyme per animal. Duration of injection course was two months (three times a week). Animals were killed after termination of experimental therapy, and mass coefficients of dry lungs and lymph nodes were determined, as well as oxyproline content in the lungs and lymph nodes, which allowed us to assess the degree of fibrosis progression. Statistical analysis of the data obtained was performed using Student’s t-test (p < 0.05). Morphological changes were determined by lung sections stained with hematoxyline and eosine, and using oxyproline-pikrofuxine staining to reveal connective tissue proteins (according to Van-Gieson).
RESULTS AND DISCUSSION Modified Envme Preparations
The present method using CAT-AD-SOD affords the highest yield according to protein and residual catalytic activity. A three-step procedure of obtaining conjugate (CAT modification by aldehyde dextran; SOD covalent coupling; reduction of
MAKSIMENKO el aL: EXPERIMENTAL SILICOSIS
121
NaBHp products) proved to be preferable as compared with a one-step synthesis procedure or preliminary modification of SOD. CAT modification increases its thermostability in CAT-AD complex and in CAT-AD-SODconjugate. SOD also exhibits enhanced thermostability in this conjugate, as well as in SOD-AD complex. Some data on comparative properties of enzyme preparations are presented in TABLE1.
TABLE1. Indices of Native and Modified Forms of CAT and SOD Preparations Parameter Protein content (%)
PreDaration
Retained activity of superoxide dismutase Retained catalase activity
U/mg protein (%I, U/mg preparation U/mg protein (961, U/mg preparation
Amino group content per 1 M protein (? 2%) Residual catalytic SOD activity activity after 3 hours of incubation of enzyme preparations at 50°C in 0.1 M K,Na-phosphate Catalase buffer pH 7.4 (% activity from initial value, ? 3%)
Native Native SOD SOD-AD CAT CAT-AD CAT-AD-SOD 80 30 57 22 29 100
86
140oh
450
100
32
75
90
< 7w 354 100
83
INTRODUCTION
Silicosis is a rather widespread occupational disease which is induced by industrial quartz-containing aerosols affecting the lungs. According to published data and our data, modified forms of enzymes with depolymerizing and antiradical action (hyaluronidase, collagenase, superoxide dismutase) are capable of inhibiting the progress of fibrosis.' The efficacy of superoxide dismutase (a scavenger for superoxide radicals) is likely to be increased with its simultaneous administration with catalase, which converts toxic hydrogen peroxide to harmless water and oxygen.2 Preparations of native and modified catalase (CAT) were used to study the possibility of enhancing the effects. Native CAT and experimentally obtained aldehyde dextran-modified catalase (CAT-AD) and catalase-aldehyde dextran-superoxide dismutase covalent conjugate (CAT-AD-SOD) were used for the investigation. The aim of the present study was to perform chemical modification and to carry out a comparative study on the properties of native and modified forms of SOD and CAT, as well as their covalent conjugate, and to test them on the model of experimental silicosis in rats.
EXPERIMENTAL WORK
Currier Dextran [m.m. 70 kilodaltons (kDa)] modified by partial periodate oxidation was used as a carrier for SOD modification. This aldehyde dextran contained 20 aldehyde groups per 100 glycoside units of polymer chain. 118
MAKSIMENKO el nl.: EXPERIMENTAL SILICOSIS
119
Preparation of SOD-AD
Reaction mixture containing 10 pM SOD from rat liver and 0.3 mM AD was incubated for 18 hours (0.05 M K,Na-phosphate buffer) at room temperature (pH 8.5). Then N a B b reduction was performed and the conjugate obtained was isolated by ultrafiltration (Amicon, XM-50) and subsequently lyophilized. Enzyme activity of SOD was defined by a spectrophotometry method (560 nm) following inhibition of nitrotetrasolium blue reduction during generation of superoxide radical in a xanthine-xanthine oxidase system at pH 7.8. Carrier and Cross-linkingAgent
Aldehyde dextran obtained as the result of partial periodate oxidation of dextran (mm. 150 kDa) was used for CAT modification and covalent cross-linking of SOD and CAT. The dextran mentioned exceeded dextran of another molecular mass in the following: catalase amount coupled to carrier, its residual catalytic activity, simplicity of the isolation of modification products. All these factors indicated the choice of this dextran (m.m. 150 kDa) as an optimal carrier or cross-linking agent (for SOD coupling) for chemical modification of CAT. The aldehyde dextran obtained contained 20-22 aldehyde groups per 100 glycoside units of polymer chain. Preparation of CAT-AD
Catalase (10 pM) from bovine liver was incubated with AD (0.02-30 pM) in 0.05 M buffer saline (pH 8.5) for 18 hours at 4°C. Then the mixture was carefully reduced by NaBH4 and the product obtained was isolated by ultracentrifugation (Amicon, XM-100) and rinsed with 0.05 M of KHzP04 (pH 4.0). Such acid washing provides dissociation of enzyme not coupled to subunits, and it can be easily separated from the CAT-AD covalent complex by ultracentrifugation. The product isolated was lyophilized. Catalase enzyme activity was determined by spectrophotometry following disappearance of hydrogen peroxide at 240 nm. Preparation of CAT-AD-SOD
AD (18 mg) was dissolved in 30 ml of 0.05 M buffer saline (pH 8.5) and supplemented with 7.2 mg of CAT (243 pl), and then the mixture was incubated with careful agitation for 3 hours at room temperature. After that 1.92 mg of SOD was added to the reaction mixture and incubation was carried out for 18 hours at 4°C. The incubation mixture was reduced with N a B h , the conjugate was isolated by ultrafiltration, and then lyophilized (Amicon, XM-300). Model of Erperimental Silicosis in Rats
Investigation was done on Wistar male rats. Intensive progression of silicosis (morphology similar to that observed in humans) was noted upon administration of
120
ANNALS NEW YORK ACADEMY OF SCIENCES
quartz dust into the lungs. Experimental silicosis was induced by a single intratracheal administration of 20 mg of quartz dust (dispersity less than 5 km). Respirable fractions of dust were isolated by sedimentation in water. Quartz dust accumulation in the lungs induced development of fibrosis, which results in an increase of lung mass and an elevation of connective tissue proteins (determined by oxyproline content). Therefore, fibrosis progression was monitored by mass of wet lungs and lymph nodes, mass of dry lungs and lymph nodes, as well as oxyproline level in dry tissue. Rats were killed at different steps of the experimental procedure and the indices mentioned above were determined calculating lung and lymph node mass coefficients (i.e., mass of the latter per 100 g of body weight). Stable fibrosis was observed during a three-week administration of quartz dust into rat lungs.
Experimental Therapy
Inhalation was applied to animals three weeks after quartz dust administration. SOD in native and modified (SOD-AD) form was administered in a dose of 200 U per animal. Inhalation continued for two months (three times a week), each duration being 1hour. Native and modified (CAT-AD) catalase preparations were inhaled into rats in a similar way. The dose of native or modified CAT was 200 U per animal, and the dose of the CAT-SOD mixture of native enzymes was 200 U of each enzyme per animal. CAT-AD-SOD bienzyme conjugate and the mixture of modified enzymes SOD-AD and CAT-AD were injected into rats intraperitoneally to study the antifibrosis effect of these preparations. CAT-AD-SOD was administered in a dose of 400 U SOD and 750 U CAT, and modified enzyme mixture was administered in a dose of 400 U of each enzyme per animal. Duration of injection course was two months (three times a week). Animals were killed after termination of experimental therapy, and mass coefficients of dry lungs and lymph nodes were determined, as well as oxyproline content in the lungs and lymph nodes, which allowed us to assess the degree of fibrosis progression. Statistical analysis of the data obtained was performed using Student’s t-test (p < 0.05). Morphological changes were determined by lung sections stained with hematoxyline and eosine, and using oxyproline-pikrofuxine staining to reveal connective tissue proteins (according to Van-Gieson).
RESULTS AND DISCUSSION Modified Envme Preparations
The present method using CAT-AD-SOD affords the highest yield according to protein and residual catalytic activity. A three-step procedure of obtaining conjugate (CAT modification by aldehyde dextran; SOD covalent coupling; reduction of
MAKSIMENKO el aL: EXPERIMENTAL SILICOSIS
121
NaBHp products) proved to be preferable as compared with a one-step synthesis procedure or preliminary modification of SOD. CAT modification increases its thermostability in CAT-AD complex and in CAT-AD-SODconjugate. SOD also exhibits enhanced thermostability in this conjugate, as well as in SOD-AD complex. Some data on comparative properties of enzyme preparations are presented in TABLE1.
TABLE1. Indices of Native and Modified Forms of CAT and SOD Preparations Parameter Protein content (%)
PreDaration
Retained activity of superoxide dismutase Retained catalase activity
U/mg protein (%I, U/mg preparation U/mg protein (961, U/mg preparation
Amino group content per 1 M protein (? 2%) Residual catalytic SOD activity activity after 3 hours of incubation of enzyme preparations at 50°C in 0.1 M K,Na-phosphate Catalase buffer pH 7.4 (% activity from initial value, ? 3%)
Native Native SOD SOD-AD CAT CAT-AD CAT-AD-SOD 80 30 57 22 29 100
86
140oh
450
100
32
75
90
< 7w 354 100
83
