- Email: [email protected]
Changes in superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and thiobarbituric acid-reactive products levels in early stages of development in dystrophic chickens
EXPERIMENTAL
NEUROLOGY
&4,58-73
(1984)
Changes in Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione Reductase Activities and Thiobarbituric Acid-Reactive Products Levels in Early Stages of Development in Dystrophic Chickens YOSHIKUNI Department
Received
of Neurology, Minamikawchi,
MIZUNO’
Jichi Medical School, 3311 Tochigi 32904, Japan
June 28. 1983; revision
received
November
Yakushiji,
18, 1983
Cu-Zn superoxide dismutase, Mn superoxide dismutase, cat&se, glutathione peroxidase, and ghnathione reductase activities and thiobarbituric acid-reactive products were assayed in the supe.rhciaI pectoral muscles of genetically dystrophic chickens (line 4 13) and their controls (line 4 12) I, 2, and 4 weeks, and 4 months after hatching. In control chickens, all these enzyme activities declined as they grew older. In dystrophic chickens, all these enzyme activities were significantly elevated at ah stages of development studied, and their developmental time courses were quite different from those in the controls. Thiobarbituric acid-reactive products were also significantly elevated in dystrophic chickens after 2 weeks of age. Invasion of macrophages and lipid cells were not manifest until 4 weeks after hatching in the dystrophic chickens studied. Therefore, observed abnormalities were considered to represent biochemical pathologies within muscle cells. Increased activities of the enzymes which are responsible for the regulation of active oxygen speciesand the elevated thiobarbituric acid-reactive products would indicate the presence of increased turnover of those active oxygen species. These findings indicated that active oxygen species were playing a significant role in the pathogenesis of muscular dystrophies. The possible mechanisms of cellular damage by active oxygen species are discussed.
Abbreviations: GSH-reduced glutathione, GSSG-oxidized glutathione. ’ The author thanks Dr. Hideo Sugita, Head of the First Research Department, the National Institute for Neurological Disorders, for his kind suggestions and advice. The author also thanks Dr. Tetsuji Atsumi, Associate Professor of Neurology, Niigata University, for preparation of histologic specimens. Technical a&stances from Miss Kumiko Hamano and Miss Yukie Noguchi are greatly acknowledged. This study was in part supported by a research grant for progressive muscular dystrophy from the Ministry of Health and Welfare of Japan. 58 0014-4886/84 $3.00 Copyright 0 1984 by Academic F’ress. Inc. All rights of reproduction in any form reserved.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
59
INTRODUCTION The mechanism of cellular damage in progressive muscular dystrophies is still unknown. Various approaches seem to be necessary to elucidate this problem. We wanted to see if free radicals were playing a role in pathogenesis of muscular dystrophies. There is much evidence in the literature to suggest involvement of active oxygen species in pathogenesis of muscular dystrophies (6, 13, 19, 22, 35, 38, 39). Active oxygen species include superoxide anion (O;), hydrogen peroxide, hydroxyl radical (OH ), and singlet oxygen (‘0~). Those species are being produced during sequential reduction of oxygen molecules and are highly cytotoxic by their strong oxidizing properties. Superoxide dismutases, catalase, glutathione peroxidase, and glutathione reductase are important enzymic systems which protect respiring cells from the hazards of these active oxygen species ( 14). It is known that activities of catalase and glutathione reductase and levels of thiobarbituric acid-reactive products are elevated in human dystrophies (22) and that activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase and levels of thiobarbituric acid-reactive products are elevated in animal dystrophies ( 18,35,38). Peroxidation of lipids composing intracellular membrane structures can induce their dysfunction (5, 15, 33, 5 1,52). Active oxygen species are known to promote lipid peroxidation (12, 25,40). Those findings suggest involvement of active oxygen species in the pathogenesis of muscular dystrophies. However, those studies used specimens of rather well developed states of the diseases. In such states, invasion of phagocytes, increase in lipid and connective tissues, and some regenerative processes were common. Macrophages are known to have high activities of glutathione peroxidase and glutathione reductase (32). Therefore, reported increases in those enzyme activities may have been, in part, due to those secondary changes. Abnormal metabolism of active oxygen species should be present From very early stages of development if they are really playing a role. To explore this problem, we took a genetically selected line of dystrophic chickens as a model, and investigated changes in activities of superoxide dismutases, catalase, glutathione peroxidase, and glutathione reductase and levels of thiobarbituric acid-reactive products during early stages of the disease. We found significant increases in these enzyme activities which were already apparent 1 week after hatching in dystrophic chickens. l
MATERIALS
AND
METHODS
Xanthine, xanthine oxidase (type III), reduced glutathione (GSH), oxidized glutathione (GSSG), NADPH, GSSG reductase, and sodium azide were purchased from Sigma (St. Louis, MO.), epinephrine from Merck (Darmstadt),
60
YOSHIKUNI
MIZUNO
EDTA from Dojin (Kumamoto), hydrogen peroxide from Santoku (Miyagi), and other reagents used from Wako (Osaka). Fertile eggs from genetically selected dystrophic (line 413) and control (line 412) chickens were obtained from the Institute of Biological Sciences, Japan (Yamanashi). After incubation and hatching, the chickens were grown in a cage appropriate for their size, and standard commercial rations and water were available ad libitum. Four chickens from each line were sacrificed 1,2, and 4 weeks and 4 months after hatching by decapitation. The superficial pectoral muscles were immediately removed, weighed, and used for enzyme assays. A portion of a central part from each superficial pectoral muscle was homogenized in 10 vol ice-cold 10 mM sodium phosphate buffer containing 127 mM NaCl, pH 7.4 using a Dounce-type homogenizer. Immediately after homogenization a sample was taken for catalase assay to which ethanol was added at a final concentration of l%, and the tube was placed in an ice bath for 30 min to prevent formation of compound II (7). The rest of the homogenate was centrifuged 60 min at 100,OOOg at 4°C. The supernatant was used for assays of other enzyme activities. Superoxide dismutase activities were assayed according to the method of Misra and Fridovich (3 1) in 50 mM phosphate buffer, pH 7.8, containing 1 mM EDTA. Because two isoenzymes of superoxide dismutases are known in avian and mammalian species (9, 14, 24, 55), total superoxide dismutase activity was assayed first and cyanide-insensitive Mn superoxide dismutase activity was assayed in the presence of 2 mM KCN (9, 55). Assay mixtures for total activity contained 0.05 to 0.15 ml supematant, 0.2 mA4 xanthine, 0.3 mM epinephrine, and 0.05 ml of appropriately diluted xanthine oxidase. The total volume was adjusted to 2 ml with the above phosphate buffer. Because some differences in activity of xanthine oxidase preparations were noted among the lots, they were diluted appropriately so that an assay mixture without superoxide dismutase source yielded 0.02 A change/min at 320 nm. This adjustment of xanthine oxidase solution was necessary to obtain a constant rate of superoxide anion generation in every experiment. For each sample six assays using three different amounts of the supematant and corresponding blanks were carried out. Supematants heated 5 min at 100°C served as blanks. Assay mixtures without xanthine oxidase were preincubated 4 min at 30°C. Reaction was started by the addition of xanthine oxidase, and absorbance change at 320 nm was followed for as long as 4 min according to the modification of Sun and Zigman (49), using a Hitachi139 spectrophotometer equipped with an isothermal cell holder. For assay of Mn superoxide dismutase activity, KCN was added to assay mixtures at 2 mM concentration. The amount of supematant which would produce 50% inhibition of epinephrine oxidation was defined as 1 unit of activity according
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
61
to Misra and Fridovich (3 1). The difference between total and Mn superoxide dismutase activities was considered to represent Cu-Zn superoxide dismutase activity. Cat&se activity was assayed according to the method of Cohen et al. (7). The homogenate containing 1% ethanol was centrifuged 10 min at 700 g at 4°C. Into the supernatant Triton X-100 was added at a final concentration of 0.5%. The solution was diluted by five times using isotonic 10 mM phosphate buffer, pH 7.4, and 0.5 ml was used for the assay. Incubation was carried out at 0°C for 8 min. other details of the methods were the same as those of Cohen et al. (7). Hydrogen peroxide solution was titrated with a standard solution of potassium permanganate. Glutathione peroxidase activity was assayed using 100,000 g supematant according to the method of Paglia and Valentine (37) with slight modifications. Assays were carried out in 50 mM potassium phosphate buffer, pH 7.0. A typical assay mixture contained 1 mM EDTA disodium salt, 1 rniW sodium azide, 0.1 mM NADPH, 2.5 mA4 GSH, 0.75 mM HzOz, 0.33 units GSSG reductase, and 0.2 ml of sample in a total volume of 2 ml. It was conlirmed that GSSG reductase was not a rate-limiting factor at this concentration. The reaction was started by the addition of H202 and absorbance change at 340 nm was followed for as long as 4 min at 25°C. Glutathione reductase activity was assayed in 50 mMpotassium phosphate buffer, pH 7.0. A typical assay mixture contained 1 mM EDTA disodium salt, 1.5 mM GSSG, 0.1 mM NADPH, and 0.2 ml of a sample in a total volume of 2 ml. The reaction was started by the addition of GSSG, and absorbance change at 340 nm was followed for as long as 4 min at 25°C. For calculation of glutathione peroxidase and glutathione reductase activities, the molar extinction coefficient of NADPH of 6270 was used. Thiobarbituric acid-reactive products were assayed according to the method of Tanizawa et al. (50) using malon dialdehyde as a standard. All assays were done in at least duplicate samples, and reproducibility was confirmed. Total protein was assayed according to the method of Lowry et al. (27). Results were analyzed statistically with Student’s t test. RESULTS The ratio of superficial pectoral muscle weight to body weight was significantly higher in dystrophic chickens at 2 weeks and 4 weeks of age, indicating the presence of hypertrophic changes at those ages. Representative photomicrographs of histologic specimens are shown in Fig. 1. Because protein concentrations in 700- and 100,000-g supernatants were significantly lower in the dystrophic chickens compared with those in the normal controls (Table I), enzyme activities are expressed in units per milligram protein and units per gram wet tissue.
FIG. 1. Representative photomicrographs of the superticial pectoral muscles of control and dystrophic chickens. Hematoxylin-eosin stain, 400X magnification. Left--control chickens, rightdystrophic chickens. From top to bottom, l-week, 2-week, Cweek, and 4-month-old chickens
OXYGEN
SPECIES ACTIVITY
IN AVIAN
63
DYSTROPHY
TABLE I Protein Concentration in IO-Vol Supematants in Normal and Dystrophic Chicken Superficial Pectoral Muscles’ 700-g Supematant Control 1 Week 2 weeks 4 Weeks 4 Months
7.41 8.38 8.60 8.17
f 0.34 + 0.30 IL 0.50 + 0.42
100,000-g Supematant Control
Dyfirophy 5.93 7.21 6.91 5.84
+ k + +
0.44** 0.34** 0.51** 0.42**
5.86 6.57 6.27 6.40
* + rt +
0.53 0.43 0.21 0.62
Dystrophy 4.28 4.95 4.85 5.03
f f + +
0.38** 0.16+* 0.21** 0.56.
a All data are the mean + SD (N = 4) mg/ml supematant. * Statistical significance at 5% level. ** Statistical significance at 1% level.
Changes in specific activities of Cu-Zn and Mn superoxide dismutases are shown in Tables 2 and 3, respectively. Approximately one-third of the total activity in the 100,000-g supematant was cyanide-insensitive Mn superoxide dismutase activity in both dystrophic and control chickens. These results are consistent with recent findings that a considerable amount of activity of cyanide-insensitive Mn superoxide dismutase was present in the supematant fraction of avian as well as mammalian tissues (9, 29). Specific activities of both Cu-Zn and Mn superoxide dismutases were significantly higher in dystrophic chickens at all stages of development studied. Standard deviations in dystrophic chickens were larger than those in the controls, indicating a greater variance in dystrophic chickens. Rather large standard deviations were not due to technical problems because standard deviations in control chickens were reasonably small. In control chickens, both Cu-Zn and Mn superoxide dismutase activities declined with age. In dystrophic chickens, Mn superoxide dismutase activity declined as they grew older, but Cu-Zn superoxide dismutase activity was highest at 4 weeks of age, attaining an approximately threefold difference compared with the controls. Changes in specific activity of catalase are shown in Table 4. Catalase activity in normal superhcial pectoral muscles was very low and it further declined as they grew older. Mean activity at 4 months of age was approximately one-tenth that at 1 week. In dystrophic chickens, catalase activity are shown. At 1 week, it was difficult to find clear morphologic abnormalities in dystrophic chickens. At 2 weeks, slight differences in fiber diameters were noted. At 4 weeks, more prominent differences in fiber diameters and some increases in interstitial connective tissues were clearly seen, however, no active necrosis nor invasions of phagocytes or lipid cells were seen. At 4 months, muscle fiber necrosis and increases in phagocytes and lipid tissue were evident.
64
YOSHIKUNI
MIZUNO
TABLE 2 Cu-Zn Superoxide Dismutase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/g tissueb
Units/mg protein b Control 1 Week 2 Weeks 4 Weeks 4 Months
0.141 0.568 0.536 0.412
k + f f
0.037 0.089 0.076 0.062
Dystrophy 1.112 0.921 1.350 0.993
+ f + f
0.240* 0.199* 0.384* 0.355*
Control 43.9 31.5 33.8 26.9
+ f + f
Dystrophy
5.1 7.3 5.9 3.6
’ All data are the mean + SD (N = 4). b One unit = amount of enzyme activity which produced 50% inhibition epinephrine. * Statistical significance at 5% level.
47.5 45.6 65.7 50.6
+ + k +
10.6 1.9 19.6* 12.5;
of oxidation of
was markedly elevated attaining 19- and 35times differences, respectively, at 4 weeks and 4 months of age compared with those in the controls. The activity declined as they grew older but to a much less extent. At 4 months of age, dystrophic chickens retained approximately 60% of catalase activity in the first week ex ova. In addition, a small peak of activity was noted at 4 weeks of age and the overall time course of catalase activity resembled that of Cu-Zn superoxide dismutase activity. Changes in glutathione peroxidase activity are shown in Table 5. Here again, the specific activity in control chickens declined as they grew older. In dystrophic chickens, it remained elevated until 4 months of age, when it TABLE 3 Mn Superoxide Dismutase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein b Age 1 Week 2 Weeks 4 Weeks 4 Months
Control 0.4 I9 0.388 0.372 0.344
f + f +
0.024 0.021 0.045 0.014
Dystrophy 0.815 0.706 0.738 0.556
f f f +
0.076***,’ 0.078*** 0.126** 0.060**
Units/g tissue b Control 24.6 25.4 23.2 23.3
-+ 2.6 + 1.2 f 2.1 + 1.6
Dystrophy 34.6 34.9 35.7 29.2
f + f k
1.6** 3.F 5.9f l.O**
’ All data are the mean + SD (N = 4). b One unit = amount of enzyme activity which produced 50% inhibition of oxidation of epinephrine. ‘Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
65
DYSTROPHY
TABLE 4 Catalase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein b Age 1 Week 2 weeks 4 Weeks 4 Months
Control 0.20 0.086 0.052 0.021
+ f + +
Units/g tissue b
Dystrophy
0.10 0.078 0.017 0.025
1 23 0:76 0.99 0.73
f + & +
0 34***’ 0:14*** 0.43** 0.19***
Control 15.1 6.14 4.44 1.82
+ k + k
Dystrophy
7.7 2.07 1.39 2.16
71.4 53.8 65.1 42.1
+ 14.8** + 9.8*** -+ 25.8** + 9.6***
a All data are the mean f SD (N = 4). b One unit = 1 pmol Hz02 catalyzed/min. ‘Asterisks indicate statistical significance at 1% (**) and 0.1% (***) levels.
was highest among the groups studied. The overall time course was not similar to any of the other enzyme activities assayed. At 4 months of age the difference in activity was more than threefold when expressed in units per milligram protein. Changes in specific activity of glutathione reductase are shown in Table 6. Here again, decline of specific activity according to development was very clear in control chickens. In dystrophic chickens, it remained elevated and only a slight reduction was noted at 4 months of age. Estimates of thiobarbituric acid-reactive products levels are shown in Table 7. They were significantly elevated in dystrophic chickens alter 2 weeks of age. However, developmental time courses of both control and dystrophic chickens did not resemble any of the enzyme activities studied. TABLE 5 Glutathione Peroxidase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein’ Age 1 Week 2 Weeks 4 Weeks 4 Months
Control 8.60 7.08 6.16 5.19
+ * + f
1.78 0.71 0.74 1.21
Units/g tissue b
Dystrophy 14.89 14.32 13.48 16.13
+ + + f
1.28**f 2.00** 3.38* 2.15***
Control 497 463 385 328
+ + f f
69 34 40 58
Dystrophy 633 706 651 802
+ + rt *
’ All data are the mean + SD (N = 4). b One unit = 1 nmol NADPH oxidized/min. c Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
41* 74** 154. 66***
66
YOSHIKUNI
MIZUNO
TABLE 6 Glutathione Reductase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles’ Units/mg protein * Control
Age 1 Week 2 Weeks 4 Weeks 4 Months
3.92 + 2.63 f 1.60 f 1.70 +
0.16 0.11 0.32 0.50
Units/g tissue*
Dystrophy 6.99 5.69 6.42 5.64
+ + + +
0.83**s 1.56* 2.76* 0.76***
Control 227 f 171 k lOOk 107 f
Dystrophy
31 46 19 25
312 279 309 279
+ 17** + 65 + 127* + 13***
‘All data are the mean f SD (N = 4). *One unit = 1 nmol NADPH oxidized/min. ‘Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
To show clearer differences in the enzyme activities between the control and dystrophic chickens, mean values were plotted against age in Fig. 2. The activities are expressed in units per milligram protein in the figure, and the results were essentially similar when expressed in units per gram wet tissue. The gradual decline of specific activities of all five enzymes responsible for metabolism of active oxygen species was very clearly shown to be related to development in control chickens. In dystrophic chickens, all these enzyme activities remained elevated, and time courses were quite different from those in the controls. Only Mn superoxide dismutase activity declined fairly clearly in dystrophic chickens as they grew older. Some similarities in developmental time courses among Cu-Zn superoxide dismutase, catalase, and glutathione reductase activities were noted. TABLE 7 Thiobarbituric
Age 1 Week 2 Weeks 4 Weeks 4 Months
Acid-Reactive Products Levels in Normal and Dystrophic Superficial Pectoral Muscles” Control 4.86 2.72 13.22 8.18
+ f f f
0.93 0.90 1.89 1.39
Dystrophy 6.59 6.90 40.91 24.45
f 2.09 + 1.51**,* + 14.56* f 3.96’-
’ All data are the mean f SD (N = 4) nmol malon dialdehydelg tissue. * Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
OXYGEN
SPECIES ACTIVITY
units/
IN AYIAN
DYSTROPHY
67
mg protein 26
16
12
8
4
-4 1w
2w
4w
IW
Dystrophy
2w
*-la* ‘4
4w
Control
FIG. 2. Changes in Cu-Zn superoxide dismutase, Mn superoxide dismutase, catalam, glutathione peroxidase, and glutathione reductase activities according to the stage of development. Mean values are shown. The ordinates indicate units of enzyme activities. *-indicates the scale for catalase activity (I unit = 1 gmol Hz4 catalyxed/min), **-indicates the scale for glutathione peroxidase and glutathione reductase activities (1 unit = 1 nmol NADPH oxidized/min), ‘indicates the scale for Cu-Zn and Mn superoxide dismutase activities (1 unit = amount of enzyme activity which produced 50% inhibition of oxidation of epinephrine). The abscissae indicate the age of the chickens in weeks (W) or months (M).
DISCUSSION Genetically selected lines of dystrophic chickens are considered to be good models of human dystrophies because of similarities of histologic findings such as fiber necrosis, phagocytosis, replacement of muscle fibers by adipose and collagenous tissues, and some regenerative changes (2 1,56). In dystrophic chickens used in this study, clinical and histologic characteristics of dystrophy become manifest gradually 1 to 2 weeks after ex uvo (56), but infiltration of muscle fibers by phagocytic cells was usually not seen until approximately 4 weeks alter hatching (36), as shown in Fig. 1. Biochemical pathologies of the primary defect may be obscured by secondary changes in adult chickens. In this study, biochemical findings of l- and 2-week-old chickens are considered to represent changes taking place within muscle cells, and also, those of 4-week-old dystrophic chickens are likely to represent muscle pathologies. We found in this study that enzyme activities responsible for regulation of active oxygen species declined in control chickens as they grew older. In dystrophic chickens, all these enzyme activities remained elevated, and normal
68
YOSHIKUNI
MIZUNO
declines were not seen until at least 4 months of age. As far as we know, this is the first report on simultaneous assays of five enzyme activities responsible for regulation of active oxygen metabolism at these early stages of development in this avian muscular dystrophy. Sustained increases in these enzyme activities would indicate increased turnover of active oxygen species, i.e., increased formation and catabolism of superoxide anion and other active oxygen species which are formed by interactions with superoxide anion. Increased turnover of active oxygen species in this line of dystrophic chickens is also supported from elevated levels of thiobarbituric acid-reactive products (Table 7) because active oxygen species are known to enhance lipid peroxidation (12, 25, 40). Involvement of active oxygen species is also supported by findings reported in the literature. Francis et al. (13) and Park et al. (38) reported increased activities of superoxide dismutase and glutathione peroxidase in 4-monthold dystrophic chickens, and the same group of investigators demonstrated decreased activity of glyceraldehyde 3-phospate dehydrogenase (39), an enzyme extremely sensitive to radical damage. Omaye and Tappel(35) reported increased activities of glutathione peroxidase and glutathione reductase, and elevation of thiobarbituric acid-reactive products in 18-month-old dystrophic chickens. Those findings were interpreted as indicating the presence of increased formation of active oxygen species. However, the latter authors admitted that increases in those enzyme activities could, in part, be due to secondary changes such as invasion of phagocytes, for phagocytes were known to have high glutathione peroxidase and reductase activities (53). Therefore, it is essential to use very young chickens in which morphologic changes are minimum, as shown in Fig. 1, to prove explicitly the elevation of these enzyme activities within muscle cells. In addition, many other biochemical changes have been known to occur from early stages of the disease, i.e., within 2 weeks ex ova, in dystrophic chickens. For instance, activities of cathepsins A, B, C, D (18, 41), other lysosomal enzymes (36), uptake of amino acids into muscle cells (54), turnover of sarcoplasmic protein (1 l), and mitochondrial succinate dehydrogenase activity (I), all of which normally declined as chickens aged, remained elevated in dystrophic chickens. On the other hand, development of phosphorylase activity, which normally rapidly increased in normal white muscles, showed a delayed increase of activity in dystrophic chickens and never attained the normal level (8). Therefore, any biochemical abnormality of possible pathogenetic significance should be present from very early stages of the disease. The exact mechanism of cellular damage by active oxygen species are still to be elucidated; however, several possibilities can be considered. Superoxide anion per se, as well as other active oxygen species formed as a consequence of its interaction with hydrogen peroxide, have been implicated as mediators of the damage to proteins including enzymes (14,20,43,47, 5 1). It is known
OXYGEN
SPECIES
ACTIVITY
IN
AVIAN
DYSTROPHY
69
that superoxide anion can oxidize thiol groups in proteins thus inducihg losses of enzymic activities (14, 20, 47). The other important mechanism of cellular damage by active oxygen species is the enhancement of lipid peroxidation (12, 25, 40). Lipid peroxidation of various membrane structures are known to induce their dysfunction (5, 15, 33, 5 1, 52). Hydroxyl radical and singlet oxygen are two main species that have been proposed to be responsible for lipid peroxidation (12, 25, 40). Hydroxyl radical and singlet oxygen are formed by the Haber-Weiss reaction (2) or the ion-catalyzed Haber-Weiss reaction (16, 30). There is ample evidence to indicate the presence of abnormalities in the plasma membranes (4,32,42), the sarcoplasmic membranes (10, 17,44,45), the lysosomal membranes (36), and in the mitochondrial membranes (34, 57) in human as well as lower animal muscular dystrophies. Impaired regulation of intracellular calcium content due to defects in the plasma membrane has been postulated as the hallmarks of the disease (42). Increased influx of calcium ion into the cells probably is responsible for intracellular calcium accumulation (4,34) and the elevation of calcium-activated neutral proteinase activities (23, 48). The exact cause and nature of these membrane abnormalities are still unknown. Some abnormalities are probably direct manifestations of genetic defects, however, it seems to be likely that the membrane abnormalities, are induced, at least in part, by peroxidation of lipids composing those membranes. One of the consequences of lipid peroxidation of the lipid bilayer is the increase in permeability, inducing an increase in influx of calcium ions among others at the plasma membrane site (5 1, 52). In lysosomes, it is known that peroxidation of those membranes by free radicals results in release of active acid hydrolases (5, 12). The latter mechanism may be responsible for the reported increase in the activities of lysosomal proteinases in muscular dystrophies (18, 24, 41). In mitochondria, it is known that lipid peroxidation of those membranes results in swelling, uncoupling of oxidative phosphorylation, and the inhibition of respiration (33, 52). Calcium overload of the mitochondria due to the increased permeability of the mitochondrial membranes has been postulated as the fundamental fault for these alterations and finally for muscle cell necrosis (34, 57). Thus, a number of biochemical abnormalities reported in muscular dystrophies could be explained, at least in part, on the basis of increased turnover of active oxygen species. Our results strongly suggest the existence of that possibility. In addition, abnormal redox states of glutathione and glutathione disulfide (oxidized form of glutathione) may also be playing a role in inducing cellular damage. It is known that enhanced glutathione peroxidase activity is accompanied by a decrease in the cellular concentration of both glutathione and NADPH (46), and that glutathione protects cell membranes from lipid peroxidation (28, 58). Inhibition of the initiation of protein synthesis by a slight
70
YOSHIKUNI
MIZUNO
increase of glutathione disulfide was also reported (26). Therefore, the marked increase in glutathione peroxidase activity in our study suggests the presence of a contribution by altered redox states of glutathione and glutathione disulfide to cellular damage in this avian muscular dystrophy. In conclusion, we reported marked increases in activities of enzymes responsible for regulation of active oxygen species and in the levels of thiobarbituric acid-reactive products in early stages of a line of genetically distinct dystrophic chickens. The results were interpreted as indicating the presence of increased turnover of active oxygen species. We do not consider these findings as direct manifestations of genetic defects, but many biochemical abnormalities could be explained on the basis of that mechanism. Further studies on this line seem to be important to elucidate the pathogenesis of muscular dystrophies. REFERENCES I. ASHMORE, C. R., AND L. DOERR. 1970. Oxidative metabolism in skeletal muscle of normal and dystrophic chickens. Biochem. Med. 4: 246-259. 2. BEAUCHAMP, C., AND I. FRIDOVICH. 1970. A mechanism for the production of ethylene from methional. J. Biol. Chem. 245: 4641-4646. 3. BERNHEIM, F., M. L. C. BERNHEIM, AND K. M. WILBUR. 1948. The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174: 257264. 4.
5. 6. 7. 8. 9. 10. Il. 12.
BODENSTEINER,J. B., AND A. G. ENGEL. 1978. Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibers in 114 biopsies. Neurology (Minneapolis) 28: 439-448. CHEN, K.-L., AND P. B. MCCAY. 1972. Lysosome disruption by a free radical-like component generated during microsomal NADPH oxidase activity. B&hem. Biophys. Rex Commun. 48: 1412-1418. CHOU, T.-H., E. J. HILL, E. BARTLE, K. WOOLLEY, V. LEQUIRE, W. OLSON, R. REOLOFS, AND J. H. PARK. 1975. Beneficial effectsof penicillamine treatment on hereditary avian muscular dystrophy. J. Clin. Invest. 56: 842-849. COHEN, G., D. DEMBIEC, AND J. MARCUS. 1970. Measurement of catalase activity in tissue extracts. Anal. B&hem. 34: 30-38. COSMOS, E. 1966. Enzymatic activity of differentiating muscle fibers. Dev. Biol. 13: 163181. DE ROSA, G., D. S. DUNCAN, C. L. DEEN, AND L. HURLEY. 1979. Evolution of negative staining technique for detection of CN--insensitive superoxide dismutase activity. B&him. Biophys. Acta 566: 32-39. ETTIENNE, E. M., AND R. H. SINGER. 1978. Ca*+ binding, ATP dependent Cat+ transport and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis. J. Membr. Biol. 44: 195-210. ETTIENNE, E. M., K. SWARTZ, AND R. H. SINGER. 1980. Increased turnover of proteins from the sarcoplasmic reticulum of dystrophic chicken muscle cells in tissue culture. J. Biol. Chem. 256: 6408-6412. FONG, K.-L., P. B. MCCAY, J. L. POYER, B. B. KEEL, AND H. MISRA. 1973. Evidence that peroxidation of lysosomal membrane is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 248: 7792-7797.
OXYGEN
SPECIES
ACTIVITY
IN
AVIAN
DYSTROPHY
71
13. FRANCIS,S. H., S. BRANDON, AND J. H. PARK. 1976. Superoxide dismutase levels in tissue of genetically dystrophic chickens. Fed. Proc. 35: 1652. 14. FRIDOVICH, I. 1974. Superoxide dismutases. Adv. Enzymol. 41: 35-97. 15. GOLDSTEIN, I. M., AND G. WEISSMAN. 1977. Effects of the generation of superoxide anion on permeability of @some. Biochem. Biophys. Rex Commun. 75: 604-609. 16. HALLIWELL, B. 1978. Superoxidedependent formation of hydroxyl radicals in the presence of ion chelates. FEBS Left. 92: 31 l-326. 17. HANNA, S., R. KAWAMOTO, M. MCNAMEE, AND R. J. BASKIN. 1981. Enxymic activity of dystrophic chicken sarcoplasmic reticulum. B&him. Biophys. Acta 643: 41-54. 18. IODICE, A. A., J. CHIN, S. PARKER, AND I. M. WEINSTOCK. 1972. Cathepsins A, B, C, D and autolysis during development of breast muscle of normal and dystrophic chickens. Arch. Biochem. Biophys. 152: 166-174. 19. IONASESCU,V., L. Z. STERN, R. IONASEXU, AND P. RUBENSTEIN. 1979. Stimulating effects of drugs for protein synthesis on muscle cell cultures in dystrophy. Ann. Neural. 5: 107110. 20. JOCELYN, P. C. 1970. The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate. Biochem. J. 117: 951-956. 21. JULIAN, L. M., AND V. S. ASMUNDSON. 1963. Muscular dystrophy of the chicken. Pages 457-498 in G. H. BOURNE, AND M. N. GOLARZ, Eds., Muscular Dystrophy of Man and Animals. Karger, Basel. 22. KAR, N. C., AND C. M. PEARSON.1979. Catalase, superoxide dismutase, glutathione reductase and thiobarbituric acid-reactive products in normal and dystrophic human muscle. Clin. Chim. Acta 94: 277-280. 23. KAR, N. C., AND C. M. PEARSON. 1980. Elevated activity of a neutral proteins.% in human muscular dystrophy. B&hem. Med. 24: 238-243. 24. KATUNUMA, N., N. YASOGAWA, K. KITO, Y. SANADA, AND K. MIYOSHI. 1978. Abnormal expression of a serine protease in human dystrophic muscle. J. Biochem. (Tokyo) 83: 625-628.
E. W. III, AND I. FRIDOVICH. 1977. Liposome oxidation and hydrogen peroxide. J. Biol. Chem. 251: 6721-6728. 26. KOSOWER,N. S., G. A. VANDERHOF’F,AND E. M. KOSOWER. 1972. Glutatbione. VIII. The effects of glutathione disulfide on initiation of protein synthesis. B&him. Biophys. Acta 25. KELLOG,
272: 623-637. 27.
28.
29. 30. 31.
32.
33.
LOWRY, 0. H., N. J. ROSENBROUGH,A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. MCCAY, P. B., D. D. GIEISON, AND K. R. HORNBROOK. 1981. Glutathionedependent inhibition of lipid peroxidation by a soluble, heat-labile factor not &tathione peroxidase. Fed. Proc. 40: 199-205. MCCORD, J. M. 1976. Iron- and manganese-containing superoxide dismutases: structure, distribution, and evolutionary relationships. Adv. Exp. Med. Biol. 74: 540-550. MCCORD, J. M., AND D. DAY, JR. 1978. Superoxidedependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Left. 86: 139-142. MISRA, H. P., AND I. FRIDOVICH. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247: 31703175. MOKRI, B., AND A. G. ENGEL. 1975. Duchenne dystrophy: electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscular fiber. Neurology (Minneapolis) 25: 11 I 1- 1120. NEUBERT, D., A. B. WFJTCZAK, AND A. L. LEHNINGER. 1962. Purification and enzymatic
72
34.
35.
36. 37. 38.
YOSHIKUNI
MIZUNO
identity of mitochondrial contraction-factors I and II. Proc. Natl. Acad. Sci. USA 48: 1651-1658. NYLEN, E. G., AND K. WROGEMANN. 1983. Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice. Exp. Neural. 80: 6980. OMAYE, S. T., AND A. L. TAPPEL. 1974. Glutathione peroxidase, glutathione reductase, and thiobarbituric acid-reactive products in muscles of chickens and mice with genetic muscular dystrophy. Life Sci. 15: 137-145. OWENS, K. 1979. Biochemical studies of dystrophy in the young chicken lysosomal and sarcolemmal enzymes Ann. N. Y. Acad. Sci. 317: 247-262. PAGLIA, D. E., AND W. N. VALENTINE. 1967. Studies on the quantitative and qualitative characterization of erythrocytes glutathione peroxidase. J. Lab. Clin. Med. 70: 158- 169. PARK, J. H., E. J. HILL, T.-H. CHOU, V. LEQUIRE, R. REOLOFS, AND C. R. PARK. 1979. Mechanism of action of penicillamine in the treatment of avian muscular dystrophy.
Ann. N.Y. Acad. Sci. 317: 356-369. 39. PATNODE, R., E. BARTLE, E. J. HILL, V. LEQUIRE, AND J. H. PARK. 1976. Enzymological studies on hereditary avian muscular dystrophy. J. Biol. Chem. 251: 4468-4475. 40. PEDERSON, T. C., AND S. D. AUST. 1973. The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase. B&hem. Biophys. Rex Commun. 52: 107 l1078. 41. PETERSON, D. W., A. L. LILYBLADE, AND D. C. BOND. 1972. Proteolytic activity during growth of hypertrophic and atrophic muscles of genetically dystrophic chickens. Proc. Sot. Exp. Biol. Med. 141: 1056-1062. 42. ROWLAND, L. P. 1980. Biochemistry of muscle membranes in Duchenne muscular dystrophy.
Muscle Nerve 3: 3-20. 43. SALIN, M. L., AND J. M. MCCORD. 1975. Free radicals and inflammation. Protection of phagocytosing leukocytes by superoxide dismutase. J. C/in. Invest. 56: 13 19-1323. 44. SAMAHA, F. J., AND J. GERGELY. 1969. Biochemical abnormalities of the sarcoplasmic reticulum in muscular dystrophy. N. Engl. J. Med. 280: 184-188. 45. SCALES, D., R. SABBADINI, AND G. INESI. 1977. The involvement of snrcotubular membranes in genetic muscular dystrophy. B&him. Biophys. Acta 465: 535-549. 46. SIES, H., C. GERSTENECKER, H. MENZEL, AND L. FLOHE. 1972. Oxidation in the NADP system and release. of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides. FEBS Lett. 27: 17 1-175. 47. SINGH, A. 1982. Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen. Can. J. Physiol. Pharmacol. 60: 1330-1345. 48. SUGITA, H., S. ISHIURA, K. SUZUKI, AND K. IMAHORI. 1980. Ca-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils. Muscle Nerve 3: 335-339. 49. SUN, M., AND S. ZIGMAN. 1978. An improved dismutase based on epinephrine autoxidation. 50. TANIZAWA, H., Y. SAZUKA, in the mouse myocardium 29: 2910-2914. 5 1. TAPPEL, A. L. 1973. Lipid 1973. 52. VLADIMIROV, peroxidation
spectrophotometric
AND Y. TAKINO. 1981. Micro-determination by thiobarbituric acid tluorometry. Chem. peroxidation
damage
assay for superoxide
Anal. Biochem. 90: 81-89.
to cell components.
Y. A., V. I. OLENEV, T. B. SUSLOVA, in mitochondrial membrane. Adv.
of lipoperoxide
Pharm. Bull. (Tokyo) Fed. Proc. 32: 1970-
AND z. P. CHEREMISINA.
1980. Lipid
Lipid Rex 17: 173-249.
53. VCK~T, M. Y., C. THOMAS, C. L. VASSALO, R. E. BASFORD, AND J. B. L. GEE. 197 1. Glutathionedependent peroxidative metabolism in the alveolar macrophage. J. Clin. Invest. 50: 401-410.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
73
54. WEINSTOCK, L. M., T. S. SOH, H. A. FREEMAN, AND M. E. CUTLER. 1969. Amino acid incorporation into proteins of developing breast muscle of the normal and dystrophic chicken. B&hem. Med. 2: 345-356. 55. WEISINGER, R. A., AND I. PRIDOVICH. 1973. Mitochondrial superoxide dismutase. J. Biol.
Chem.248:4793-4796. 56. WILSON, B. W., W. R. RANDALL, G. T. PATI-ERSON, AND R. K. ENTRIKIN. 1979. Major histologic and hi&chemical characterixation of inherited dystrophy of the chicken. Ann.
N.Y. Acad.Sci.317:224-246. 57. WROGEMANN, K., AND S. D. J. PENA. 1976. Mitochondrial calcium overload: a general mechanism for cell necrosis in muscle disease. Luncet1: 672-674. 58. YOUNES, M., AND C.-P. SIEGERS.1980. Lipid peroxidation as a consequence of glutathione depletion in rat and mouse liver. Rex Commun. Chem.Pathol.Pharmacol. 27: 119-128.
NEUROLOGY
&4,58-73
(1984)
Changes in Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione Reductase Activities and Thiobarbituric Acid-Reactive Products Levels in Early Stages of Development in Dystrophic Chickens YOSHIKUNI Department
Received
of Neurology, Minamikawchi,
MIZUNO’
Jichi Medical School, 3311 Tochigi 32904, Japan
June 28. 1983; revision
received
November
Yakushiji,
18, 1983
Cu-Zn superoxide dismutase, Mn superoxide dismutase, cat&se, glutathione peroxidase, and ghnathione reductase activities and thiobarbituric acid-reactive products were assayed in the supe.rhciaI pectoral muscles of genetically dystrophic chickens (line 4 13) and their controls (line 4 12) I, 2, and 4 weeks, and 4 months after hatching. In control chickens, all these enzyme activities declined as they grew older. In dystrophic chickens, all these enzyme activities were significantly elevated at ah stages of development studied, and their developmental time courses were quite different from those in the controls. Thiobarbituric acid-reactive products were also significantly elevated in dystrophic chickens after 2 weeks of age. Invasion of macrophages and lipid cells were not manifest until 4 weeks after hatching in the dystrophic chickens studied. Therefore, observed abnormalities were considered to represent biochemical pathologies within muscle cells. Increased activities of the enzymes which are responsible for the regulation of active oxygen speciesand the elevated thiobarbituric acid-reactive products would indicate the presence of increased turnover of those active oxygen species. These findings indicated that active oxygen species were playing a significant role in the pathogenesis of muscular dystrophies. The possible mechanisms of cellular damage by active oxygen species are discussed.
Abbreviations: GSH-reduced glutathione, GSSG-oxidized glutathione. ’ The author thanks Dr. Hideo Sugita, Head of the First Research Department, the National Institute for Neurological Disorders, for his kind suggestions and advice. The author also thanks Dr. Tetsuji Atsumi, Associate Professor of Neurology, Niigata University, for preparation of histologic specimens. Technical a&stances from Miss Kumiko Hamano and Miss Yukie Noguchi are greatly acknowledged. This study was in part supported by a research grant for progressive muscular dystrophy from the Ministry of Health and Welfare of Japan. 58 0014-4886/84 $3.00 Copyright 0 1984 by Academic F’ress. Inc. All rights of reproduction in any form reserved.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
59
INTRODUCTION The mechanism of cellular damage in progressive muscular dystrophies is still unknown. Various approaches seem to be necessary to elucidate this problem. We wanted to see if free radicals were playing a role in pathogenesis of muscular dystrophies. There is much evidence in the literature to suggest involvement of active oxygen species in pathogenesis of muscular dystrophies (6, 13, 19, 22, 35, 38, 39). Active oxygen species include superoxide anion (O;), hydrogen peroxide, hydroxyl radical (OH ), and singlet oxygen (‘0~). Those species are being produced during sequential reduction of oxygen molecules and are highly cytotoxic by their strong oxidizing properties. Superoxide dismutases, catalase, glutathione peroxidase, and glutathione reductase are important enzymic systems which protect respiring cells from the hazards of these active oxygen species ( 14). It is known that activities of catalase and glutathione reductase and levels of thiobarbituric acid-reactive products are elevated in human dystrophies (22) and that activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase and levels of thiobarbituric acid-reactive products are elevated in animal dystrophies ( 18,35,38). Peroxidation of lipids composing intracellular membrane structures can induce their dysfunction (5, 15, 33, 5 1,52). Active oxygen species are known to promote lipid peroxidation (12, 25,40). Those findings suggest involvement of active oxygen species in the pathogenesis of muscular dystrophies. However, those studies used specimens of rather well developed states of the diseases. In such states, invasion of phagocytes, increase in lipid and connective tissues, and some regenerative processes were common. Macrophages are known to have high activities of glutathione peroxidase and glutathione reductase (32). Therefore, reported increases in those enzyme activities may have been, in part, due to those secondary changes. Abnormal metabolism of active oxygen species should be present From very early stages of development if they are really playing a role. To explore this problem, we took a genetically selected line of dystrophic chickens as a model, and investigated changes in activities of superoxide dismutases, catalase, glutathione peroxidase, and glutathione reductase and levels of thiobarbituric acid-reactive products during early stages of the disease. We found significant increases in these enzyme activities which were already apparent 1 week after hatching in dystrophic chickens. l
MATERIALS
AND
METHODS
Xanthine, xanthine oxidase (type III), reduced glutathione (GSH), oxidized glutathione (GSSG), NADPH, GSSG reductase, and sodium azide were purchased from Sigma (St. Louis, MO.), epinephrine from Merck (Darmstadt),
60
YOSHIKUNI
MIZUNO
EDTA from Dojin (Kumamoto), hydrogen peroxide from Santoku (Miyagi), and other reagents used from Wako (Osaka). Fertile eggs from genetically selected dystrophic (line 413) and control (line 412) chickens were obtained from the Institute of Biological Sciences, Japan (Yamanashi). After incubation and hatching, the chickens were grown in a cage appropriate for their size, and standard commercial rations and water were available ad libitum. Four chickens from each line were sacrificed 1,2, and 4 weeks and 4 months after hatching by decapitation. The superficial pectoral muscles were immediately removed, weighed, and used for enzyme assays. A portion of a central part from each superficial pectoral muscle was homogenized in 10 vol ice-cold 10 mM sodium phosphate buffer containing 127 mM NaCl, pH 7.4 using a Dounce-type homogenizer. Immediately after homogenization a sample was taken for catalase assay to which ethanol was added at a final concentration of l%, and the tube was placed in an ice bath for 30 min to prevent formation of compound II (7). The rest of the homogenate was centrifuged 60 min at 100,OOOg at 4°C. The supernatant was used for assays of other enzyme activities. Superoxide dismutase activities were assayed according to the method of Misra and Fridovich (3 1) in 50 mM phosphate buffer, pH 7.8, containing 1 mM EDTA. Because two isoenzymes of superoxide dismutases are known in avian and mammalian species (9, 14, 24, 55), total superoxide dismutase activity was assayed first and cyanide-insensitive Mn superoxide dismutase activity was assayed in the presence of 2 mM KCN (9, 55). Assay mixtures for total activity contained 0.05 to 0.15 ml supematant, 0.2 mA4 xanthine, 0.3 mM epinephrine, and 0.05 ml of appropriately diluted xanthine oxidase. The total volume was adjusted to 2 ml with the above phosphate buffer. Because some differences in activity of xanthine oxidase preparations were noted among the lots, they were diluted appropriately so that an assay mixture without superoxide dismutase source yielded 0.02 A change/min at 320 nm. This adjustment of xanthine oxidase solution was necessary to obtain a constant rate of superoxide anion generation in every experiment. For each sample six assays using three different amounts of the supematant and corresponding blanks were carried out. Supematants heated 5 min at 100°C served as blanks. Assay mixtures without xanthine oxidase were preincubated 4 min at 30°C. Reaction was started by the addition of xanthine oxidase, and absorbance change at 320 nm was followed for as long as 4 min according to the modification of Sun and Zigman (49), using a Hitachi139 spectrophotometer equipped with an isothermal cell holder. For assay of Mn superoxide dismutase activity, KCN was added to assay mixtures at 2 mM concentration. The amount of supematant which would produce 50% inhibition of epinephrine oxidation was defined as 1 unit of activity according
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
61
to Misra and Fridovich (3 1). The difference between total and Mn superoxide dismutase activities was considered to represent Cu-Zn superoxide dismutase activity. Cat&se activity was assayed according to the method of Cohen et al. (7). The homogenate containing 1% ethanol was centrifuged 10 min at 700 g at 4°C. Into the supernatant Triton X-100 was added at a final concentration of 0.5%. The solution was diluted by five times using isotonic 10 mM phosphate buffer, pH 7.4, and 0.5 ml was used for the assay. Incubation was carried out at 0°C for 8 min. other details of the methods were the same as those of Cohen et al. (7). Hydrogen peroxide solution was titrated with a standard solution of potassium permanganate. Glutathione peroxidase activity was assayed using 100,000 g supematant according to the method of Paglia and Valentine (37) with slight modifications. Assays were carried out in 50 mM potassium phosphate buffer, pH 7.0. A typical assay mixture contained 1 mM EDTA disodium salt, 1 rniW sodium azide, 0.1 mM NADPH, 2.5 mA4 GSH, 0.75 mM HzOz, 0.33 units GSSG reductase, and 0.2 ml of sample in a total volume of 2 ml. It was conlirmed that GSSG reductase was not a rate-limiting factor at this concentration. The reaction was started by the addition of H202 and absorbance change at 340 nm was followed for as long as 4 min at 25°C. Glutathione reductase activity was assayed in 50 mMpotassium phosphate buffer, pH 7.0. A typical assay mixture contained 1 mM EDTA disodium salt, 1.5 mM GSSG, 0.1 mM NADPH, and 0.2 ml of a sample in a total volume of 2 ml. The reaction was started by the addition of GSSG, and absorbance change at 340 nm was followed for as long as 4 min at 25°C. For calculation of glutathione peroxidase and glutathione reductase activities, the molar extinction coefficient of NADPH of 6270 was used. Thiobarbituric acid-reactive products were assayed according to the method of Tanizawa et al. (50) using malon dialdehyde as a standard. All assays were done in at least duplicate samples, and reproducibility was confirmed. Total protein was assayed according to the method of Lowry et al. (27). Results were analyzed statistically with Student’s t test. RESULTS The ratio of superficial pectoral muscle weight to body weight was significantly higher in dystrophic chickens at 2 weeks and 4 weeks of age, indicating the presence of hypertrophic changes at those ages. Representative photomicrographs of histologic specimens are shown in Fig. 1. Because protein concentrations in 700- and 100,000-g supernatants were significantly lower in the dystrophic chickens compared with those in the normal controls (Table I), enzyme activities are expressed in units per milligram protein and units per gram wet tissue.
FIG. 1. Representative photomicrographs of the superticial pectoral muscles of control and dystrophic chickens. Hematoxylin-eosin stain, 400X magnification. Left--control chickens, rightdystrophic chickens. From top to bottom, l-week, 2-week, Cweek, and 4-month-old chickens
OXYGEN
SPECIES ACTIVITY
IN AVIAN
63
DYSTROPHY
TABLE I Protein Concentration in IO-Vol Supematants in Normal and Dystrophic Chicken Superficial Pectoral Muscles’ 700-g Supematant Control 1 Week 2 weeks 4 Weeks 4 Months
7.41 8.38 8.60 8.17
f 0.34 + 0.30 IL 0.50 + 0.42
100,000-g Supematant Control
Dyfirophy 5.93 7.21 6.91 5.84
+ k + +
0.44** 0.34** 0.51** 0.42**
5.86 6.57 6.27 6.40
* + rt +
0.53 0.43 0.21 0.62
Dystrophy 4.28 4.95 4.85 5.03
f f + +
0.38** 0.16+* 0.21** 0.56.
a All data are the mean + SD (N = 4) mg/ml supematant. * Statistical significance at 5% level. ** Statistical significance at 1% level.
Changes in specific activities of Cu-Zn and Mn superoxide dismutases are shown in Tables 2 and 3, respectively. Approximately one-third of the total activity in the 100,000-g supematant was cyanide-insensitive Mn superoxide dismutase activity in both dystrophic and control chickens. These results are consistent with recent findings that a considerable amount of activity of cyanide-insensitive Mn superoxide dismutase was present in the supematant fraction of avian as well as mammalian tissues (9, 29). Specific activities of both Cu-Zn and Mn superoxide dismutases were significantly higher in dystrophic chickens at all stages of development studied. Standard deviations in dystrophic chickens were larger than those in the controls, indicating a greater variance in dystrophic chickens. Rather large standard deviations were not due to technical problems because standard deviations in control chickens were reasonably small. In control chickens, both Cu-Zn and Mn superoxide dismutase activities declined with age. In dystrophic chickens, Mn superoxide dismutase activity declined as they grew older, but Cu-Zn superoxide dismutase activity was highest at 4 weeks of age, attaining an approximately threefold difference compared with the controls. Changes in specific activity of catalase are shown in Table 4. Catalase activity in normal superhcial pectoral muscles was very low and it further declined as they grew older. Mean activity at 4 months of age was approximately one-tenth that at 1 week. In dystrophic chickens, catalase activity are shown. At 1 week, it was difficult to find clear morphologic abnormalities in dystrophic chickens. At 2 weeks, slight differences in fiber diameters were noted. At 4 weeks, more prominent differences in fiber diameters and some increases in interstitial connective tissues were clearly seen, however, no active necrosis nor invasions of phagocytes or lipid cells were seen. At 4 months, muscle fiber necrosis and increases in phagocytes and lipid tissue were evident.
64
YOSHIKUNI
MIZUNO
TABLE 2 Cu-Zn Superoxide Dismutase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/g tissueb
Units/mg protein b Control 1 Week 2 Weeks 4 Weeks 4 Months
0.141 0.568 0.536 0.412
k + f f
0.037 0.089 0.076 0.062
Dystrophy 1.112 0.921 1.350 0.993
+ f + f
0.240* 0.199* 0.384* 0.355*
Control 43.9 31.5 33.8 26.9
+ f + f
Dystrophy
5.1 7.3 5.9 3.6
’ All data are the mean + SD (N = 4). b One unit = amount of enzyme activity which produced 50% inhibition epinephrine. * Statistical significance at 5% level.
47.5 45.6 65.7 50.6
+ + k +
10.6 1.9 19.6* 12.5;
of oxidation of
was markedly elevated attaining 19- and 35times differences, respectively, at 4 weeks and 4 months of age compared with those in the controls. The activity declined as they grew older but to a much less extent. At 4 months of age, dystrophic chickens retained approximately 60% of catalase activity in the first week ex ova. In addition, a small peak of activity was noted at 4 weeks of age and the overall time course of catalase activity resembled that of Cu-Zn superoxide dismutase activity. Changes in glutathione peroxidase activity are shown in Table 5. Here again, the specific activity in control chickens declined as they grew older. In dystrophic chickens, it remained elevated until 4 months of age, when it TABLE 3 Mn Superoxide Dismutase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein b Age 1 Week 2 Weeks 4 Weeks 4 Months
Control 0.4 I9 0.388 0.372 0.344
f + f +
0.024 0.021 0.045 0.014
Dystrophy 0.815 0.706 0.738 0.556
f f f +
0.076***,’ 0.078*** 0.126** 0.060**
Units/g tissue b Control 24.6 25.4 23.2 23.3
-+ 2.6 + 1.2 f 2.1 + 1.6
Dystrophy 34.6 34.9 35.7 29.2
f + f k
1.6** 3.F 5.9f l.O**
’ All data are the mean + SD (N = 4). b One unit = amount of enzyme activity which produced 50% inhibition of oxidation of epinephrine. ‘Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
65
DYSTROPHY
TABLE 4 Catalase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein b Age 1 Week 2 weeks 4 Weeks 4 Months
Control 0.20 0.086 0.052 0.021
+ f + +
Units/g tissue b
Dystrophy
0.10 0.078 0.017 0.025
1 23 0:76 0.99 0.73
f + & +
0 34***’ 0:14*** 0.43** 0.19***
Control 15.1 6.14 4.44 1.82
+ k + k
Dystrophy
7.7 2.07 1.39 2.16
71.4 53.8 65.1 42.1
+ 14.8** + 9.8*** -+ 25.8** + 9.6***
a All data are the mean f SD (N = 4). b One unit = 1 pmol Hz02 catalyzed/min. ‘Asterisks indicate statistical significance at 1% (**) and 0.1% (***) levels.
was highest among the groups studied. The overall time course was not similar to any of the other enzyme activities assayed. At 4 months of age the difference in activity was more than threefold when expressed in units per milligram protein. Changes in specific activity of glutathione reductase are shown in Table 6. Here again, decline of specific activity according to development was very clear in control chickens. In dystrophic chickens, it remained elevated and only a slight reduction was noted at 4 months of age. Estimates of thiobarbituric acid-reactive products levels are shown in Table 7. They were significantly elevated in dystrophic chickens alter 2 weeks of age. However, developmental time courses of both control and dystrophic chickens did not resemble any of the enzyme activities studied. TABLE 5 Glutathione Peroxidase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles” Units/mg protein’ Age 1 Week 2 Weeks 4 Weeks 4 Months
Control 8.60 7.08 6.16 5.19
+ * + f
1.78 0.71 0.74 1.21
Units/g tissue b
Dystrophy 14.89 14.32 13.48 16.13
+ + + f
1.28**f 2.00** 3.38* 2.15***
Control 497 463 385 328
+ + f f
69 34 40 58
Dystrophy 633 706 651 802
+ + rt *
’ All data are the mean + SD (N = 4). b One unit = 1 nmol NADPH oxidized/min. c Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
41* 74** 154. 66***
66
YOSHIKUNI
MIZUNO
TABLE 6 Glutathione Reductase Activity at Different Stages of Development in Normal and Dystrophic Chicken Superficial Pectoral Muscles’ Units/mg protein * Control
Age 1 Week 2 Weeks 4 Weeks 4 Months
3.92 + 2.63 f 1.60 f 1.70 +
0.16 0.11 0.32 0.50
Units/g tissue*
Dystrophy 6.99 5.69 6.42 5.64
+ + + +
0.83**s 1.56* 2.76* 0.76***
Control 227 f 171 k lOOk 107 f
Dystrophy
31 46 19 25
312 279 309 279
+ 17** + 65 + 127* + 13***
‘All data are the mean f SD (N = 4). *One unit = 1 nmol NADPH oxidized/min. ‘Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
To show clearer differences in the enzyme activities between the control and dystrophic chickens, mean values were plotted against age in Fig. 2. The activities are expressed in units per milligram protein in the figure, and the results were essentially similar when expressed in units per gram wet tissue. The gradual decline of specific activities of all five enzymes responsible for metabolism of active oxygen species was very clearly shown to be related to development in control chickens. In dystrophic chickens, all these enzyme activities remained elevated, and time courses were quite different from those in the controls. Only Mn superoxide dismutase activity declined fairly clearly in dystrophic chickens as they grew older. Some similarities in developmental time courses among Cu-Zn superoxide dismutase, catalase, and glutathione reductase activities were noted. TABLE 7 Thiobarbituric
Age 1 Week 2 Weeks 4 Weeks 4 Months
Acid-Reactive Products Levels in Normal and Dystrophic Superficial Pectoral Muscles” Control 4.86 2.72 13.22 8.18
+ f f f
0.93 0.90 1.89 1.39
Dystrophy 6.59 6.90 40.91 24.45
f 2.09 + 1.51**,* + 14.56* f 3.96’-
’ All data are the mean f SD (N = 4) nmol malon dialdehydelg tissue. * Asterisks indicate statistical significance at 5% (*), 1% (**), and 0.1% (***) levels.
OXYGEN
SPECIES ACTIVITY
units/
IN AYIAN
DYSTROPHY
67
mg protein 26
16
12
8
4
-4 1w
2w
4w
IW
Dystrophy
2w
*-la* ‘4
4w
Control
FIG. 2. Changes in Cu-Zn superoxide dismutase, Mn superoxide dismutase, catalam, glutathione peroxidase, and glutathione reductase activities according to the stage of development. Mean values are shown. The ordinates indicate units of enzyme activities. *-indicates the scale for catalase activity (I unit = 1 gmol Hz4 catalyxed/min), **-indicates the scale for glutathione peroxidase and glutathione reductase activities (1 unit = 1 nmol NADPH oxidized/min), ‘indicates the scale for Cu-Zn and Mn superoxide dismutase activities (1 unit = amount of enzyme activity which produced 50% inhibition of oxidation of epinephrine). The abscissae indicate the age of the chickens in weeks (W) or months (M).
DISCUSSION Genetically selected lines of dystrophic chickens are considered to be good models of human dystrophies because of similarities of histologic findings such as fiber necrosis, phagocytosis, replacement of muscle fibers by adipose and collagenous tissues, and some regenerative changes (2 1,56). In dystrophic chickens used in this study, clinical and histologic characteristics of dystrophy become manifest gradually 1 to 2 weeks after ex uvo (56), but infiltration of muscle fibers by phagocytic cells was usually not seen until approximately 4 weeks alter hatching (36), as shown in Fig. 1. Biochemical pathologies of the primary defect may be obscured by secondary changes in adult chickens. In this study, biochemical findings of l- and 2-week-old chickens are considered to represent changes taking place within muscle cells, and also, those of 4-week-old dystrophic chickens are likely to represent muscle pathologies. We found in this study that enzyme activities responsible for regulation of active oxygen species declined in control chickens as they grew older. In dystrophic chickens, all these enzyme activities remained elevated, and normal
68
YOSHIKUNI
MIZUNO
declines were not seen until at least 4 months of age. As far as we know, this is the first report on simultaneous assays of five enzyme activities responsible for regulation of active oxygen metabolism at these early stages of development in this avian muscular dystrophy. Sustained increases in these enzyme activities would indicate increased turnover of active oxygen species, i.e., increased formation and catabolism of superoxide anion and other active oxygen species which are formed by interactions with superoxide anion. Increased turnover of active oxygen species in this line of dystrophic chickens is also supported from elevated levels of thiobarbituric acid-reactive products (Table 7) because active oxygen species are known to enhance lipid peroxidation (12, 25, 40). Involvement of active oxygen species is also supported by findings reported in the literature. Francis et al. (13) and Park et al. (38) reported increased activities of superoxide dismutase and glutathione peroxidase in 4-monthold dystrophic chickens, and the same group of investigators demonstrated decreased activity of glyceraldehyde 3-phospate dehydrogenase (39), an enzyme extremely sensitive to radical damage. Omaye and Tappel(35) reported increased activities of glutathione peroxidase and glutathione reductase, and elevation of thiobarbituric acid-reactive products in 18-month-old dystrophic chickens. Those findings were interpreted as indicating the presence of increased formation of active oxygen species. However, the latter authors admitted that increases in those enzyme activities could, in part, be due to secondary changes such as invasion of phagocytes, for phagocytes were known to have high glutathione peroxidase and reductase activities (53). Therefore, it is essential to use very young chickens in which morphologic changes are minimum, as shown in Fig. 1, to prove explicitly the elevation of these enzyme activities within muscle cells. In addition, many other biochemical changes have been known to occur from early stages of the disease, i.e., within 2 weeks ex ova, in dystrophic chickens. For instance, activities of cathepsins A, B, C, D (18, 41), other lysosomal enzymes (36), uptake of amino acids into muscle cells (54), turnover of sarcoplasmic protein (1 l), and mitochondrial succinate dehydrogenase activity (I), all of which normally declined as chickens aged, remained elevated in dystrophic chickens. On the other hand, development of phosphorylase activity, which normally rapidly increased in normal white muscles, showed a delayed increase of activity in dystrophic chickens and never attained the normal level (8). Therefore, any biochemical abnormality of possible pathogenetic significance should be present from very early stages of the disease. The exact mechanism of cellular damage by active oxygen species are still to be elucidated; however, several possibilities can be considered. Superoxide anion per se, as well as other active oxygen species formed as a consequence of its interaction with hydrogen peroxide, have been implicated as mediators of the damage to proteins including enzymes (14,20,43,47, 5 1). It is known
OXYGEN
SPECIES
ACTIVITY
IN
AVIAN
DYSTROPHY
69
that superoxide anion can oxidize thiol groups in proteins thus inducihg losses of enzymic activities (14, 20, 47). The other important mechanism of cellular damage by active oxygen species is the enhancement of lipid peroxidation (12, 25, 40). Lipid peroxidation of various membrane structures are known to induce their dysfunction (5, 15, 33, 5 1, 52). Hydroxyl radical and singlet oxygen are two main species that have been proposed to be responsible for lipid peroxidation (12, 25, 40). Hydroxyl radical and singlet oxygen are formed by the Haber-Weiss reaction (2) or the ion-catalyzed Haber-Weiss reaction (16, 30). There is ample evidence to indicate the presence of abnormalities in the plasma membranes (4,32,42), the sarcoplasmic membranes (10, 17,44,45), the lysosomal membranes (36), and in the mitochondrial membranes (34, 57) in human as well as lower animal muscular dystrophies. Impaired regulation of intracellular calcium content due to defects in the plasma membrane has been postulated as the hallmarks of the disease (42). Increased influx of calcium ion into the cells probably is responsible for intracellular calcium accumulation (4,34) and the elevation of calcium-activated neutral proteinase activities (23, 48). The exact cause and nature of these membrane abnormalities are still unknown. Some abnormalities are probably direct manifestations of genetic defects, however, it seems to be likely that the membrane abnormalities, are induced, at least in part, by peroxidation of lipids composing those membranes. One of the consequences of lipid peroxidation of the lipid bilayer is the increase in permeability, inducing an increase in influx of calcium ions among others at the plasma membrane site (5 1, 52). In lysosomes, it is known that peroxidation of those membranes by free radicals results in release of active acid hydrolases (5, 12). The latter mechanism may be responsible for the reported increase in the activities of lysosomal proteinases in muscular dystrophies (18, 24, 41). In mitochondria, it is known that lipid peroxidation of those membranes results in swelling, uncoupling of oxidative phosphorylation, and the inhibition of respiration (33, 52). Calcium overload of the mitochondria due to the increased permeability of the mitochondrial membranes has been postulated as the fundamental fault for these alterations and finally for muscle cell necrosis (34, 57). Thus, a number of biochemical abnormalities reported in muscular dystrophies could be explained, at least in part, on the basis of increased turnover of active oxygen species. Our results strongly suggest the existence of that possibility. In addition, abnormal redox states of glutathione and glutathione disulfide (oxidized form of glutathione) may also be playing a role in inducing cellular damage. It is known that enhanced glutathione peroxidase activity is accompanied by a decrease in the cellular concentration of both glutathione and NADPH (46), and that glutathione protects cell membranes from lipid peroxidation (28, 58). Inhibition of the initiation of protein synthesis by a slight
70
YOSHIKUNI
MIZUNO
increase of glutathione disulfide was also reported (26). Therefore, the marked increase in glutathione peroxidase activity in our study suggests the presence of a contribution by altered redox states of glutathione and glutathione disulfide to cellular damage in this avian muscular dystrophy. In conclusion, we reported marked increases in activities of enzymes responsible for regulation of active oxygen species and in the levels of thiobarbituric acid-reactive products in early stages of a line of genetically distinct dystrophic chickens. The results were interpreted as indicating the presence of increased turnover of active oxygen species. We do not consider these findings as direct manifestations of genetic defects, but many biochemical abnormalities could be explained on the basis of that mechanism. Further studies on this line seem to be important to elucidate the pathogenesis of muscular dystrophies. REFERENCES I. ASHMORE, C. R., AND L. DOERR. 1970. Oxidative metabolism in skeletal muscle of normal and dystrophic chickens. Biochem. Med. 4: 246-259. 2. BEAUCHAMP, C., AND I. FRIDOVICH. 1970. A mechanism for the production of ethylene from methional. J. Biol. Chem. 245: 4641-4646. 3. BERNHEIM, F., M. L. C. BERNHEIM, AND K. M. WILBUR. 1948. The reaction between thiobarbituric acid and the oxidation products of certain lipids. J. Biol. Chem. 174: 257264. 4.
5. 6. 7. 8. 9. 10. Il. 12.
BODENSTEINER,J. B., AND A. G. ENGEL. 1978. Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibers in 114 biopsies. Neurology (Minneapolis) 28: 439-448. CHEN, K.-L., AND P. B. MCCAY. 1972. Lysosome disruption by a free radical-like component generated during microsomal NADPH oxidase activity. B&hem. Biophys. Rex Commun. 48: 1412-1418. CHOU, T.-H., E. J. HILL, E. BARTLE, K. WOOLLEY, V. LEQUIRE, W. OLSON, R. REOLOFS, AND J. H. PARK. 1975. Beneficial effectsof penicillamine treatment on hereditary avian muscular dystrophy. J. Clin. Invest. 56: 842-849. COHEN, G., D. DEMBIEC, AND J. MARCUS. 1970. Measurement of catalase activity in tissue extracts. Anal. B&hem. 34: 30-38. COSMOS, E. 1966. Enzymatic activity of differentiating muscle fibers. Dev. Biol. 13: 163181. DE ROSA, G., D. S. DUNCAN, C. L. DEEN, AND L. HURLEY. 1979. Evolution of negative staining technique for detection of CN--insensitive superoxide dismutase activity. B&him. Biophys. Acta 566: 32-39. ETTIENNE, E. M., AND R. H. SINGER. 1978. Ca*+ binding, ATP dependent Cat+ transport and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis. J. Membr. Biol. 44: 195-210. ETTIENNE, E. M., K. SWARTZ, AND R. H. SINGER. 1980. Increased turnover of proteins from the sarcoplasmic reticulum of dystrophic chicken muscle cells in tissue culture. J. Biol. Chem. 256: 6408-6412. FONG, K.-L., P. B. MCCAY, J. L. POYER, B. B. KEEL, AND H. MISRA. 1973. Evidence that peroxidation of lysosomal membrane is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 248: 7792-7797.
OXYGEN
SPECIES
ACTIVITY
IN
AVIAN
DYSTROPHY
71
13. FRANCIS,S. H., S. BRANDON, AND J. H. PARK. 1976. Superoxide dismutase levels in tissue of genetically dystrophic chickens. Fed. Proc. 35: 1652. 14. FRIDOVICH, I. 1974. Superoxide dismutases. Adv. Enzymol. 41: 35-97. 15. GOLDSTEIN, I. M., AND G. WEISSMAN. 1977. Effects of the generation of superoxide anion on permeability of @some. Biochem. Biophys. Rex Commun. 75: 604-609. 16. HALLIWELL, B. 1978. Superoxidedependent formation of hydroxyl radicals in the presence of ion chelates. FEBS Left. 92: 31 l-326. 17. HANNA, S., R. KAWAMOTO, M. MCNAMEE, AND R. J. BASKIN. 1981. Enxymic activity of dystrophic chicken sarcoplasmic reticulum. B&him. Biophys. Acta 643: 41-54. 18. IODICE, A. A., J. CHIN, S. PARKER, AND I. M. WEINSTOCK. 1972. Cathepsins A, B, C, D and autolysis during development of breast muscle of normal and dystrophic chickens. Arch. Biochem. Biophys. 152: 166-174. 19. IONASESCU,V., L. Z. STERN, R. IONASEXU, AND P. RUBENSTEIN. 1979. Stimulating effects of drugs for protein synthesis on muscle cell cultures in dystrophy. Ann. Neural. 5: 107110. 20. JOCELYN, P. C. 1970. The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate. Biochem. J. 117: 951-956. 21. JULIAN, L. M., AND V. S. ASMUNDSON. 1963. Muscular dystrophy of the chicken. Pages 457-498 in G. H. BOURNE, AND M. N. GOLARZ, Eds., Muscular Dystrophy of Man and Animals. Karger, Basel. 22. KAR, N. C., AND C. M. PEARSON.1979. Catalase, superoxide dismutase, glutathione reductase and thiobarbituric acid-reactive products in normal and dystrophic human muscle. Clin. Chim. Acta 94: 277-280. 23. KAR, N. C., AND C. M. PEARSON. 1980. Elevated activity of a neutral proteins.% in human muscular dystrophy. B&hem. Med. 24: 238-243. 24. KATUNUMA, N., N. YASOGAWA, K. KITO, Y. SANADA, AND K. MIYOSHI. 1978. Abnormal expression of a serine protease in human dystrophic muscle. J. Biochem. (Tokyo) 83: 625-628.
E. W. III, AND I. FRIDOVICH. 1977. Liposome oxidation and hydrogen peroxide. J. Biol. Chem. 251: 6721-6728. 26. KOSOWER,N. S., G. A. VANDERHOF’F,AND E. M. KOSOWER. 1972. Glutatbione. VIII. The effects of glutathione disulfide on initiation of protein synthesis. B&him. Biophys. Acta 25. KELLOG,
272: 623-637. 27.
28.
29. 30. 31.
32.
33.
LOWRY, 0. H., N. J. ROSENBROUGH,A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. MCCAY, P. B., D. D. GIEISON, AND K. R. HORNBROOK. 1981. Glutathionedependent inhibition of lipid peroxidation by a soluble, heat-labile factor not &tathione peroxidase. Fed. Proc. 40: 199-205. MCCORD, J. M. 1976. Iron- and manganese-containing superoxide dismutases: structure, distribution, and evolutionary relationships. Adv. Exp. Med. Biol. 74: 540-550. MCCORD, J. M., AND D. DAY, JR. 1978. Superoxidedependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Left. 86: 139-142. MISRA, H. P., AND I. FRIDOVICH. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247: 31703175. MOKRI, B., AND A. G. ENGEL. 1975. Duchenne dystrophy: electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscular fiber. Neurology (Minneapolis) 25: 11 I 1- 1120. NEUBERT, D., A. B. WFJTCZAK, AND A. L. LEHNINGER. 1962. Purification and enzymatic
72
34.
35.
36. 37. 38.
YOSHIKUNI
MIZUNO
identity of mitochondrial contraction-factors I and II. Proc. Natl. Acad. Sci. USA 48: 1651-1658. NYLEN, E. G., AND K. WROGEMANN. 1983. Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice. Exp. Neural. 80: 6980. OMAYE, S. T., AND A. L. TAPPEL. 1974. Glutathione peroxidase, glutathione reductase, and thiobarbituric acid-reactive products in muscles of chickens and mice with genetic muscular dystrophy. Life Sci. 15: 137-145. OWENS, K. 1979. Biochemical studies of dystrophy in the young chicken lysosomal and sarcolemmal enzymes Ann. N. Y. Acad. Sci. 317: 247-262. PAGLIA, D. E., AND W. N. VALENTINE. 1967. Studies on the quantitative and qualitative characterization of erythrocytes glutathione peroxidase. J. Lab. Clin. Med. 70: 158- 169. PARK, J. H., E. J. HILL, T.-H. CHOU, V. LEQUIRE, R. REOLOFS, AND C. R. PARK. 1979. Mechanism of action of penicillamine in the treatment of avian muscular dystrophy.
Ann. N.Y. Acad. Sci. 317: 356-369. 39. PATNODE, R., E. BARTLE, E. J. HILL, V. LEQUIRE, AND J. H. PARK. 1976. Enzymological studies on hereditary avian muscular dystrophy. J. Biol. Chem. 251: 4468-4475. 40. PEDERSON, T. C., AND S. D. AUST. 1973. The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase. B&hem. Biophys. Rex Commun. 52: 107 l1078. 41. PETERSON, D. W., A. L. LILYBLADE, AND D. C. BOND. 1972. Proteolytic activity during growth of hypertrophic and atrophic muscles of genetically dystrophic chickens. Proc. Sot. Exp. Biol. Med. 141: 1056-1062. 42. ROWLAND, L. P. 1980. Biochemistry of muscle membranes in Duchenne muscular dystrophy.
Muscle Nerve 3: 3-20. 43. SALIN, M. L., AND J. M. MCCORD. 1975. Free radicals and inflammation. Protection of phagocytosing leukocytes by superoxide dismutase. J. C/in. Invest. 56: 13 19-1323. 44. SAMAHA, F. J., AND J. GERGELY. 1969. Biochemical abnormalities of the sarcoplasmic reticulum in muscular dystrophy. N. Engl. J. Med. 280: 184-188. 45. SCALES, D., R. SABBADINI, AND G. INESI. 1977. The involvement of snrcotubular membranes in genetic muscular dystrophy. B&him. Biophys. Acta 465: 535-549. 46. SIES, H., C. GERSTENECKER, H. MENZEL, AND L. FLOHE. 1972. Oxidation in the NADP system and release. of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides. FEBS Lett. 27: 17 1-175. 47. SINGH, A. 1982. Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen. Can. J. Physiol. Pharmacol. 60: 1330-1345. 48. SUGITA, H., S. ISHIURA, K. SUZUKI, AND K. IMAHORI. 1980. Ca-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils. Muscle Nerve 3: 335-339. 49. SUN, M., AND S. ZIGMAN. 1978. An improved dismutase based on epinephrine autoxidation. 50. TANIZAWA, H., Y. SAZUKA, in the mouse myocardium 29: 2910-2914. 5 1. TAPPEL, A. L. 1973. Lipid 1973. 52. VLADIMIROV, peroxidation
spectrophotometric
AND Y. TAKINO. 1981. Micro-determination by thiobarbituric acid tluorometry. Chem. peroxidation
damage
assay for superoxide
Anal. Biochem. 90: 81-89.
to cell components.
Y. A., V. I. OLENEV, T. B. SUSLOVA, in mitochondrial membrane. Adv.
of lipoperoxide
Pharm. Bull. (Tokyo) Fed. Proc. 32: 1970-
AND z. P. CHEREMISINA.
1980. Lipid
Lipid Rex 17: 173-249.
53. VCK~T, M. Y., C. THOMAS, C. L. VASSALO, R. E. BASFORD, AND J. B. L. GEE. 197 1. Glutathionedependent peroxidative metabolism in the alveolar macrophage. J. Clin. Invest. 50: 401-410.
OXYGEN
SPECIES ACTIVITY
IN AVIAN
DYSTROPHY
73
54. WEINSTOCK, L. M., T. S. SOH, H. A. FREEMAN, AND M. E. CUTLER. 1969. Amino acid incorporation into proteins of developing breast muscle of the normal and dystrophic chicken. B&hem. Med. 2: 345-356. 55. WEISINGER, R. A., AND I. PRIDOVICH. 1973. Mitochondrial superoxide dismutase. J. Biol.
Chem.248:4793-4796. 56. WILSON, B. W., W. R. RANDALL, G. T. PATI-ERSON, AND R. K. ENTRIKIN. 1979. Major histologic and hi&chemical characterixation of inherited dystrophy of the chicken. Ann.
N.Y. Acad.Sci.317:224-246. 57. WROGEMANN, K., AND S. D. J. PENA. 1976. Mitochondrial calcium overload: a general mechanism for cell necrosis in muscle disease. Luncet1: 672-674. 58. YOUNES, M., AND C.-P. SIEGERS.1980. Lipid peroxidation as a consequence of glutathione depletion in rat and mouse liver. Rex Commun. Chem.Pathol.Pharmacol. 27: 119-128.