83
Original Articles
0 1992
The Japanese Society o f Pathology
Immunoreactive Copper-Zinc Superoxide Dismutase in Damaged Human Myocardium
Ryugo Sato, Kenji Kashima, Shigeo Yokoyama, and lwao Nakayama The myocardium i n 50 autopsy cases was studied using immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and standard histochemical procedures. Mucinous degeneration observed in 42 cases showed moderately enhanced expression of immunoreactive CuZn-SOD i n lesions which were stained strongly by periodic acidSchiff but negative with Heidenhain iron-hematoxylin (HIH), von Kossa and luxol fast blue (LFB) stains, whereas coagulation necrosis in 4 cases revealed almost identical immunostaining for CuZn-SOD and HIH t o that of contraction band necrosis, i.e. strongly positive HIH staining but negative immunostaining. Basophilic alteration of the myocardial cells in sections fixed with 4% formalin i n 2% calcium acetate was seen i n 29cases, being identified frequently i n isolated cells as well as in several foci varying considerably i n size. This type of alteration demonstrated significantly enhanced expression of immunoreactive CuZn-SOD and was strongly positive with von Kossa and LFB stains. The present study indicates that the myocardium can be affected by free radicals produced i n any organ of the body, and that subsequently, insoluble phospholipids react with calcium ions i n the fixative and accumulate i n the basophilic sarcoplasm. Acta Pathol Jpn 42: 83-90, 1992. Key words : Immunostaining, CuZn--superoxidedismutase, Myocardia I damage
It is well known that at least three different types of superoxide dismutase (SOD), i.e. cytosolic copper-zinc SOD (CuZn-SOD), mitochondria1 manganese SOD (MnSOD) (1) and extracellular SOD (2), exist in mammalian tissues. This enzyme catalyzes dismutation of the superoxide anion, a toxic free radical produced by
Received July 1, 1991. Accepted for publication October 14, 1991. First Department of Pathology, Medical College of Oita, Oita. Mailing address: Ryugo Sato. First Department of Pathology, Medical College of Oita, 1-1 Idaigaoka, Hasama-machi, Oita-gun, Oita 879-55, Japan.
monovalent metabolic reduction of oxygen and implicated in a variety of pathologic processes (3-6). Assays of bioactivity have revealed that the content of SOD varies considerably in different tissues, the highest activity being present in the liver, followed in descending order by the kidney, muscle and brain (7-9). However, the results of biochemical analyses are difficult to compare with those of immunostaining for CuZn-SOD, because of the three types of SOD that are present in mammalian tissues. Therefore, until the reports of Thaete et a/. in 1 9 8 3 ( 1 0 ) and 1 9 8 5 ( l l ) , the specific cell type containing CuZn-SOD in mammalian tissues remained largely undetermined. They reported immunohistochemical localization of CuZn-SOD in various organs except for the myocardium in rat and dog. Recently, Dobashi et a/. (12) studied the distribution of both CuZn-SOD and Mn-SOD in normal rat myocardium, and Watanabe et a/. (13) reported intense expression of immunoreactive CuZn-SOD in the canine myocardium, probably indicative of myocardial damage caused by free radicals produced in extracardiac regions, after re-establishment of arterial flow in an experimentally induced myonephropathic metabolic syndrome. Therefore, it is thought that myocardial damage in humans might be induced by free radicals produced in various organs under severe pathological conditions during a life-ordeath crisis. The present study describes the results of immunostaining for CuZn-SOD, and of histochemical staining with Heidenha in iron- hematoxylin (HI H), periodic acidSchiff (PAS), luxol fast blue (LFB) and von Kossa in human myocardium obtained at autopsy.
MATERIALS AND METHODS Myocardial specimens used in this study were obtained from 50 consecutive autopsied patients aged over 40 years, except for one 12-year-old girl, with various
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.)
84
diseases, including epithelial and non-epithelial malignant neoplasms untreated or treated with adriamycin (total dose less than 240 mg), at the Medical College of Oita. The myocardium, 3 mm in thickness, was removed transversely at a level 2 cm from the atrioventricular valves and fixed with either 4% formalin in 2% calcium acetate for 5 h at room temperature or 10% neutral formalin for 2 4 h and embedded in paraffin. Serial 4 - p m sections were cut from all blocks and stained with hematoxylin-eosin (HE), PAS, HIH, von Kossa, LFB and an immunohistochemical stain for CuZn-
SOD. For the immunostaining of CuZn-SOD, endogenous peroxidase activity was blocked by incubation with 3% H,O, for 1 5 m i n . After washing in three changes of 1 / 1 5 M phosphate-buffered saline (PBS, pH 7.4) for 5 min each, the sections were preincubated in 0.1% protease (Sigma Chemical Co., St. Louis, MO) in PBS for 3 m i n at room temperature. They were incubated at room temperature in 20% normal goat serum diluted with PBS for 1 5 min and then treated with anti-human CuZn-SOD rabbit IgG diluted 1 : 200 for 30 min followed by the avidin-biotin peroxidase complex (BioGenex, Dublin, CA) method (14). After washing in 0.05 M Tris-HCI buffer (pH 7.6), the sections were reacted with a solution containing 2 0 m g of 3,3'-diaminobenzidine tetrahydrochloride in 100 mi of 0.05 M Tris-HCI buffer with 0.1 ml of 5% H202for 10 min. The nuclei were counterstained for 5 min with hematoxylin. Control sections incubated in either normal rabbit IgG or the primary antibody absorbed with the corresponding antigen at 37°C for 2 h instead of the primary antibody showed no nonspecific staining. Purified human CuZn-SOD (Sigma Chemical Co.) was emulsified in an equal volume of Freund's complete adjuvant (FCA). Rabbits were then injected subcutaneously with 0.5 mg of CuZn-SOD in FCA. One month after the first injection, the rabbits were given another injection in the same manner using an emulsion of CuZn-SOD in Freund's incomplete adjuvant. Antisera obtained from the rabbits 14 days after the second injection were applied to a Protein A Sepharose CL4B column (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) for 4 h at 4°C and eluted with 0.1 M phosphate buffer (pH -
-_ _
-
_______
Figure 1. Sodium dodecyl sulfate-20% polyacrylamide gel electrophoresis and western blotting. kDa, kilodaltons ; a, low - molecula r-weig ht k i t E (Pha rmacia LKB Biotechnology AB) ; b, purified human CuZn-SOD, calculated molecular mass: 16 kDa ; c, western blotting using anti-CuZn-SOD rabbit IgG.
8.0) followed by 1 M acetic acid solution. The specificity of the antibody was established by western blotting (Fig. 1) and enzyme-linked immunosorbent assay. Statistica I analysis (Fisher's exact probability test) was performed to test the differences in the developmental incidence of basophilic alteration among the cases of malignant epithelial and non-epithelial neoplasms, and non-neoplastic diseases.
RESULTS Apart from fibrous scars caused by old myocardial infarcts and interstitial fibrosis, several different types of myocardial disorders were observed histologically in the sections of myocardium obtained from the 50 autopsy cases. These included mucinous degeneration in 42 cases, contraction band necrosis in 4, coagulation ne-
-~-
Figure 2. Micrographs showing mucinous degeneration of the myocardium. Areas of mucinous degeneration show moderately enhanced expression of immunoreactive CuZn-SOD (b), no staining with HIH ( c ) or LFB (d). a-d, serial sections; a : HE, h : immunostaining for CuZn SOD, c : HIH staining, d : LFB staining. Figures 3. Contraction band necrosis, demonstrating strongly positive HIH staining (a). The contraction bands are completely negative with CuZn-SOD immunostaining (b, arrow), but the intercalated discs and sarcoplasm of the neighboring cells show weak to moderate deposits of immunoreaction product (b, double arrows). a and b, serial sections; a : HIH staining. b : immunostaining for CuZn-SOD. Figures4. Micrographs showing a focus of myocardial infarction. The myocardial cells exhibiting coagulation necrosis are dark black in color by HIH staining (a), but contain no immunoreactive CuZn-SOD (b). a and b, serial sections; a : HIH staining, b : immunostaining for CuZn-SOD.
Acta Pathologica Japonica 42 (2) : 1992
85
86
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato ef at.)
87
Acta Pathologica Japonica 42 (2) : 1992
crosis in 4 and basophilic alteration in 29. The results obtained from the histochemical and immunohistochemical staining are summarized in Table 1. Mucinous degeneration was observed sporadically in isolated myocardial cells as large vesicles filled with mucinous substances occupying almost the entire sarcoplasm, surrounding the nucleus and stretching along the greater axis of the cell (Fig. 2a). The mucinous substance was slightly basophilic with HE staining in sections fixed with both 10% neutral formalin and 4% formalin in 2% calcium acetate. Even in the most severely affected fibers, a rim of sarcoplasm with preserved myofibrils could be seen (Fig. 2a) and the nucleus, located either inside the lesion or at the rim, was usually deformed by compression (Fig. 2b). The areas of mucinous degeneration were strongly PAS-positive and contained a more intense reaction product for CuZn-SOD with a diffuse distribution than that at the rim and in neighboring myocardial cells (Fig. 2b), whereas they were not stained by the HIH and LFB methods (Figs. 2c, d), in contrast to normal myocardium, which showed speckled dark spots with the former staining and a negative result with the latter (Figs. 2c, d). Contraction band necrosis observed in 4 patients who had shown no clinical sign of cardiac disorder during hospitalization was characterized by hypercontraction of the myocardial fibers, accompanied by a considerable number of irregularly shaped acidophilic bands by HE staining and numerous dark spots with HIH (Fig. 3a). These myocardial cells did not stain with LFB, and the contraction bands were completely negative for CuZnSOD immunostaining (Fig. 3b, arrow), although the intercalated discs and sarcoplasm of these cells showed weak deposits of CuZn-SOD immunoreaction product (Fig. 3b, double arrows).
Myocardial cells showing coagulation necrosis due to either acute myocardial infarction (1 case) or sepsis (3 cases) were characterized by an acidophilic sarcoplasm without nuclei, which stained light blue with LFB and a dark color with HIH (Fig. 4a), but showed no immunoreaction for CuZn-SOD (Fig. 4b). On the other hand, myocardial cells surrounding areas of coagulation necrosis demonstrated vacuolate degeneration in their sarcoplasm in a case of acute myocardial infarction, where a moderately enhanced immunoreaction for CuZnSOD was seen, especially at the rim (Fig. 4b). The most conspicuous alteration was seen in the myocardium fixed with 4% formalin in 2% calcium acetate, in comparison with that fixed in 10% neutral formalin. The myocardium fixed with the former mixture demonstrated basophilic alteration in 2 9 of the 5 0 cases. This basophilic alteration was observed in the right or left ventricle, or both. The affected cells were either single isolated ones (Fig. 5a) or appeared in several foci each composed of a considerable number of myocardial cells (Fig. 6a). Furthermore, the basophilic alteration was usually present throughout the entire sarcoplasm of the myocardial cells, but occasionally was observed only in a segment of each cell (Fig. 5a). The immunoreactivity for CuZn-SOD and the results obtained from histochemical observations using LFB and von Kossa stains in areas of basophilic alteration were quite different from those in normal adjacent myocardial cells. Areas of basophilic alteration exhibited significantly enhanced expression of immunoreactive CuZn-SOD, as revealed by intense deposition of reaction product either diffusely (Figs. 5b, 6b) or segmentally in the sarcoplasm (Fig. 5b, arrow). However, endothelial cells of capillaries or relatively large vessels located adjacent to the myocardial cells showing the basophilic
Table 1. Results of Histochemical and lmmunohistochemical Staining Normal rnvocardium
HIH PAS von Kossa LFB CuZu-SOD
+
-
Mucinous denenera t ion
Basophilic alteration
Contract ion band necrosis
Coagulation necrosis
-
+ -
-
-
-----c
+
+,
weakly positive staining ; +, moderately positive staining or considerably enhanced expression -, negative staining ; of immunoreactive CuZn-SOD ; it, strongly positive staining or intensely enhanced expression of immunoreactive CuZnSOD. Figures 5. Myocardial cells showing basophilic alteration are distributed sporadically (a) and the majority show diffuse and significantly enhanced expression of immunoreactive CuZn-SOD (b). However, some cells demonstrate a segmental distribution of the reaction product (b, arrow). These cells show strongly positive staining with von Kossa (c) and LFB (d). a-d, serial sections; a : HE, b : imrnunostaining for CuZn-SOD, c : von Kossa staining, d : LFB staining. Figures 6. Myocardial cells showing basophilic alteration distributed as small foci each containing a considerable number of cells (a). The majority show significantly enhanced expression of immunoreactive CuZn-SOD (b). These cells show strongly positive von Kossa (c) and LFB (d) staining. a-d, serial sections; a : HE, b : imrnunostaining for CuZn-SOD, c : von Kossa staining, d : LFB staining.
88
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.)
Table 2. Relationship between Background Diseases and Basophilic Alteration Background diseases
Number of cases
A) Malignant epithelial neoplasms 8 ) Malignant non-epithelial neoplasms C) Non-neoplastic diseases Total
28 9 13
50
Number of cases showing basophilic alteration 18 (64%) 4 (44%) 7 (54%) 29 (58%)
There were no significant differences among A, B and C (p>0.05).
alteration and sarcolemmal cells surrounding them exhibited no enhancement of the CuZn-SOD immunoreaction (Fig. 5b). Only myocardial cells staining brown with von Kossa (Figs. 5c, 6c) and deep blue with LFB (Figs. 5d, 6d) demonstrated significant enhancement of the CuZn-SOD immunoreaction. Basophilic alteration in the myocardium showed no relationship with background diseases, which included malignant epithelial and nonepithelial neoplasms, and other non-neoplastic conditions (Table 2). Furthermore, in the cases of malignant neoplasm, the incidence of basophilic alteration did not differ significantly (p>0.05) between those untreated and those treated with ad ria mycin. The myocardium without any basophilic alteration seen in 2 9 of the 50cases was weakly to moderately positive for immunoreactive CuZn-SOD, especially in intercalated discs, as shown in Fig. 3b, but only weakly positive or negative in the sarcoplasm (see Figs. 2b, 5 b and 6b). In addition, these cases were not stained with von Kossa and LFB.
DISCUSSION It has been clearly demonstrated using biochemical procedures that SOD is detectable over a wide concentration range in various mammalian tissues, even in normal or damaged myocardium (7-9, 15). However, such biochemical results cannot be compared with those of immunostaining for CuZn-SOD. In the present study, immunostaining for CuZn-SOD in myocardium fixed with 4% formalin in 2% calcium acetate showed a specific reaction product in the sarcoplasm. Moderately to markedly enhanced expression or complete loss of immunoreactive CuZn-SOD was seen in human myocardial cells under certain pat holog ica I conditions. M yoca rdia I damage other than basophilic alteration was observed in sections fixed with both 10% neutral formalin and 4% formalin in 2% calcium acetate. However, immunostaining for CuZn-SOD was clearer in sections fixed with the latter than with the former. Thus there is some difficulty in explaining the absence of the reaction product for CuZn-SOD in some sections fixed with 10% formalin. It is hypothesized that CuZn-SOD in the myocardium easily loses its antigenicity during the proc-
ess of tissue preparation. Therefore, in previous studies describing the immunohistochemical procedure for demonstration of CuZn-SOD, all tissues have been fixed with 4% formalin in 2% calcium acetate (10, 11, 13), and the specimens in the present study were also fixed in this way. Myocardial tissue judged to be normal with HE, HIH and LFB staining exhibited a more distinct reaction product in intercalated discs than in the sarcoplasm, which showed weaker o r negative staining, although Thaete et a/. (16) reported that immunostained sections of canine skeletal muscle showed a strongly striated staining pattern, which was interpreted as evidence for the presence of CuZn-SOD in the Z line and I band. Some variations in the immunoreactivity for CuZn-SOD might occur due to differences in species or specificity of the staining for cardiac or skeletal muscle, although the intercalated discs observed in the myocardium correspond to the Z lines of skeletal muscle. The myocardium with coagulation necrosis due to acute myocardial infarction or sepsis, and that with contraction band necrosis completely lacked immunoreactive CuZn-SOD in the sarcoplasm and contraction bands, indicating leakage of the enzyme from the dead cells. Watanabe et a/. (13), using an experimentally induced myonephropathic metabolic syndrome in dogs, reported that the fibers of the adductor muscle showed conspicuous alterations after re-establishment of arterial flow, which were characterized by a loss of transverse striations in association with dissolution of the sarcoplasm and negative immunostaining for both myoglobin and CuZn-SOD. On the other hand, myocardium showing mucinous degeneration, which has been referred to as basophilic, mucoid and mucinous degeneration, or cardiac colloid (17-20), demonstrated enhanced expression of immunoreactive CuZn-SOD in the degenerated sarcoplasm, which was strongly stained with PAS but negative with LFB and von Kossa stains. The basophilic alteration appeared to have no direct relationship with administration of the cardiotoxic drug, adriamycin (21,22), and demonstrated intense deposits of the immunoreaction product for CuZn-SOD, associated with deep blue staining with LFB and brown staining with von Kossa.
Acta Pathologica Japonica 42 (2) : 1992
Mucinous degeneration appears to be quite different in its distribution, histochemical features, intensity of CuZn-SOD immunoreactivity and mechanism of metabolic abnormality in comparison with basophilic alteration of the myocardium. From the results of biochemical analysis and positivity obtained with all glycogen stains that are digested with amyloglucosidase, malt diastase and pectinase, it is postulated that mucinous degeneration is composed of a glucan representing a byproduct of glycogen metabolism (19). A previous study found that mucinous degeneration was present in isolated myocardial cells from almost all individuals more than 11 years of age, although it was not seen in any patients who had died in the first decade of life (19). In the present study, myocardial tissue from a 12-year-old girl showed no mucinous degeneration in the myocardium. After sufficient discoloration with 0.05% lithium carbonate followed by 70% ethanol, phospholipids were not stained by LFB in normal myocardium fixed with 4% formalin in 2% calcium acetate, and von Kossa staining was also negative, since it is likely that calcium ions react only slightly with phosphatidylcholine (23), the principal phospholipid component of mammalian cell membranes. The basophilic alteration observed in the myocardium was considered to result from conversion of a soluble form of phospholipid to an insoluble one, possibly due to the effect of free radicals, since there was an intense reaction for CuZn-SOD and reaction with calcium ions in the fixative. There are several hypotheses related to free-radical production under conditions of cellular damage. Using a biochemical approach, Granger et a/. (24) and McCord (25) demonstrated that oxygen-derived free radicals appeared to be produced by xanthine oxidase in postischemic tissue. Membranebound nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) in neutrophils may play an important role in sterilization through production of its own free radical (26,27). Free radicals are also produced by the mitochondria1 electron transport system (28), the enzymic oxidation of arachidonic and linoleic acids (29) and the NADPH-P-450 reductase system in microsomes and nuclear membranes (30, 31). In addition, lipid peroxide, which has a long half-life but weaker reactivity than that of superoxide, can be induced directly and/or through production of free radicals. It is difficult to determine the direct source of peroxide production, whether it be certain organelles of affected myocardial cells, other cells in the vicinity, or other organs secondary to abnormal metabolism during a lifeor-death crisis. Nishigaki et a/. (32) reported an increase of lipid peroxide in the spleen, liver and kidney, as well as elevation of enzymes released from these
89
organs following an increase of lipid peroxide in burned skin. It is thought that several factors contribute to basophilic alteration, including ischemic change in the myocardium, contraction of myocardial fibers, arteriosclerosis of the coronary arteries, reduced supply of oxygen to the myocardium and abnormal metabolism. In the present study, the effects of these factors differed considerably from one case to another, because all the materials examined were obtained from autopsy cases. Therefore, it is considered that the myocardium with or without basophilic alteration reflects the presence or absence of the severe pathological conditions described above, which alter the amounts of free radicals produced in a life-or-death crisis. In myocardial specimens showing markedly enhanced expression of CuZn-SOD with basophilic alteration, it is thought that more CuZn-SOD was produced in the sarcoplasm in order to protect against cellular damage induced by free radicals. The basophilic alteration observed in the myocardium was distributed either diffusely or segmentally in the sarcoplasm. Takasaki et a/. (33) described in experimentally induced myonephropathic metabolic syndrome that the adductors showed severe ischemic change characterized by the separation of muscle fibers due to an increase of interstitial tissue fluid, and partial or total degeneration of the individual muscle fibers. Therefore, myocardium affected by free radicals may undergo either segmental or total degeneration of the sarcoplasm. Basophilic alteration is considered to be a cellular response to free radicals and certain degenerative changes in the myocardium, since the myocardium affected by free radicals shows increased amounts of CuZn-SOD and accumulation of phospholipids reactive with calcium ions in the sarcoplasm.
REFERENCES 1. Weisiger RA and Fridovich I. Mitochondria1superoxide dismutase : Site of synthesis and intramitochondrial localization. J Biol Chem 248: 4793-4796, 1973. 2. Marklund SL, Holme E, and Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 126: 41-51, 1982. 3. Grankvist K, Marklund SL, Sehlin J, and Taljedal IB. Superoxide dismutase, catalase and scavengers of
hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 182 : 17-25, 1979. 4.
Fridovich I. Superoxide radical : An endogenous toxicant. Annu Rev Pharmacol Toxicol 23: 239-257,
1983. 5. Jolly SR, Kane WJ, Bailie MB, Abrams GD, and Lucchesi BR. Canine myocardial reperfusion injury : Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 54: 277-285,
90
6.
7.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17. 18. 19.
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.) 1984. Girotti AW and Thomas JP. Superoxide and hydrogen peroxide-dependent lipid peroxidation in intact and Triton-dispersed erythrocyte membranes. Biochem Biophys Res Commun 118: 474-480, 1984. Peeters-Joris C, Vandevoorde AM, and Baudhuin P. Subcellular localization of superoxide dismutase in rat liver. Biochem J 150: 31-39, 1975. Westman NG and Marklund SL. Copper- and zinccontaining superoxide dismutase and manganesecontaining superoxide dismutase in human tissues and human malignant tumors. Cancer Res 41: 29622966, 1981. Marklund SL, Westman NG, Lundgren E, and Roos G. Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res 42: 1955-1961, 1982. Thaete LG, Crouch RK, Schulte BA, and Spicer SS. The immunolocalization of copper-zinc superoxide dismutase in canine tissues. J Histochem Cytochem 31 : 1399.-1406, 1983. Thaete LG, Crouch RK, and Spicer SS. Immunolocalization of copper-zinc superoxide dismutase : 11. Rat. J Histochem Cytochem 33 : 803-808, 1985. Dobashi K, Asayama K, Kato K, Kobayashi M, and Kawaoi A. lmmunohistochemical localization of copper-zinc and manganese superoxide dismutases in rat tissues. Acta Histochem Cytochem 22 : 351 -365, 1989. Watanabe S, Hadama T, Takasaki H, et a/. Myocardial damage caused by free radicals in experimentally induced myonephropathic metabolic syndrome in dogs. Jpn J Surg ( i n press). Guesdon JL, Ternynck T, and Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27: 1131-1139, 1979. Sazuka Y, Tanizawa H, and Takino Y. Effect of adriamycin on the activities of superoxide dismutase, glutathione peroxidase and catalase in tissues of mice. Jpn J Cancer Res 80: 89-94, 1989. Thaete LG, Crouch RK, and Spicer SS. Immunolocalization of CuZn superoxide dismutase in striated muscle. Histochem J 17: 259-262, 1985. Scotti TM. Basophilic (mucinous) degeneration of the myocardium. Am J Clin Pathol25 : 994-101 1, 1955. Haust MD, Rowlands DT Jr, Garancis JC, and Landing BH. Histochemical studies on cardiac "colloid". Am J Pathol 40: 185-197, 1962. Rosai J and Lascano EF. Basophilic (mucoid) degeneration of myocardium. A disorder of glycogen metabolism. Am J Pathol 61 : 99-112, 1970.
20. 21. 22.
2 3.
24.
25. 26.
27.
28.
29.
30.
31,
32.
3 3.
Kawamoto S and Wakabayashi T. Basophilic degeneration with special reference to its pathophysiological aspects. Acta Pathol Jpn 33: 619-628, 1983. Myers CE, McGuire WP, Liss RH, et a/. Adriamycin: The role of lipid peroxidation in cardiac toxicity and tumor response. Science 197 : 165-167, 1977. Politi PM and Rothlin RP. Acute effects of doxorubicin on chronotropic and inotropic mechanisms in guinea pig atria. Cancer Treat Rep 69 : 859-865, 1985. Maeda M, Nishijima M, Takenaka Y, Kuge 0, and Akamatsu Y. pHdependent Ca2+ interaction with phospholipids related to phosphatidylethanolamine Nmethylation. Biochim Biophys Acta 794 : 298-306, 1984. Granger DN, Rutili G, and McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 81 : 22-29, 1981. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159-163, 1985. Babior BM, Kipnes RS, and Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52: 741-744, 1973. Petrone WF, English DK, Wong K, and McCord JM. Free radicals and inflammation : Superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 77: 1159-1163, 1980. Cadenas E, Boveris A, Ragan C1, and Stoppani AOM. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch Biochem Biophys 180: 248-257, 1977. Porter NA, Lehman LS, Weber BA, and Smith KJ. Unified mechanism for polyunsaturated fatty acid autoxidation. Competition of peroxy radical hydrogen atom abstraction, p-scission, and cyclization. J Am Chem SOC 103: 6447-6455, 1981. Strobel HW and Coon MJ. Effect of superoxide generation and dismutation on hydroxylation reactions catalized by liver microsomal cytochrome P-450. J Biol Chem 246: 7826-7829, 1971. Patton SE, Rosen GM, and Rauckman EJ. Superoxide generation by purified hamster hepatic nuclei. Mol Pharmacol 18: 588-593, 1980. Nishigaki I, Hagihara M, Hiramatsu M, lzawa Y, and Yagi K. Effect of thermal injury on lipid peroxide levels of rat. Biochem Med 24: 185-189, 1980. Takasaki H, Hadama T, Matsushima R, Watanabe S, and Shirabe J. Extracorporeal circulation for the removal of serum myoglobin in experimentally induced myonephropathic metabolic syndrome in dogs. Jpn J Surg 21 : 201-209, 1991.
Original Articles
0 1992
The Japanese Society o f Pathology
Immunoreactive Copper-Zinc Superoxide Dismutase in Damaged Human Myocardium
Ryugo Sato, Kenji Kashima, Shigeo Yokoyama, and lwao Nakayama The myocardium i n 50 autopsy cases was studied using immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and standard histochemical procedures. Mucinous degeneration observed in 42 cases showed moderately enhanced expression of immunoreactive CuZn-SOD i n lesions which were stained strongly by periodic acidSchiff but negative with Heidenhain iron-hematoxylin (HIH), von Kossa and luxol fast blue (LFB) stains, whereas coagulation necrosis in 4 cases revealed almost identical immunostaining for CuZn-SOD and HIH t o that of contraction band necrosis, i.e. strongly positive HIH staining but negative immunostaining. Basophilic alteration of the myocardial cells in sections fixed with 4% formalin i n 2% calcium acetate was seen i n 29cases, being identified frequently i n isolated cells as well as in several foci varying considerably i n size. This type of alteration demonstrated significantly enhanced expression of immunoreactive CuZn-SOD and was strongly positive with von Kossa and LFB stains. The present study indicates that the myocardium can be affected by free radicals produced i n any organ of the body, and that subsequently, insoluble phospholipids react with calcium ions i n the fixative and accumulate i n the basophilic sarcoplasm. Acta Pathol Jpn 42: 83-90, 1992. Key words : Immunostaining, CuZn--superoxidedismutase, Myocardia I damage
It is well known that at least three different types of superoxide dismutase (SOD), i.e. cytosolic copper-zinc SOD (CuZn-SOD), mitochondria1 manganese SOD (MnSOD) (1) and extracellular SOD (2), exist in mammalian tissues. This enzyme catalyzes dismutation of the superoxide anion, a toxic free radical produced by
Received July 1, 1991. Accepted for publication October 14, 1991. First Department of Pathology, Medical College of Oita, Oita. Mailing address: Ryugo Sato. First Department of Pathology, Medical College of Oita, 1-1 Idaigaoka, Hasama-machi, Oita-gun, Oita 879-55, Japan.
monovalent metabolic reduction of oxygen and implicated in a variety of pathologic processes (3-6). Assays of bioactivity have revealed that the content of SOD varies considerably in different tissues, the highest activity being present in the liver, followed in descending order by the kidney, muscle and brain (7-9). However, the results of biochemical analyses are difficult to compare with those of immunostaining for CuZn-SOD, because of the three types of SOD that are present in mammalian tissues. Therefore, until the reports of Thaete et a/. in 1 9 8 3 ( 1 0 ) and 1 9 8 5 ( l l ) , the specific cell type containing CuZn-SOD in mammalian tissues remained largely undetermined. They reported immunohistochemical localization of CuZn-SOD in various organs except for the myocardium in rat and dog. Recently, Dobashi et a/. (12) studied the distribution of both CuZn-SOD and Mn-SOD in normal rat myocardium, and Watanabe et a/. (13) reported intense expression of immunoreactive CuZn-SOD in the canine myocardium, probably indicative of myocardial damage caused by free radicals produced in extracardiac regions, after re-establishment of arterial flow in an experimentally induced myonephropathic metabolic syndrome. Therefore, it is thought that myocardial damage in humans might be induced by free radicals produced in various organs under severe pathological conditions during a life-ordeath crisis. The present study describes the results of immunostaining for CuZn-SOD, and of histochemical staining with Heidenha in iron- hematoxylin (HI H), periodic acidSchiff (PAS), luxol fast blue (LFB) and von Kossa in human myocardium obtained at autopsy.
MATERIALS AND METHODS Myocardial specimens used in this study were obtained from 50 consecutive autopsied patients aged over 40 years, except for one 12-year-old girl, with various
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.)
84
diseases, including epithelial and non-epithelial malignant neoplasms untreated or treated with adriamycin (total dose less than 240 mg), at the Medical College of Oita. The myocardium, 3 mm in thickness, was removed transversely at a level 2 cm from the atrioventricular valves and fixed with either 4% formalin in 2% calcium acetate for 5 h at room temperature or 10% neutral formalin for 2 4 h and embedded in paraffin. Serial 4 - p m sections were cut from all blocks and stained with hematoxylin-eosin (HE), PAS, HIH, von Kossa, LFB and an immunohistochemical stain for CuZn-
SOD. For the immunostaining of CuZn-SOD, endogenous peroxidase activity was blocked by incubation with 3% H,O, for 1 5 m i n . After washing in three changes of 1 / 1 5 M phosphate-buffered saline (PBS, pH 7.4) for 5 min each, the sections were preincubated in 0.1% protease (Sigma Chemical Co., St. Louis, MO) in PBS for 3 m i n at room temperature. They were incubated at room temperature in 20% normal goat serum diluted with PBS for 1 5 min and then treated with anti-human CuZn-SOD rabbit IgG diluted 1 : 200 for 30 min followed by the avidin-biotin peroxidase complex (BioGenex, Dublin, CA) method (14). After washing in 0.05 M Tris-HCI buffer (pH 7.6), the sections were reacted with a solution containing 2 0 m g of 3,3'-diaminobenzidine tetrahydrochloride in 100 mi of 0.05 M Tris-HCI buffer with 0.1 ml of 5% H202for 10 min. The nuclei were counterstained for 5 min with hematoxylin. Control sections incubated in either normal rabbit IgG or the primary antibody absorbed with the corresponding antigen at 37°C for 2 h instead of the primary antibody showed no nonspecific staining. Purified human CuZn-SOD (Sigma Chemical Co.) was emulsified in an equal volume of Freund's complete adjuvant (FCA). Rabbits were then injected subcutaneously with 0.5 mg of CuZn-SOD in FCA. One month after the first injection, the rabbits were given another injection in the same manner using an emulsion of CuZn-SOD in Freund's incomplete adjuvant. Antisera obtained from the rabbits 14 days after the second injection were applied to a Protein A Sepharose CL4B column (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) for 4 h at 4°C and eluted with 0.1 M phosphate buffer (pH -
-_ _
-
_______
Figure 1. Sodium dodecyl sulfate-20% polyacrylamide gel electrophoresis and western blotting. kDa, kilodaltons ; a, low - molecula r-weig ht k i t E (Pha rmacia LKB Biotechnology AB) ; b, purified human CuZn-SOD, calculated molecular mass: 16 kDa ; c, western blotting using anti-CuZn-SOD rabbit IgG.
8.0) followed by 1 M acetic acid solution. The specificity of the antibody was established by western blotting (Fig. 1) and enzyme-linked immunosorbent assay. Statistica I analysis (Fisher's exact probability test) was performed to test the differences in the developmental incidence of basophilic alteration among the cases of malignant epithelial and non-epithelial neoplasms, and non-neoplastic diseases.
RESULTS Apart from fibrous scars caused by old myocardial infarcts and interstitial fibrosis, several different types of myocardial disorders were observed histologically in the sections of myocardium obtained from the 50 autopsy cases. These included mucinous degeneration in 42 cases, contraction band necrosis in 4, coagulation ne-
-~-
Figure 2. Micrographs showing mucinous degeneration of the myocardium. Areas of mucinous degeneration show moderately enhanced expression of immunoreactive CuZn-SOD (b), no staining with HIH ( c ) or LFB (d). a-d, serial sections; a : HE, h : immunostaining for CuZn SOD, c : HIH staining, d : LFB staining. Figures 3. Contraction band necrosis, demonstrating strongly positive HIH staining (a). The contraction bands are completely negative with CuZn-SOD immunostaining (b, arrow), but the intercalated discs and sarcoplasm of the neighboring cells show weak to moderate deposits of immunoreaction product (b, double arrows). a and b, serial sections; a : HIH staining. b : immunostaining for CuZn-SOD. Figures4. Micrographs showing a focus of myocardial infarction. The myocardial cells exhibiting coagulation necrosis are dark black in color by HIH staining (a), but contain no immunoreactive CuZn-SOD (b). a and b, serial sections; a : HIH staining, b : immunostaining for CuZn-SOD.
Acta Pathologica Japonica 42 (2) : 1992
85
86
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato ef at.)
87
Acta Pathologica Japonica 42 (2) : 1992
crosis in 4 and basophilic alteration in 29. The results obtained from the histochemical and immunohistochemical staining are summarized in Table 1. Mucinous degeneration was observed sporadically in isolated myocardial cells as large vesicles filled with mucinous substances occupying almost the entire sarcoplasm, surrounding the nucleus and stretching along the greater axis of the cell (Fig. 2a). The mucinous substance was slightly basophilic with HE staining in sections fixed with both 10% neutral formalin and 4% formalin in 2% calcium acetate. Even in the most severely affected fibers, a rim of sarcoplasm with preserved myofibrils could be seen (Fig. 2a) and the nucleus, located either inside the lesion or at the rim, was usually deformed by compression (Fig. 2b). The areas of mucinous degeneration were strongly PAS-positive and contained a more intense reaction product for CuZn-SOD with a diffuse distribution than that at the rim and in neighboring myocardial cells (Fig. 2b), whereas they were not stained by the HIH and LFB methods (Figs. 2c, d), in contrast to normal myocardium, which showed speckled dark spots with the former staining and a negative result with the latter (Figs. 2c, d). Contraction band necrosis observed in 4 patients who had shown no clinical sign of cardiac disorder during hospitalization was characterized by hypercontraction of the myocardial fibers, accompanied by a considerable number of irregularly shaped acidophilic bands by HE staining and numerous dark spots with HIH (Fig. 3a). These myocardial cells did not stain with LFB, and the contraction bands were completely negative for CuZnSOD immunostaining (Fig. 3b, arrow), although the intercalated discs and sarcoplasm of these cells showed weak deposits of CuZn-SOD immunoreaction product (Fig. 3b, double arrows).
Myocardial cells showing coagulation necrosis due to either acute myocardial infarction (1 case) or sepsis (3 cases) were characterized by an acidophilic sarcoplasm without nuclei, which stained light blue with LFB and a dark color with HIH (Fig. 4a), but showed no immunoreaction for CuZn-SOD (Fig. 4b). On the other hand, myocardial cells surrounding areas of coagulation necrosis demonstrated vacuolate degeneration in their sarcoplasm in a case of acute myocardial infarction, where a moderately enhanced immunoreaction for CuZnSOD was seen, especially at the rim (Fig. 4b). The most conspicuous alteration was seen in the myocardium fixed with 4% formalin in 2% calcium acetate, in comparison with that fixed in 10% neutral formalin. The myocardium fixed with the former mixture demonstrated basophilic alteration in 2 9 of the 5 0 cases. This basophilic alteration was observed in the right or left ventricle, or both. The affected cells were either single isolated ones (Fig. 5a) or appeared in several foci each composed of a considerable number of myocardial cells (Fig. 6a). Furthermore, the basophilic alteration was usually present throughout the entire sarcoplasm of the myocardial cells, but occasionally was observed only in a segment of each cell (Fig. 5a). The immunoreactivity for CuZn-SOD and the results obtained from histochemical observations using LFB and von Kossa stains in areas of basophilic alteration were quite different from those in normal adjacent myocardial cells. Areas of basophilic alteration exhibited significantly enhanced expression of immunoreactive CuZn-SOD, as revealed by intense deposition of reaction product either diffusely (Figs. 5b, 6b) or segmentally in the sarcoplasm (Fig. 5b, arrow). However, endothelial cells of capillaries or relatively large vessels located adjacent to the myocardial cells showing the basophilic
Table 1. Results of Histochemical and lmmunohistochemical Staining Normal rnvocardium
HIH PAS von Kossa LFB CuZu-SOD
+
-
Mucinous denenera t ion
Basophilic alteration
Contract ion band necrosis
Coagulation necrosis
-
+ -
-
-
-----c
+
+,
weakly positive staining ; +, moderately positive staining or considerably enhanced expression -, negative staining ; of immunoreactive CuZn-SOD ; it, strongly positive staining or intensely enhanced expression of immunoreactive CuZnSOD. Figures 5. Myocardial cells showing basophilic alteration are distributed sporadically (a) and the majority show diffuse and significantly enhanced expression of immunoreactive CuZn-SOD (b). However, some cells demonstrate a segmental distribution of the reaction product (b, arrow). These cells show strongly positive staining with von Kossa (c) and LFB (d). a-d, serial sections; a : HE, b : imrnunostaining for CuZn-SOD, c : von Kossa staining, d : LFB staining. Figures 6. Myocardial cells showing basophilic alteration distributed as small foci each containing a considerable number of cells (a). The majority show significantly enhanced expression of immunoreactive CuZn-SOD (b). These cells show strongly positive von Kossa (c) and LFB (d) staining. a-d, serial sections; a : HE, b : imrnunostaining for CuZn-SOD, c : von Kossa staining, d : LFB staining.
88
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.)
Table 2. Relationship between Background Diseases and Basophilic Alteration Background diseases
Number of cases
A) Malignant epithelial neoplasms 8 ) Malignant non-epithelial neoplasms C) Non-neoplastic diseases Total
28 9 13
50
Number of cases showing basophilic alteration 18 (64%) 4 (44%) 7 (54%) 29 (58%)
There were no significant differences among A, B and C (p>0.05).
alteration and sarcolemmal cells surrounding them exhibited no enhancement of the CuZn-SOD immunoreaction (Fig. 5b). Only myocardial cells staining brown with von Kossa (Figs. 5c, 6c) and deep blue with LFB (Figs. 5d, 6d) demonstrated significant enhancement of the CuZn-SOD immunoreaction. Basophilic alteration in the myocardium showed no relationship with background diseases, which included malignant epithelial and nonepithelial neoplasms, and other non-neoplastic conditions (Table 2). Furthermore, in the cases of malignant neoplasm, the incidence of basophilic alteration did not differ significantly (p>0.05) between those untreated and those treated with ad ria mycin. The myocardium without any basophilic alteration seen in 2 9 of the 50cases was weakly to moderately positive for immunoreactive CuZn-SOD, especially in intercalated discs, as shown in Fig. 3b, but only weakly positive or negative in the sarcoplasm (see Figs. 2b, 5 b and 6b). In addition, these cases were not stained with von Kossa and LFB.
DISCUSSION It has been clearly demonstrated using biochemical procedures that SOD is detectable over a wide concentration range in various mammalian tissues, even in normal or damaged myocardium (7-9, 15). However, such biochemical results cannot be compared with those of immunostaining for CuZn-SOD. In the present study, immunostaining for CuZn-SOD in myocardium fixed with 4% formalin in 2% calcium acetate showed a specific reaction product in the sarcoplasm. Moderately to markedly enhanced expression or complete loss of immunoreactive CuZn-SOD was seen in human myocardial cells under certain pat holog ica I conditions. M yoca rdia I damage other than basophilic alteration was observed in sections fixed with both 10% neutral formalin and 4% formalin in 2% calcium acetate. However, immunostaining for CuZn-SOD was clearer in sections fixed with the latter than with the former. Thus there is some difficulty in explaining the absence of the reaction product for CuZn-SOD in some sections fixed with 10% formalin. It is hypothesized that CuZn-SOD in the myocardium easily loses its antigenicity during the proc-
ess of tissue preparation. Therefore, in previous studies describing the immunohistochemical procedure for demonstration of CuZn-SOD, all tissues have been fixed with 4% formalin in 2% calcium acetate (10, 11, 13), and the specimens in the present study were also fixed in this way. Myocardial tissue judged to be normal with HE, HIH and LFB staining exhibited a more distinct reaction product in intercalated discs than in the sarcoplasm, which showed weaker o r negative staining, although Thaete et a/. (16) reported that immunostained sections of canine skeletal muscle showed a strongly striated staining pattern, which was interpreted as evidence for the presence of CuZn-SOD in the Z line and I band. Some variations in the immunoreactivity for CuZn-SOD might occur due to differences in species or specificity of the staining for cardiac or skeletal muscle, although the intercalated discs observed in the myocardium correspond to the Z lines of skeletal muscle. The myocardium with coagulation necrosis due to acute myocardial infarction or sepsis, and that with contraction band necrosis completely lacked immunoreactive CuZn-SOD in the sarcoplasm and contraction bands, indicating leakage of the enzyme from the dead cells. Watanabe et a/. (13), using an experimentally induced myonephropathic metabolic syndrome in dogs, reported that the fibers of the adductor muscle showed conspicuous alterations after re-establishment of arterial flow, which were characterized by a loss of transverse striations in association with dissolution of the sarcoplasm and negative immunostaining for both myoglobin and CuZn-SOD. On the other hand, myocardium showing mucinous degeneration, which has been referred to as basophilic, mucoid and mucinous degeneration, or cardiac colloid (17-20), demonstrated enhanced expression of immunoreactive CuZn-SOD in the degenerated sarcoplasm, which was strongly stained with PAS but negative with LFB and von Kossa stains. The basophilic alteration appeared to have no direct relationship with administration of the cardiotoxic drug, adriamycin (21,22), and demonstrated intense deposits of the immunoreaction product for CuZn-SOD, associated with deep blue staining with LFB and brown staining with von Kossa.
Acta Pathologica Japonica 42 (2) : 1992
Mucinous degeneration appears to be quite different in its distribution, histochemical features, intensity of CuZn-SOD immunoreactivity and mechanism of metabolic abnormality in comparison with basophilic alteration of the myocardium. From the results of biochemical analysis and positivity obtained with all glycogen stains that are digested with amyloglucosidase, malt diastase and pectinase, it is postulated that mucinous degeneration is composed of a glucan representing a byproduct of glycogen metabolism (19). A previous study found that mucinous degeneration was present in isolated myocardial cells from almost all individuals more than 11 years of age, although it was not seen in any patients who had died in the first decade of life (19). In the present study, myocardial tissue from a 12-year-old girl showed no mucinous degeneration in the myocardium. After sufficient discoloration with 0.05% lithium carbonate followed by 70% ethanol, phospholipids were not stained by LFB in normal myocardium fixed with 4% formalin in 2% calcium acetate, and von Kossa staining was also negative, since it is likely that calcium ions react only slightly with phosphatidylcholine (23), the principal phospholipid component of mammalian cell membranes. The basophilic alteration observed in the myocardium was considered to result from conversion of a soluble form of phospholipid to an insoluble one, possibly due to the effect of free radicals, since there was an intense reaction for CuZn-SOD and reaction with calcium ions in the fixative. There are several hypotheses related to free-radical production under conditions of cellular damage. Using a biochemical approach, Granger et a/. (24) and McCord (25) demonstrated that oxygen-derived free radicals appeared to be produced by xanthine oxidase in postischemic tissue. Membranebound nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) in neutrophils may play an important role in sterilization through production of its own free radical (26,27). Free radicals are also produced by the mitochondria1 electron transport system (28), the enzymic oxidation of arachidonic and linoleic acids (29) and the NADPH-P-450 reductase system in microsomes and nuclear membranes (30, 31). In addition, lipid peroxide, which has a long half-life but weaker reactivity than that of superoxide, can be induced directly and/or through production of free radicals. It is difficult to determine the direct source of peroxide production, whether it be certain organelles of affected myocardial cells, other cells in the vicinity, or other organs secondary to abnormal metabolism during a lifeor-death crisis. Nishigaki et a/. (32) reported an increase of lipid peroxide in the spleen, liver and kidney, as well as elevation of enzymes released from these
89
organs following an increase of lipid peroxide in burned skin. It is thought that several factors contribute to basophilic alteration, including ischemic change in the myocardium, contraction of myocardial fibers, arteriosclerosis of the coronary arteries, reduced supply of oxygen to the myocardium and abnormal metabolism. In the present study, the effects of these factors differed considerably from one case to another, because all the materials examined were obtained from autopsy cases. Therefore, it is considered that the myocardium with or without basophilic alteration reflects the presence or absence of the severe pathological conditions described above, which alter the amounts of free radicals produced in a life-or-death crisis. In myocardial specimens showing markedly enhanced expression of CuZn-SOD with basophilic alteration, it is thought that more CuZn-SOD was produced in the sarcoplasm in order to protect against cellular damage induced by free radicals. The basophilic alteration observed in the myocardium was distributed either diffusely or segmentally in the sarcoplasm. Takasaki et a/. (33) described in experimentally induced myonephropathic metabolic syndrome that the adductors showed severe ischemic change characterized by the separation of muscle fibers due to an increase of interstitial tissue fluid, and partial or total degeneration of the individual muscle fibers. Therefore, myocardium affected by free radicals may undergo either segmental or total degeneration of the sarcoplasm. Basophilic alteration is considered to be a cellular response to free radicals and certain degenerative changes in the myocardium, since the myocardium affected by free radicals shows increased amounts of CuZn-SOD and accumulation of phospholipids reactive with calcium ions in the sarcoplasm.
REFERENCES 1. Weisiger RA and Fridovich I. Mitochondria1superoxide dismutase : Site of synthesis and intramitochondrial localization. J Biol Chem 248: 4793-4796, 1973. 2. Marklund SL, Holme E, and Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 126: 41-51, 1982. 3. Grankvist K, Marklund SL, Sehlin J, and Taljedal IB. Superoxide dismutase, catalase and scavengers of
hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 182 : 17-25, 1979. 4.
Fridovich I. Superoxide radical : An endogenous toxicant. Annu Rev Pharmacol Toxicol 23: 239-257,
1983. 5. Jolly SR, Kane WJ, Bailie MB, Abrams GD, and Lucchesi BR. Canine myocardial reperfusion injury : Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 54: 277-285,
90
6.
7.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17. 18. 19.
CuZn-Superoxide Dismutase in Damaged Myocardium (Sato et a/.) 1984. Girotti AW and Thomas JP. Superoxide and hydrogen peroxide-dependent lipid peroxidation in intact and Triton-dispersed erythrocyte membranes. Biochem Biophys Res Commun 118: 474-480, 1984. Peeters-Joris C, Vandevoorde AM, and Baudhuin P. Subcellular localization of superoxide dismutase in rat liver. Biochem J 150: 31-39, 1975. Westman NG and Marklund SL. Copper- and zinccontaining superoxide dismutase and manganesecontaining superoxide dismutase in human tissues and human malignant tumors. Cancer Res 41: 29622966, 1981. Marklund SL, Westman NG, Lundgren E, and Roos G. Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res 42: 1955-1961, 1982. Thaete LG, Crouch RK, Schulte BA, and Spicer SS. The immunolocalization of copper-zinc superoxide dismutase in canine tissues. J Histochem Cytochem 31 : 1399.-1406, 1983. Thaete LG, Crouch RK, and Spicer SS. Immunolocalization of copper-zinc superoxide dismutase : 11. Rat. J Histochem Cytochem 33 : 803-808, 1985. Dobashi K, Asayama K, Kato K, Kobayashi M, and Kawaoi A. lmmunohistochemical localization of copper-zinc and manganese superoxide dismutases in rat tissues. Acta Histochem Cytochem 22 : 351 -365, 1989. Watanabe S, Hadama T, Takasaki H, et a/. Myocardial damage caused by free radicals in experimentally induced myonephropathic metabolic syndrome in dogs. Jpn J Surg ( i n press). Guesdon JL, Ternynck T, and Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27: 1131-1139, 1979. Sazuka Y, Tanizawa H, and Takino Y. Effect of adriamycin on the activities of superoxide dismutase, glutathione peroxidase and catalase in tissues of mice. Jpn J Cancer Res 80: 89-94, 1989. Thaete LG, Crouch RK, and Spicer SS. Immunolocalization of CuZn superoxide dismutase in striated muscle. Histochem J 17: 259-262, 1985. Scotti TM. Basophilic (mucinous) degeneration of the myocardium. Am J Clin Pathol25 : 994-101 1, 1955. Haust MD, Rowlands DT Jr, Garancis JC, and Landing BH. Histochemical studies on cardiac "colloid". Am J Pathol 40: 185-197, 1962. Rosai J and Lascano EF. Basophilic (mucoid) degeneration of myocardium. A disorder of glycogen metabolism. Am J Pathol 61 : 99-112, 1970.
20. 21. 22.
2 3.
24.
25. 26.
27.
28.
29.
30.
31,
32.
3 3.
Kawamoto S and Wakabayashi T. Basophilic degeneration with special reference to its pathophysiological aspects. Acta Pathol Jpn 33: 619-628, 1983. Myers CE, McGuire WP, Liss RH, et a/. Adriamycin: The role of lipid peroxidation in cardiac toxicity and tumor response. Science 197 : 165-167, 1977. Politi PM and Rothlin RP. Acute effects of doxorubicin on chronotropic and inotropic mechanisms in guinea pig atria. Cancer Treat Rep 69 : 859-865, 1985. Maeda M, Nishijima M, Takenaka Y, Kuge 0, and Akamatsu Y. pHdependent Ca2+ interaction with phospholipids related to phosphatidylethanolamine Nmethylation. Biochim Biophys Acta 794 : 298-306, 1984. Granger DN, Rutili G, and McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 81 : 22-29, 1981. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159-163, 1985. Babior BM, Kipnes RS, and Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52: 741-744, 1973. Petrone WF, English DK, Wong K, and McCord JM. Free radicals and inflammation : Superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 77: 1159-1163, 1980. Cadenas E, Boveris A, Ragan C1, and Stoppani AOM. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch Biochem Biophys 180: 248-257, 1977. Porter NA, Lehman LS, Weber BA, and Smith KJ. Unified mechanism for polyunsaturated fatty acid autoxidation. Competition of peroxy radical hydrogen atom abstraction, p-scission, and cyclization. J Am Chem SOC 103: 6447-6455, 1981. Strobel HW and Coon MJ. Effect of superoxide generation and dismutation on hydroxylation reactions catalized by liver microsomal cytochrome P-450. J Biol Chem 246: 7826-7829, 1971. Patton SE, Rosen GM, and Rauckman EJ. Superoxide generation by purified hamster hepatic nuclei. Mol Pharmacol 18: 588-593, 1980. Nishigaki I, Hagihara M, Hiramatsu M, lzawa Y, and Yagi K. Effect of thermal injury on lipid peroxide levels of rat. Biochem Med 24: 185-189, 1980. Takasaki H, Hadama T, Matsushima R, Watanabe S, and Shirabe J. Extracorporeal circulation for the removal of serum myoglobin in experimentally induced myonephropathic metabolic syndrome in dogs. Jpn J Surg 21 : 201-209, 1991.
