In skeletal muscles from rats treated with germanium for 23 weeks, there were numerous ragged-red fibers and cytochrome-c oxidase (COX)-deficient fibers. Biochemically, germanium reduced the enzyme activities in the mitochondrial respiratory chain. Rotenone-sensitive NADH-cytochrome-c reductase as well as COX activities were markedly reduced, while succinate-cytochrome-c reductase was less severely, but significantly, affected. The histopathologicalfindings in these muscles were similar to those seen in patients with mitochondrial encephalomyopathy, suggesting that germanium-induced myopathy may be a useful experimental model. Coenzyme Q,, administration appeared to be ineffective in preventing this experimental myopathy. 0 1992 John Wiley & Sons, Inc. Key words: germanium intoxication mitochondrial myopathy rat muscle muscle fiber type coenzyme Q,, MUSCLE & NERVE 1511258-12641992
AN EXPERIMENTAL MODEL OF MITOCHONDRIAL MYOPATHY: GERMANIUM4NDUCED MYOPATHY AND COENZYME Qqo ADMINISTRATION CHIEN-MING WU, MD, TARO MATSUOKA, MD, MASAKAZU TAKEMITSU, MD, YU-ICHI GOTO, MD, and IKUYA NONAKA, MD
T h e heavy metal germanium is known to have antineoplastic and anti-infectious actions, particularly when given as an organic ~ a 1 t . l ’However, ~ in approximately 20 patients, long-term administration of high-dose germanium caused renal failure, anemia, emaciation, and muscle weakness.4,7,13,14,16,19-22 Three children died from multiple organ failure. Higuchi and coworkers demonstrated the myotoxic effects of germanium in After 4 months of germanium dioxide administration, there was a marked histochemical decrease in cytochrome-c oxidase (COX) activity similar to that seen in biopsied muscle from a patient with ger-
manium into~ication.~ The details of histopathologic analysis and biochemical studies on mitochondrial enzymes were, however, not given. To determine the pathomechanism of mitochondrial damage in germanium intoxication, we carried out a detailed morphometric and biochemical analysis in a rat model. In addition, coenzyme (Coy), which is sometimes given for the treatment of patients with mitochondrial encephalomyopathies, was administered to determine whether CoQ would prevent the progression of germanium-induced mitochondrial disorder in these animals.
al,,
MATERIALS AND METHODS From the Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan (Drs. Wu, Matsuoka, Takemitsu, Goto, and Nonaka); and the Department of Pediatrics, Far Eastern Memorial Hospital. Taipei, Taiwan, Republic of China (Dr Wu). Acknowledgments: The authors express their thanks to Professor S.M. Sumi, Department of Pathology, University of Washington, Seattle, and Dr. I. Higuchi, Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, for their helpful suggestions and advice on this study. This work was partially supported by the Ministry of Health and Welfare, Japan. Address reprint requests to Dr. Nonaka. Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi. Kodaira, Tokyo 187, Japan. Accepted for publication April 6, 1992.
CCC 0148-639X1921111258-07 0 1992 John Wiley & Sons, Inc.
1258
Mitochondria1 Myopathy in Rats
Sixty female Wistar rats, aged 3 weeks, and weighing 70 to 80 g, were divided into three groups. In groups 1 and 2, their feed contained 0.15% germanium dioxide. The animals in group 1 had no additional treatment. Group 2 received CoQ at approximately 100 mg/kg per day orally (100 mg/kg feed). The animals in group 3 served as controls. The body weight of all rats was measured every week. At experimental week 23, all rats were killed. The wet weights of the extensor digitorum longus (EDL), tibialis anterior (TA), and soleus muscles were measured, and the muscles were submitted for histopathologic examination. In addition, biochemical examination was Animals.
MUSCLE & NERVE
November 1992
performed on the EDL muscle, which was frozen in liquid nitrogen and stored at -70°C until use for mitochondria1 isolation.
-t
300[
E
For histochemical examination, the muscle specimens were frozen in isopentane chilled in liquid nitrogen. Serial frozen sections, 10 pm in thickness, were stained with hematoxylin-eosin (H&E), modified Gomori trichrome (mGt), and a battery of histochemical methods, including NADH- tetrazolium reductase, succinate dehydrogenase, COX and adenosine triphoshatase (ATPase). In each specimen, approximately 500 fibers were chosen randomly, their diameters were measured, and the percentage of ragged-red fibers (RRFs) as well as COXdeficient fibers were recorded. Histopathologic Studies.
Small parts of EDL muscles were fixed in 2% glutaraldehyde buffered with 0.1 mol/L sodium cacodylate, postfixed in 1% osmium tetroxide, dehydrated, and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead nitrate, and examined with an H-7000 electron microscope (Hitachi). Electron-Microscopic Examination.
Skeletal muscle mitochondria were isolated and the final crude mitochondrial pellet was resuspended, as previously described.2"* The isolated mitochondria were immediately stored at - 70°C until biochemical analysis. Mitochondria1 Isolation.
SpectrophotometricAssays of Respiratory Chain Enzyme Activity. Spectrophotometric determina-
,o 2 0 0 EQ
-
: z 4 .
a
W Group 1 Group 2 A Group 3
- 100-
+
0
z
C
0
5
10 15 duration (w8.k.)
20
FIGURE 1. Change in whole body weight. The body weight showed significant difference (P < 0.05) as compared with group 3 (controls; triangles) from week 3 in group 1 (germanium only; squares) and from week 6 in group 2 (germanium and oral CoQ; diamonds). After week 3, the body weight in group 1 also showed a significant difference (P < 0.05) as compared with group 2.
in groups 1 and 2 progressively declined after the weeks 10 and 12, respectively. After the week 3 , the animals in group 2 had higher body weights than those in group 1 (P < 0.05), indicating minimal, but insignificant, effect of CoQ administration on the body weight. By the week 23, most of the rats in groups 1 and 2 were severely emaciated (Table 1). Except for inactivity, none of the rats showed apparent neurologic abnormality such as ataxia, convulsions, or dragging of the hindlegs. The wet weight of skeletal muscles from groups 1 and 2 was significantly less than that in the controls (group 3 ) (Table 1). CoQ administration did not prevent this loss in muscle weight. In
tions of enzyme activity included rotenone-sensitive NADH-cytochrome-c reductase (rotenonesensitive NCCR, complex I III)," succinatecytochrome-c reductase (SCCR, complex I1 + III)," COX (complex IV),23 and citrate synthase.26 The respiratory chain enzyme activity was expressed as the value corrected for citrate synthase activity.
+
Statistical comparisons were made using Student's t-test.
Statistical Methods.
RESULTS
The growth curves showed significant deviation ( P < 0.05) from that of the controls (group 3) after week 3 in group 1 and week 6 in group 2 (Fig. 1). The body weight Body and Muscle Weight.
Mitochondrial Myopathy in Rats
Table 1. Weight of whole body and skeletal muscles at experiment week 23.
Group 1 Whole body (9) 168.7 2 Muscles (rng) 78.2 2 EDL 186 6 2 TA Soleus 9892
2 15.3*t 186.6 2 26.1' 290.7
3 2
22.5
11.8' 86.1 t 21.4* 150.3 t 11.9 30 3*t 212 0 t 43 9' 506 3 2 43 1 107* 9 5 6 ? 138* 1 2 0 7 2 11 9
Values are expressed as mean IT SD Group 1 germanium on/y group germanium and oral COQ, group 3 controls. EDL extensor digltorum longus TA tib/a'lsanferm 'Signif/cant difference (f < 0 001) as compared wlth the controls (gioup3j tsignifmnt difference (f c 0 05) as compared w/rh group 2
z
MUSCLE & NERVE
November 1992
1259
FIGURE 2. Frozen sections from soleus muscle (a, b) and extensor digitorum longus (EDL) muscle (c, d) from group 1 [germanium only; (a) and (c)] and grup 3 [controls; (b) and (d)] stained with modified Gomori trichrome. The ragged-red fibers (asterisks) were only present in group 1 animals and were more frequent in EDL muscle. Bar = 100 pm.
groups 1 and 2, the EDL and TA were more significantly affected than the soleus muscles: 43%48% weight loss in EDL, 55%-63% in TA, while only 14%-21% in soleus muscle. Histopathologic Studies. All animals in groups 1 and 2 showed significant myopathic changes which consisted of numerous RRFs and moderate variation in fiber size. There were some necrotic fibers in the EDL and TA, but not in the soleus muscles. Muscle fiber diameter was decreased to 68%-76% of the controls in the EDL, and 91%94% in the soleus muscles (Table 2). There was no suggestion of neurogenic atrophy in any specimen. On mGt stain, RRFs were found in all specimens in groups 1 and 2, predominantly in the EDL and TA (Table 2, Fig. 2). RRFs in the EDL, TA, and soleus muscles were mostly type 2A fibers (Fig. 3). There were no RRFs in control muscles. Intramuscular nerves were well-myelinated in all the experimental rats. Oxidative enzyme stain showed disorganized intermyofibrillar network and strong enzyme reac-
1260
Mitochondria1 Myopathy in Rats
tion in many fibers in groups 1 and 2, which were usually RRFs (Fig. 3). Muscle fiber type distribution in groups 1 and 2 did not differ from that in the controls (group 3). Approximately 90% of fibers were type 1 fibers
~
Table 2. Muscle fiber size and the incidence of RRFs and COX-deficient fibers Group
2
1
3
Fiber diameter (pm)
EDL Soleus RRFs (%)
EDL Soleus COX-deficient fibers (%)
EDL Soleus
36.6 t 3.2' 39.3 5 5.0' 51.6
5 5.2 70.7 i 4.9* 71.9 t 4.2' 76.3 t 2.9
38.9 t 7.0 35.1 t 6.0 7.2 t 4.1 8.4 t 4.1
0.0
100.0 17.8 5 4.2
0.0
100.0 17.3 3.6
*
0.0 0.0
Values are expressed as mean 2 SD. Group I : germanium only; group 2: germanium and oral COO; group 3: controls; EDL: extensor digitorurn longus; RRF: ragged-red fiber; COX: cytochrome-c oxidase. *Significant difference (P < 0.007) as compared with controls (group 3).
MUSCLE & NERVE
November 1992
FIGURE 3. Serial frozen sections from SOleUS muscle of rat in group 1 (germanium only) stained with modified Gomori trichrome (a), succinate dehydrogenase (b), ATPase preincubated at pH 4.6 (c), and cytochrome-c oxidase (COX). (d). In addition to variation in fiber size, there are scattered ragged-red fibers (RRFs; asterisks) which stain dark with oxidative enzyme [(b), arrows] and behave as type 2A fibers [(c), arrows]. Not only RRFs, but normal-appearing fibers occasionally have no COX activity (arrowheads). Type 2A fiber (A), type 2 8 fiber (B), and type 1 fiber (1). Bar = 200 pm.
in the soleus and 95% were type 2 fibers in EDL muscles. Type 2 fibers were more atrophic than type 1 fibers in both EDL and soleus muscles. There was no increase in the number of type 2C fibers. Almost all muscle fibers in the EDL and T A of the animals in groups 1 and 2 failed to show any COX activity, while approximately 83% of the fibers in the soleus muscles were COX-positive, indicating focal COX deficiency (Table 2, Figs. 3 and 4). Most of the type 2A and 2B fibers did not stain with COX, while only 5%-8% of type 1 fibers were COX-negative (Fig. 3 ) . All RRFs were COX-negative (Fig. 3). All muscle fibers in the controls (group 3 ) were COX-positive (Fig. 4). In the EDL muscles from groups 1 and 2, many large mitochondria with increased numbers of cristae were observed. Some abnormal mitochondria contained electron-dense granules (Fig. 5). Electron Microscopic Examination.
Mitochondria1 Myopathy in Rats
Biochemical Studies. There was a significant difference in the activity of rotenone-sensitive NCCR ( P < 0.001), SCCR ( P < 0.001), and COX ( P < 0.001) between groups 1 and 2 and controls (group 3 ) . CoQ treatment showed only minimal effect on the activity of these enzymes (Table 3 ) .
DISCUSSION
As in humans with germanium intoxication, large doses of germanium inhibited weight gain and caused a myopathy with abnormal mitochondria in rats. There were several similarities between our findings and those in human mitochondria1 disorders: in addition to RRF, there were moderate variations in fiber size, a few necrotic fibers, and a marked decrease in COX activity. In terms of RRF formation, mitochondrial-rich type 2A fibers were more susceptible to the toxic effect of germanium than other fiber types. This preferential damage of oxidative fibers is also seen in human muscles in which type 1 fibers have high oxidative activity. Moreover, scattered COX-negative
MUSCLE & NERVE
November 1992
1261
FIGURE 4. Frozen sections of extensor digitorum longus muscle from group 1 [germanium only, (a)] and group 3 [controls, (b)] stained with cytochrome-c oxidase (COX). Almost all fibers in group 1 lack enzyme activity. Bar = 100 p.m.
fibers were present among normal fibers in the soleus muscle, representing focal COX deficiency. Focal COX deficiency is a characteristic finding in human mitochondrial encephalomyopathies, including chronic progressive external ophthalmoplegia (CPEO); myoclonus epilepsy with raggedred fibers (MERRF); and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). Recent molecular studies revealed that these three entities had specific mitochondrial DNA mutation^.^^^'^^ Therefore, the elucidation of the pathogenetic mechanisms of focal COX deficiency in relation to the molecular
defect is considered to be the central problem in these mitochondrial disorder^.^ In this context, this germanium-induced myopathy should be a useful experimental model of human mitochondrial encephalomyopathy. From our histochemical findings, it would appear that complex IV deficiency precedes deficiencies in the other complexes because RRFs were, without exception, COX-negative. In CPEO or MERRF in which COX is preferential1 involved, almost all RRFs had no COX activity,”’’’whereas a considerable number of RRFs had normal COX activity in MELAS with complex I deficiency (unpub-
Table 3. Mitochondrial respiratory chain enzyme activity in extensor digitorum longus muscle.
Group Complexes
+
I 111 II + 111 IV
1
2
3
5.2 2 4.0* 14.1 ? 4.2*t 4.9 5 2.8*$
2.9 + 4.8* 19.6 ? 7.0* 9.2 ? 4.4*
131.6 36.2 92.3 2 13.9 202.0 2 60.7
+
~~~
~
~
_
_
_
_
h z y m e activity is expressed as: enzyme activity ( n m o l h i n per milligram mitochondrial protein) loo citrate synthase activity (nmolimin per milligram mitochondrial protein) Values are expressed as mean ? SD. Group 1. germanium only; group 2: germanium and oral CoQ; group 3: controls; / + 111. rotenonesensitive NADH-cytochrome-c reductase (NCCR); / / + ///. succinafe- cytochrome-c reductase (SCCR); IV: cytochrome-c oxidase (COX). ‘Significant difference (P < 0.007) as compared with the controls (group 3). fSignificant difference ( P < 0.01) as compared with group 2. #Significant difference (P < 0.001) as compared with group 2
1262
Mitochondrial Myopathy in Rats
FIGURE 5. Ultrastructural findings in extensor digitorum longus muscle from group 1 (germanium only). Note many enlarged mitochondria with proliferated cristae, occasionally containing electron-dense materials. Bar = 1 pm.
MUSCLE & NERVE
November 1992
lished observations). In addition, it has been shown that the decline in complex I activity could be secondary to progressive decline in complex I V activity." It is possible that inhibition of COX activity by germanium leads to RRF formation and secondary decrease in the activities of other enzymes. Further chronologial study is necessary to clarify the primary inhibitory site of germanium in the mitochondrial respiratory chain. There have been several reports of the beneficial effects of large doses of CoQ in atients with mitochondrial encephalomyopathies. 17,18,27 Coy is a mobile electron carrier which functions during the transfer of electrons between dehydrogenases and cytochrome bc, complex, and mediates electron transfer by a diffusion process across the mitochondrial membrane.24CoQ had only minimal and less than satisfactory effect on body weight and the biochemical findings, and it had no effefct on the histopathologic changes in our animals with germanium-induced mitochondrial myopathy. If the primary effect of germanium intoxication is on COX activity, then CoQ administration would not be expected to be effective in these animals. It is possible that the amount of germanium used in the present study was too large for any beneficial effect of CoQ administration to be demonstrated.
"
REFERENCES
1. Aso H, Suzuki F, Ebina T, Ishida N: Antiviral activity of carboxyethylgermanium sesquioxide (Ge-132) in mice infected with influenza virus. J Biol Response Mod 1989;8: 180- 189. 2. Bookelman H, Trijbels JMF, Sengers RCA, Janssen AJM: Measurement of cytochromes in human skeletal muscle mitochondria, isolated from fresh and frozen stored muscle specimens. Biochem Med 1978;19:366-373. 3. Brutkicwicz RR, Suzuki F: Biological activities and antitumor mechanism of an immunopotentiating organogermanium compound, Ge-132 (review). In Vivo 1987;l: 189204. 4. Chiba M, Shinohara A, Inaba Y, Nakayama S, Rinno H, Koide H: T w o cases of patients taking germanium-containing stuffs (in Japanese). Juntendo Igaku (Tokyo) 1990;36: 406-4 10. 5. Goto Y, Koga Y, Horai S, Nonaka I: Chronic progressive external ophthalmolegia: a correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies. J Neurol Scz 1990;100:63-69. 6. Goto Y, Nonaka I , Hordi S: A mutation in the tRNALe u ( ~ gene ~ ~ associated ) with the MELAS subgroup in mitochondrial encephalomyopathies. Nature 1990;348:651653. 7 . Higuchi I , Izumo S, Kuriyama M, Suehara M, Nakagawa M, Fukunaga H, Osame M, Ohtsubo S, Miyata K: Germanium myopathy: clinical and experimental pathological studies. Acta Neuropathol (Bed) 1989;79:300- 304. 8. Higuchi I, Takahashi K, Nakahara K, Izumo S, Nakagawa
Mitochondria1 Myopathy in Rats
M, Osame M: Experimental germanium myopathy. Acta Neuropathol (Berl) 1991;82:55-59. 9. Holt IJ, Harding AE, Morgan-Hughes JA: Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988;33 1:717-719. 10. Koga Y, Nonaka I, Sunohara N, Yamanaka R, Kumagai K: Variability in the activity of respiratory chain enzymes in mitochondrial myopathies. Acta Neuropathol (Berl) 1988;76: 135- 141. 11. Mackler B: Microsomal DPNH-cytochrome c reductase, in Estabrook RW, Pullman ME (eds): Method< in Enzymology. New York, Academic Press, 1967, vol 10, p p 551-553. 12. Matsuoka T, Goto Y, Yoneda M, Nonaka I: Muscle histopathology in myoclonus epilepsy with ragged-red fibers (MERRF). J Neurol Sci 1991;106:193-198. 13. Nagata N, Yoneyama T, Yanagida K, Ushio K, Yanagihara S, Matsubara 0, Eishi Y: Accumulation of germanium in the tissues of a long-term user of germanium preparation died of acute renal failure. ,J Toxic01 Sci 1985; 10:333341. 14. Nakamura R, Yajima H, Yamamoto T , Imamura Y, Watabe S, Hase H, Nakai s, Uchiyama T, Kato N, Aikawa K: A case of suspected germanium intoxication (in Japanese). Prog Med (Tokyo) 1988;8:2037-2040. 15. Nishikawa Y, Takahashi M, Yorifuji S, Nakamura Y, Ueno S, Tarui S, Kozuka T , Nishimura T : Long-term coenzyme Qlo therapy for a mitochondrial encephalomyopathy with cytochrome c oxidase deficiency: a 31P NMR study. Neurology 1989;39:399-403. 16. Obara K, Akiu N, Sato H, Saito T , Miura Y, Yoshinaga K, Kakizawa M, Tada K, Tsukamoto T, Hongo M: Two cases with various symptoms and renal dysfunction induced by long term germanium intake (in Japanese). Nzppon Nazka Gakkai Zasshi (Tokyo) 1988;77:1704- 1709. 17. Ogasahara S, Nishikawa Y, Yorifuji S, Soga F, Nakamura Y, Takahashi M, Hashimoto S, Kono N, Tdrui S: Treatment of Kearns- Sayre syndrome with coenzyme Qln.Neurology 1986;36:45-53. 18. Ogasahara S, Yorifuji S, Nishikawa Y, Takahashi M, Wada K, Hazama T, Nakamura Y, Hashimoto S, Kono N, Tarui S: Improvement of abnormal pyruvate metablism and cardiac conduction defect with coenzyme Qlo in KearnsSayre syndrome. Neurology 1985;35:372-377. 19. Ohta S, Sat0 T, Narabayashi H, Rinno H , Ohno J: Acute myoglobinuria with lipid storage myopathy caused by longterm taking of alcohol and germanium (in Japanese). Clzn Neurol (Tokyo) 1985;25:845. 20. Ohtsubo S, Nakamura M, Nomura Y, lnoue H, Miyata K: Multiple organ failure caused by a long-term taking of germanium preparation: a case report (in Japanese). Nippon Shonzka Gakkuz Zasshi (Tokyo) 1988;92: 19941999. 21. Okagawa K, Kosaka M , Sakikawa T , Hiasa M, Sato H , Morizumi K, Morita H, Shimomura S: Renal hypofunction, normochromic anemia, muscle weakness and peripheral neuropathy occurred in three patients taking germanium-containing water (in Japanese). Naikn (Tokyo) 1'386;58:1210- 1214. 22. Okuda S, Kiyama S, Oh Y, Shimamatsu K, Oochi N , Kobayashi K, Nanishi F, Fujimi S, Onoyama K, Fujishima M: Persistent renal dysfunction induced by chronic intake of germanium-containing compounds. Curr Ther Res Clin ExP 1987;41:265-275. 23. Orii Y, Okunuki K: Studies on cytochrome a. XV.
AN EXPERIMENTAL MODEL OF MITOCHONDRIAL MYOPATHY: GERMANIUM4NDUCED MYOPATHY AND COENZYME Qqo ADMINISTRATION CHIEN-MING WU, MD, TARO MATSUOKA, MD, MASAKAZU TAKEMITSU, MD, YU-ICHI GOTO, MD, and IKUYA NONAKA, MD
T h e heavy metal germanium is known to have antineoplastic and anti-infectious actions, particularly when given as an organic ~ a 1 t . l ’However, ~ in approximately 20 patients, long-term administration of high-dose germanium caused renal failure, anemia, emaciation, and muscle weakness.4,7,13,14,16,19-22 Three children died from multiple organ failure. Higuchi and coworkers demonstrated the myotoxic effects of germanium in After 4 months of germanium dioxide administration, there was a marked histochemical decrease in cytochrome-c oxidase (COX) activity similar to that seen in biopsied muscle from a patient with ger-
manium into~ication.~ The details of histopathologic analysis and biochemical studies on mitochondrial enzymes were, however, not given. To determine the pathomechanism of mitochondrial damage in germanium intoxication, we carried out a detailed morphometric and biochemical analysis in a rat model. In addition, coenzyme (Coy), which is sometimes given for the treatment of patients with mitochondrial encephalomyopathies, was administered to determine whether CoQ would prevent the progression of germanium-induced mitochondrial disorder in these animals.
al,,
MATERIALS AND METHODS From the Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan (Drs. Wu, Matsuoka, Takemitsu, Goto, and Nonaka); and the Department of Pediatrics, Far Eastern Memorial Hospital. Taipei, Taiwan, Republic of China (Dr Wu). Acknowledgments: The authors express their thanks to Professor S.M. Sumi, Department of Pathology, University of Washington, Seattle, and Dr. I. Higuchi, Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, for their helpful suggestions and advice on this study. This work was partially supported by the Ministry of Health and Welfare, Japan. Address reprint requests to Dr. Nonaka. Division of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi. Kodaira, Tokyo 187, Japan. Accepted for publication April 6, 1992.
CCC 0148-639X1921111258-07 0 1992 John Wiley & Sons, Inc.
1258
Mitochondria1 Myopathy in Rats
Sixty female Wistar rats, aged 3 weeks, and weighing 70 to 80 g, were divided into three groups. In groups 1 and 2, their feed contained 0.15% germanium dioxide. The animals in group 1 had no additional treatment. Group 2 received CoQ at approximately 100 mg/kg per day orally (100 mg/kg feed). The animals in group 3 served as controls. The body weight of all rats was measured every week. At experimental week 23, all rats were killed. The wet weights of the extensor digitorum longus (EDL), tibialis anterior (TA), and soleus muscles were measured, and the muscles were submitted for histopathologic examination. In addition, biochemical examination was Animals.
MUSCLE & NERVE
November 1992
performed on the EDL muscle, which was frozen in liquid nitrogen and stored at -70°C until use for mitochondria1 isolation.
-t
300[
E
For histochemical examination, the muscle specimens were frozen in isopentane chilled in liquid nitrogen. Serial frozen sections, 10 pm in thickness, were stained with hematoxylin-eosin (H&E), modified Gomori trichrome (mGt), and a battery of histochemical methods, including NADH- tetrazolium reductase, succinate dehydrogenase, COX and adenosine triphoshatase (ATPase). In each specimen, approximately 500 fibers were chosen randomly, their diameters were measured, and the percentage of ragged-red fibers (RRFs) as well as COXdeficient fibers were recorded. Histopathologic Studies.
Small parts of EDL muscles were fixed in 2% glutaraldehyde buffered with 0.1 mol/L sodium cacodylate, postfixed in 1% osmium tetroxide, dehydrated, and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead nitrate, and examined with an H-7000 electron microscope (Hitachi). Electron-Microscopic Examination.
Skeletal muscle mitochondria were isolated and the final crude mitochondrial pellet was resuspended, as previously described.2"* The isolated mitochondria were immediately stored at - 70°C until biochemical analysis. Mitochondria1 Isolation.
SpectrophotometricAssays of Respiratory Chain Enzyme Activity. Spectrophotometric determina-
,o 2 0 0 EQ
-
: z 4 .
a
W Group 1 Group 2 A Group 3
- 100-
+
0
z
C
0
5
10 15 duration (w8.k.)
20
FIGURE 1. Change in whole body weight. The body weight showed significant difference (P < 0.05) as compared with group 3 (controls; triangles) from week 3 in group 1 (germanium only; squares) and from week 6 in group 2 (germanium and oral CoQ; diamonds). After week 3, the body weight in group 1 also showed a significant difference (P < 0.05) as compared with group 2.
in groups 1 and 2 progressively declined after the weeks 10 and 12, respectively. After the week 3 , the animals in group 2 had higher body weights than those in group 1 (P < 0.05), indicating minimal, but insignificant, effect of CoQ administration on the body weight. By the week 23, most of the rats in groups 1 and 2 were severely emaciated (Table 1). Except for inactivity, none of the rats showed apparent neurologic abnormality such as ataxia, convulsions, or dragging of the hindlegs. The wet weight of skeletal muscles from groups 1 and 2 was significantly less than that in the controls (group 3 ) (Table 1). CoQ administration did not prevent this loss in muscle weight. In
tions of enzyme activity included rotenone-sensitive NADH-cytochrome-c reductase (rotenonesensitive NCCR, complex I III)," succinatecytochrome-c reductase (SCCR, complex I1 + III)," COX (complex IV),23 and citrate synthase.26 The respiratory chain enzyme activity was expressed as the value corrected for citrate synthase activity.
+
Statistical comparisons were made using Student's t-test.
Statistical Methods.
RESULTS
The growth curves showed significant deviation ( P < 0.05) from that of the controls (group 3) after week 3 in group 1 and week 6 in group 2 (Fig. 1). The body weight Body and Muscle Weight.
Mitochondrial Myopathy in Rats
Table 1. Weight of whole body and skeletal muscles at experiment week 23.
Group 1 Whole body (9) 168.7 2 Muscles (rng) 78.2 2 EDL 186 6 2 TA Soleus 9892
2 15.3*t 186.6 2 26.1' 290.7
3 2
22.5
11.8' 86.1 t 21.4* 150.3 t 11.9 30 3*t 212 0 t 43 9' 506 3 2 43 1 107* 9 5 6 ? 138* 1 2 0 7 2 11 9
Values are expressed as mean IT SD Group 1 germanium on/y group germanium and oral COQ, group 3 controls. EDL extensor digltorum longus TA tib/a'lsanferm 'Signif/cant difference (f < 0 001) as compared wlth the controls (gioup3j tsignifmnt difference (f c 0 05) as compared w/rh group 2
z
MUSCLE & NERVE
November 1992
1259
FIGURE 2. Frozen sections from soleus muscle (a, b) and extensor digitorum longus (EDL) muscle (c, d) from group 1 [germanium only; (a) and (c)] and grup 3 [controls; (b) and (d)] stained with modified Gomori trichrome. The ragged-red fibers (asterisks) were only present in group 1 animals and were more frequent in EDL muscle. Bar = 100 pm.
groups 1 and 2, the EDL and TA were more significantly affected than the soleus muscles: 43%48% weight loss in EDL, 55%-63% in TA, while only 14%-21% in soleus muscle. Histopathologic Studies. All animals in groups 1 and 2 showed significant myopathic changes which consisted of numerous RRFs and moderate variation in fiber size. There were some necrotic fibers in the EDL and TA, but not in the soleus muscles. Muscle fiber diameter was decreased to 68%-76% of the controls in the EDL, and 91%94% in the soleus muscles (Table 2). There was no suggestion of neurogenic atrophy in any specimen. On mGt stain, RRFs were found in all specimens in groups 1 and 2, predominantly in the EDL and TA (Table 2, Fig. 2). RRFs in the EDL, TA, and soleus muscles were mostly type 2A fibers (Fig. 3). There were no RRFs in control muscles. Intramuscular nerves were well-myelinated in all the experimental rats. Oxidative enzyme stain showed disorganized intermyofibrillar network and strong enzyme reac-
1260
Mitochondria1 Myopathy in Rats
tion in many fibers in groups 1 and 2, which were usually RRFs (Fig. 3). Muscle fiber type distribution in groups 1 and 2 did not differ from that in the controls (group 3). Approximately 90% of fibers were type 1 fibers
~
Table 2. Muscle fiber size and the incidence of RRFs and COX-deficient fibers Group
2
1
3
Fiber diameter (pm)
EDL Soleus RRFs (%)
EDL Soleus COX-deficient fibers (%)
EDL Soleus
36.6 t 3.2' 39.3 5 5.0' 51.6
5 5.2 70.7 i 4.9* 71.9 t 4.2' 76.3 t 2.9
38.9 t 7.0 35.1 t 6.0 7.2 t 4.1 8.4 t 4.1
0.0
100.0 17.8 5 4.2
0.0
100.0 17.3 3.6
*
0.0 0.0
Values are expressed as mean 2 SD. Group I : germanium only; group 2: germanium and oral COO; group 3: controls; EDL: extensor digitorurn longus; RRF: ragged-red fiber; COX: cytochrome-c oxidase. *Significant difference (P < 0.007) as compared with controls (group 3).
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November 1992
FIGURE 3. Serial frozen sections from SOleUS muscle of rat in group 1 (germanium only) stained with modified Gomori trichrome (a), succinate dehydrogenase (b), ATPase preincubated at pH 4.6 (c), and cytochrome-c oxidase (COX). (d). In addition to variation in fiber size, there are scattered ragged-red fibers (RRFs; asterisks) which stain dark with oxidative enzyme [(b), arrows] and behave as type 2A fibers [(c), arrows]. Not only RRFs, but normal-appearing fibers occasionally have no COX activity (arrowheads). Type 2A fiber (A), type 2 8 fiber (B), and type 1 fiber (1). Bar = 200 pm.
in the soleus and 95% were type 2 fibers in EDL muscles. Type 2 fibers were more atrophic than type 1 fibers in both EDL and soleus muscles. There was no increase in the number of type 2C fibers. Almost all muscle fibers in the EDL and T A of the animals in groups 1 and 2 failed to show any COX activity, while approximately 83% of the fibers in the soleus muscles were COX-positive, indicating focal COX deficiency (Table 2, Figs. 3 and 4). Most of the type 2A and 2B fibers did not stain with COX, while only 5%-8% of type 1 fibers were COX-negative (Fig. 3 ) . All RRFs were COX-negative (Fig. 3). All muscle fibers in the controls (group 3 ) were COX-positive (Fig. 4). In the EDL muscles from groups 1 and 2, many large mitochondria with increased numbers of cristae were observed. Some abnormal mitochondria contained electron-dense granules (Fig. 5). Electron Microscopic Examination.
Mitochondria1 Myopathy in Rats
Biochemical Studies. There was a significant difference in the activity of rotenone-sensitive NCCR ( P < 0.001), SCCR ( P < 0.001), and COX ( P < 0.001) between groups 1 and 2 and controls (group 3 ) . CoQ treatment showed only minimal effect on the activity of these enzymes (Table 3 ) .
DISCUSSION
As in humans with germanium intoxication, large doses of germanium inhibited weight gain and caused a myopathy with abnormal mitochondria in rats. There were several similarities between our findings and those in human mitochondria1 disorders: in addition to RRF, there were moderate variations in fiber size, a few necrotic fibers, and a marked decrease in COX activity. In terms of RRF formation, mitochondrial-rich type 2A fibers were more susceptible to the toxic effect of germanium than other fiber types. This preferential damage of oxidative fibers is also seen in human muscles in which type 1 fibers have high oxidative activity. Moreover, scattered COX-negative
MUSCLE & NERVE
November 1992
1261
FIGURE 4. Frozen sections of extensor digitorum longus muscle from group 1 [germanium only, (a)] and group 3 [controls, (b)] stained with cytochrome-c oxidase (COX). Almost all fibers in group 1 lack enzyme activity. Bar = 100 p.m.
fibers were present among normal fibers in the soleus muscle, representing focal COX deficiency. Focal COX deficiency is a characteristic finding in human mitochondrial encephalomyopathies, including chronic progressive external ophthalmoplegia (CPEO); myoclonus epilepsy with raggedred fibers (MERRF); and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). Recent molecular studies revealed that these three entities had specific mitochondrial DNA mutation^.^^^'^^ Therefore, the elucidation of the pathogenetic mechanisms of focal COX deficiency in relation to the molecular
defect is considered to be the central problem in these mitochondrial disorder^.^ In this context, this germanium-induced myopathy should be a useful experimental model of human mitochondrial encephalomyopathy. From our histochemical findings, it would appear that complex IV deficiency precedes deficiencies in the other complexes because RRFs were, without exception, COX-negative. In CPEO or MERRF in which COX is preferential1 involved, almost all RRFs had no COX activity,”’’’whereas a considerable number of RRFs had normal COX activity in MELAS with complex I deficiency (unpub-
Table 3. Mitochondrial respiratory chain enzyme activity in extensor digitorum longus muscle.
Group Complexes
+
I 111 II + 111 IV
1
2
3
5.2 2 4.0* 14.1 ? 4.2*t 4.9 5 2.8*$
2.9 + 4.8* 19.6 ? 7.0* 9.2 ? 4.4*
131.6 36.2 92.3 2 13.9 202.0 2 60.7
+
~~~
~
~
_
_
_
_
h z y m e activity is expressed as: enzyme activity ( n m o l h i n per milligram mitochondrial protein) loo citrate synthase activity (nmolimin per milligram mitochondrial protein) Values are expressed as mean ? SD. Group 1. germanium only; group 2: germanium and oral CoQ; group 3: controls; / + 111. rotenonesensitive NADH-cytochrome-c reductase (NCCR); / / + ///. succinafe- cytochrome-c reductase (SCCR); IV: cytochrome-c oxidase (COX). ‘Significant difference (P < 0.007) as compared with the controls (group 3). fSignificant difference ( P < 0.01) as compared with group 2. #Significant difference (P < 0.001) as compared with group 2
1262
Mitochondrial Myopathy in Rats
FIGURE 5. Ultrastructural findings in extensor digitorum longus muscle from group 1 (germanium only). Note many enlarged mitochondria with proliferated cristae, occasionally containing electron-dense materials. Bar = 1 pm.
MUSCLE & NERVE
November 1992
lished observations). In addition, it has been shown that the decline in complex I activity could be secondary to progressive decline in complex I V activity." It is possible that inhibition of COX activity by germanium leads to RRF formation and secondary decrease in the activities of other enzymes. Further chronologial study is necessary to clarify the primary inhibitory site of germanium in the mitochondrial respiratory chain. There have been several reports of the beneficial effects of large doses of CoQ in atients with mitochondrial encephalomyopathies. 17,18,27 Coy is a mobile electron carrier which functions during the transfer of electrons between dehydrogenases and cytochrome bc, complex, and mediates electron transfer by a diffusion process across the mitochondrial membrane.24CoQ had only minimal and less than satisfactory effect on body weight and the biochemical findings, and it had no effefct on the histopathologic changes in our animals with germanium-induced mitochondrial myopathy. If the primary effect of germanium intoxication is on COX activity, then CoQ administration would not be expected to be effective in these animals. It is possible that the amount of germanium used in the present study was too large for any beneficial effect of CoQ administration to be demonstrated.
"
REFERENCES
1. Aso H, Suzuki F, Ebina T, Ishida N: Antiviral activity of carboxyethylgermanium sesquioxide (Ge-132) in mice infected with influenza virus. J Biol Response Mod 1989;8: 180- 189. 2. Bookelman H, Trijbels JMF, Sengers RCA, Janssen AJM: Measurement of cytochromes in human skeletal muscle mitochondria, isolated from fresh and frozen stored muscle specimens. Biochem Med 1978;19:366-373. 3. Brutkicwicz RR, Suzuki F: Biological activities and antitumor mechanism of an immunopotentiating organogermanium compound, Ge-132 (review). In Vivo 1987;l: 189204. 4. Chiba M, Shinohara A, Inaba Y, Nakayama S, Rinno H, Koide H: T w o cases of patients taking germanium-containing stuffs (in Japanese). Juntendo Igaku (Tokyo) 1990;36: 406-4 10. 5. Goto Y, Koga Y, Horai S, Nonaka I: Chronic progressive external ophthalmolegia: a correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies. J Neurol Scz 1990;100:63-69. 6. Goto Y, Nonaka I , Hordi S: A mutation in the tRNALe u ( ~ gene ~ ~ associated ) with the MELAS subgroup in mitochondrial encephalomyopathies. Nature 1990;348:651653. 7 . Higuchi I , Izumo S, Kuriyama M, Suehara M, Nakagawa M, Fukunaga H, Osame M, Ohtsubo S, Miyata K: Germanium myopathy: clinical and experimental pathological studies. Acta Neuropathol (Bed) 1989;79:300- 304. 8. Higuchi I, Takahashi K, Nakahara K, Izumo S, Nakagawa
Mitochondria1 Myopathy in Rats
M, Osame M: Experimental germanium myopathy. Acta Neuropathol (Berl) 1991;82:55-59. 9. Holt IJ, Harding AE, Morgan-Hughes JA: Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988;33 1:717-719. 10. Koga Y, Nonaka I, Sunohara N, Yamanaka R, Kumagai K: Variability in the activity of respiratory chain enzymes in mitochondrial myopathies. Acta Neuropathol (Berl) 1988;76: 135- 141. 11. Mackler B: Microsomal DPNH-cytochrome c reductase, in Estabrook RW, Pullman ME (eds): Method< in Enzymology. New York, Academic Press, 1967, vol 10, p p 551-553. 12. Matsuoka T, Goto Y, Yoneda M, Nonaka I: Muscle histopathology in myoclonus epilepsy with ragged-red fibers (MERRF). J Neurol Sci 1991;106:193-198. 13. Nagata N, Yoneyama T, Yanagida K, Ushio K, Yanagihara S, Matsubara 0, Eishi Y: Accumulation of germanium in the tissues of a long-term user of germanium preparation died of acute renal failure. ,J Toxic01 Sci 1985; 10:333341. 14. Nakamura R, Yajima H, Yamamoto T , Imamura Y, Watabe S, Hase H, Nakai s, Uchiyama T, Kato N, Aikawa K: A case of suspected germanium intoxication (in Japanese). Prog Med (Tokyo) 1988;8:2037-2040. 15. Nishikawa Y, Takahashi M, Yorifuji S, Nakamura Y, Ueno S, Tarui S, Kozuka T , Nishimura T : Long-term coenzyme Qlo therapy for a mitochondrial encephalomyopathy with cytochrome c oxidase deficiency: a 31P NMR study. Neurology 1989;39:399-403. 16. Obara K, Akiu N, Sato H, Saito T , Miura Y, Yoshinaga K, Kakizawa M, Tada K, Tsukamoto T, Hongo M: Two cases with various symptoms and renal dysfunction induced by long term germanium intake (in Japanese). Nzppon Nazka Gakkai Zasshi (Tokyo) 1988;77:1704- 1709. 17. Ogasahara S, Nishikawa Y, Yorifuji S, Soga F, Nakamura Y, Takahashi M, Hashimoto S, Kono N, Tdrui S: Treatment of Kearns- Sayre syndrome with coenzyme Qln.Neurology 1986;36:45-53. 18. Ogasahara S, Yorifuji S, Nishikawa Y, Takahashi M, Wada K, Hazama T, Nakamura Y, Hashimoto S, Kono N, Tarui S: Improvement of abnormal pyruvate metablism and cardiac conduction defect with coenzyme Qlo in KearnsSayre syndrome. Neurology 1985;35:372-377. 19. Ohta S, Sat0 T, Narabayashi H, Rinno H , Ohno J: Acute myoglobinuria with lipid storage myopathy caused by longterm taking of alcohol and germanium (in Japanese). Clzn Neurol (Tokyo) 1985;25:845. 20. Ohtsubo S, Nakamura M, Nomura Y, lnoue H, Miyata K: Multiple organ failure caused by a long-term taking of germanium preparation: a case report (in Japanese). Nippon Shonzka Gakkuz Zasshi (Tokyo) 1988;92: 19941999. 21. Okagawa K, Kosaka M , Sakikawa T , Hiasa M, Sato H , Morizumi K, Morita H, Shimomura S: Renal hypofunction, normochromic anemia, muscle weakness and peripheral neuropathy occurred in three patients taking germanium-containing water (in Japanese). Naikn (Tokyo) 1'386;58:1210- 1214. 22. Okuda S, Kiyama S, Oh Y, Shimamatsu K, Oochi N , Kobayashi K, Nanishi F, Fujimi S, Onoyama K, Fujishima M: Persistent renal dysfunction induced by chronic intake of germanium-containing compounds. Curr Ther Res Clin ExP 1987;41:265-275. 23. Orii Y, Okunuki K: Studies on cytochrome a. XV.
