Estudo morfológico no músculo gastrocnêmio de camundongos C57 BL10 submetidos à ingestão prolongada de etanol

Silva, João Batista Guedes e; Lisboa, Jorge A. da Costa; Freitas, Marcos R. G.; Nascimento, Osvaldo J. M.

doi:10.1590/S0004-282X1996000200013

Resumos

Os efeitos do alcoolismo crônico no músculo gastrocnêmio de camundongos bem nutridos foram estudados morfologicamente com a finalidade de se avaliar a hipótese de que o etanol exerce um papel tóxico direto sobre o músculo esquelético. Foram usados 30 camundongos C57BL10 machos, adultos jovens, divididos em dois grupos: Grupo A (controle) consistindo de 10 camundongos que beberam água e Grupo B (alcoólico) de 20 camundongos que beberam etanol a 25%. Todos os animais receberam" uma ração balanceada de laboratório e foram mantidos nesse regime ad libitum nas mesmas condições ambientais durante 48 semanas e pesados uma vez por semana. O consumo diário de dieta e a ingestão calórica foram calculados tendo os animais apresentado substancial ganho de peso, não mostrando qualquer sinal de desnutrição. Ao final do experimento os animais foram sacrificados para estudos morfológicos. Pela microscopia óptica não foram encontradas alterações. Importantes anormalidades foram observadas pela microscopia eletrônica em todos os espécimens.O retículo sarcoplasmático mostrou-se freqüentemente dilatado, resultando na formação de grandes vesículas e envolvendo as cisternas terminais com deslocamento das tríades. Áreas de estreitamento, ruptura e perda de miofibrilas ocorreram assim como zonas de completa desorganização de miofibrilas com perda do padrão estriado normal. As mitocondrias estavam em linhas gerais dentro dos limites da normalidade. Agregados tubulares peculiares vistos comumente na paralisia periódica e outras condições patológicas humanas, foram encontrados tanto no músculo dos camundongos controles como no dos alcoólicos. Os nervos intramusculares assim como as junções neuromusculares mostravam-se normais. Importantes anormalidades nos capilares musculares foram observadas, consistindo de tumefação das células endoteliais e de espessamento da lâmina basal. Difusa infiltração lipídica microvesicular foi vista no citoplasma dos hepatócitos parecendo constituir uma evidência a mais da ação tóxica do etanol sobre o organismo. Concluímos que a ingestão prolongada de etanol, representando 14,4% de calorias totais, produz no músculo gastrocnêmio de camundongos C57BL10 bem nutridos um elenco de alterações ultraestruturais que refletem um efeito tóxico direto sobre o músculo esquelético. As alterações constatadas são semelhantes àquelas descritas na miopatia alcoólica crônica humana

ingestão prolongada de etanol; camundongos C57BL10; músculo gastrocnêmio; alterações ultraestruturais

The effects of chronic alcoholism on gastrocnemius muscle of well-nourished mice were morphologically studied to test the direct toxic role of ethanol on skeletal muscle. Thirty male young adult C57BL10 mice were divided in two groups: Group A (control) consisting of ten mice that drank water and Group B (alcoholic) consisting of twenty mice that drank 25% ethanol. All mice were allowed a balanced laboratory chow. The animals were kept on this ad libitum regimen under the same conditions of environment for 48 weeks and were weighed once a week. The daily dietary consumption and caloric intake were estimated, the animals having had a substantial weight gain, showing no signs of malnutrition. At the end of the experiment the animals were killed for morphological studies. No abnormalities were observed by conventional microscopy.Striking deviations from normal were verified by electron microscopy in all specimens. Dilatation of sarcoplasmic reticulum was a common feature, sometimes resulting in the formation of large vesicles and involving the terminal cisternae with the displacement of the triads. Areas of narrowing, splitting and loss of myofibrils were seen. Zones of complete disorganization of miofibrils could be occasionally observed. Mitochondria were generally normal. Peculiar tubular aggregates seen commonly in periodic paralysis and other human pathological conditions, were encountered in both control and alcoholic mice. Intramuscular nerves and neuromuscular junctions were normal. Important abnormalities in muscle capillaries were observed, consisting of swelling of endothelial cells and thickening of the basal lamina. A diffuse microvesicular lipid infiltration was seen in the cytoplasm of the hepatocytes which seems to be a further evidence of the toxic role played by ethanol. We concluded that prolonged ingestion of ethanol, representing 14.4% of total calories, produces in the gastrocnemius muscle of well-nourished C57BL10 mice a distinct spectrum of ultrastructural changes which reflects a direct toxic effect on the skeletal muscle. These alterations are similar to those described in human chronic alcoholic myopathy.

prolonged ethanol ingestion; C53BL10 mice; gastrocnemius muscle; ultrastructural changes

Estudo morfológico no músculo gastrocnêmio de camundongos C57 BL10 submetidos à ingestão prolongada de etanol

Study of ultrastructural alterations in gastrocnemius muscle of C57 BL10 mice after prolonged ethanol ingestion

João Batista Guedes e Silva^I; Jorge A. da Costa Lisboa^II; Marcos R. G. Freitas^III; Osvaldo J. M. Nascimento^III

^IProfessor Titular de Anatomia Patológica do Departamento de Patologia

^IIProfessor Adjunto do Departamento de Patologia

^IIIProfessor Titular da Disciplina de Neurologia

RESUMO

Os efeitos do alcoolismo crônico no músculo gastrocnêmio de camundongos bem nutridos foram estudados morfologicamente com a finalidade de se avaliar a hipótese de que o etanol exerce um papel tóxico direto sobre o músculo esquelético. Foram usados 30 camundongos C57BL10 machos, adultos jovens, divididos em dois grupos: Grupo A (controle) consistindo de 10 camundongos que beberam água e Grupo B (alcoólico) de 20 camundongos que beberam etanol a 25%. Todos os animais receberam" uma ração balanceada de laboratório e foram mantidos nesse regime ad libitum nas mesmas condições ambientais durante 48 semanas e pesados uma vez por semana. O consumo diário de dieta e a ingestão calórica foram calculados tendo os animais apresentado substancial ganho de peso, não mostrando qualquer sinal de desnutrição. Ao final do experimento os animais foram sacrificados para estudos morfológicos. Pela microscopia óptica não foram encontradas alterações. Importantes anormalidades foram observadas pela microscopia eletrônica em todos os espécimens.O retículo sarcoplasmático mostrou-se freqüentemente dilatado, resultando na formação de grandes vesículas e envolvendo as cisternas terminais com deslocamento das tríades. Áreas de estreitamento, ruptura e perda de miofibrilas ocorreram assim como zonas de completa desorganização de miofibrilas com perda do padrão estriado normal. As mitocondrias estavam em linhas gerais dentro dos limites da normalidade. Agregados tubulares peculiares vistos comumente na paralisia periódica e outras condições patológicas humanas, foram encontrados tanto no músculo dos camundongos controles como no dos alcoólicos. Os nervos intramusculares assim como as junções neuromusculares mostravam-se normais. Importantes anormalidades nos capilares musculares foram observadas, consistindo de tumefação das células endoteliais e de espessamento da lâmina basal. Difusa infiltração lipídica microvesicular foi vista no citoplasma dos hepatócitos parecendo constituir uma evidência a mais da ação tóxica do etanol sobre o organismo. Concluímos que a ingestão prolongada de etanol, representando 14,4% de calorias totais, produz no músculo gastrocnêmio de camundongos C57BL10 bem nutridos um elenco de alterações ultraestruturais que refletem um efeito tóxico direto sobre o músculo esquelético. As alterações constatadas são semelhantes àquelas descritas na miopatia alcoólica crônica humana

Palavras-chave: ingestão prolongada de etanol, camundongos C57BL10, músculo gastrocnêmio, alterações ultraestruturais.

ABSTRACT

The effects of chronic alcoholism on gastrocnemius muscle of well-nourished mice were morphologically studied to test the direct toxic role of ethanol on skeletal muscle. Thirty male young adult C57BL10 mice were divided in two groups: Group A (control) consisting of ten mice that drank water and Group B (alcoholic) consisting of twenty mice that drank 25% ethanol. All mice were allowed a balanced laboratory chow. The animals were kept on this ad libitum regimen under the same conditions of environment for 48 weeks and were weighed once a week. The daily dietary consumption and caloric intake were estimated, the animals having had a substantial weight gain, showing no signs of malnutrition. At the end of the experiment the animals were killed for morphological studies. No abnormalities were observed by conventional microscopy.Striking deviations from normal were verified by electron microscopy in all specimens. Dilatation of sarcoplasmic reticulum was a common feature, sometimes resulting in the formation of large vesicles and involving the terminal cisternae with the displacement of the triads. Areas of narrowing, splitting and loss of myofibrils were seen. Zones of complete disorganization of miofibrils could be occasionally observed. Mitochondria were generally normal. Peculiar tubular aggregates seen commonly in periodic paralysis and other human pathological conditions, were encountered in both control and alcoholic mice. Intramuscular nerves and neuromuscular junctions were normal. Important abnormalities in muscle capillaries were observed, consisting of swelling of endothelial cells and thickening of the basal lamina. A diffuse microvesicular lipid infiltration was seen in the cytoplasm of the hepatocytes which seems to be a further evidence of the toxic role played by ethanol. We concluded that prolonged ingestion of ethanol, representing 14.4% of total calories, produces in the gastrocnemius muscle of well-nourished C57BL10 mice a distinct spectrum of ultrastructural changes which reflects a direct toxic effect on the skeletal muscle. These alterations are similar to those described in human chronic alcoholic myopathy.

Key words: prolonged ethanol ingestion, C53BL10 mice, gastrocnemius muscle, ultrastructural changes.

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Full text available only in PDF format.

Agradecimento - Aos técnicos do Setor de Microscopia Eletrônica Suely Menezes da Cunha, Rita de Cássia da Costa Cunha, Rosely da Costa Cunha e Ricardo Pascual Barreto pela colaboração nos trabalhos de uitramicrotomia e de fotografia.

Aceite: 17-janeiro-1996.

Estudo do Departamento de Patologia da Faculdade de Medicina da Universidade Federal Fluminense (UFF)

Dr. J.B. Guedes e Silva - Rua Miguel Pereira Sarmento 240 - 24310-420 Niterói RJ - Brasil

1. Baruah J.K, Washington M, Kinder D. Ethanol induced skeletal muscle degeneration: role of calcium. Exp Pathol 1988;33:207-212.
2. Bosshardt DK, Paul WJ, O'Doherty K, Barnes R.H. Caloric restriction and protein metabolism in the growing mouse. J Nutr 1948;36:773-782.
3. Canolty NL, Koong LJ. Utilization of energy for maintenance and for fat lean gains by mice selected for rapid postweaning growth rate. J Nutr 1976;106:1202-1208.
4. Chui LA, Neustein H, Munsat TL. Tubular aggregates in subclinical alcoholic myopathy. Neurology 1975;25:405-412.
5. Coiro JRR, Brunner A Jr. Comportamento do polylite 8001 com aditivos plastificantes. An Acad Bras Ciênc 1973;45:679-680.
6. Cullen MJ, Johnson MA, Mastaglia FL. Pathological reactions of skeletal muscle.In: Mastaglia FL, Walton J. (eds). Skeletal muscle pathology Ed 2. Edinburgh: Churchill Livingstone, 1992;! 23-184.
7. Del Villar Negrio A, Merino Angulo J, Rivera Pomar JM, Aguirre Errasti C. Tubular aggregates in skeletal muscle of chronic alcoholic patients. Acta Neuropathol (Berl) 1982;56:250-254.
8. Del Villar Negro A, Merino Angulo J, Rivera Pomar JM. Skeletal muscle changes in chronic alcoholic patients: a conventional, histochemical, ultrastructural and morphometric study. Acta Neurol Scand 1984;70:185-196.
9. Ekbom K, Hed R, Kirstein L, Astrom K-E.:Muscular affections in chronic alcoholism. Arch Neurol 1964;10:449-458.
10. Engel WK, Bishop DW, Cunnigham GG. Tubular aggregates in type II muscle fibers:ultrastructural and histochemical correlation. J UltrastrRes, 1970;31:507-525.
11. Ferraz ML, Gabbai AA, Oliveira ASB, Ferrari APJ, Miszputen SJ, Ferreira Neto A, Castelo Filho A, Schimdt B. Estudo histoquímico do músculo esquelético no alcoolismo crônico. Arq Neuropsiquiatr 1989,47:139-149.
12. Fisher ER, Puntereri AJ, Jung Y, Corredor DG, Danowski TS. Alcoholism and other concomitants of mitochondrial inclusion in skeletal muscle. Amer J Med Sci, 1971;261:85-99.
13. Freitas MR, Marques HA, Guedes e Silva JB. Associação de miopatia e miocardiopatia alcoólicas: estudo clinico, eletroneuromiográflco e histopatológico do músculo esquelético em 10 casos de miocardiopatia alcoólica. Arq Neuropsiquiatr 1988;46:339-350.
14. Hand A, Slavin G, Mair W, Sowter C, Ward P, Webb J, Levi J. Fibre type changes in striated muscle of alcoholics. J Clin Pathol, 1981;34:991-995.
15. Humphrey CD, Pittman FE. Azure II: basic fuchsin stain for epoxy-embedded tissue sections. Stain Technol 1974;49:9-14.
16. Klinkerfuss G, Bleisch V, Dioso MM, Perkoff GT. A spectrum of myopathy associated with alcoholism. II-light and electron microscopic observations. Ann Intern Med 1967;67:493-510.
17. Knapka JJ, Smith KP, Judge FJ. Effect of crude fat and crude protein on reproduction and weanling growth in four strains of inbred mice. J Nutr 1977;107:61-69.
18. Kunel RW, Peshonk A, Lane J, Alexander E. The MRL +/+ mouse: a new model of tubular aggregates which are gender and age-related. Acta Neuropathol 1989;78:615-620.
19. Langohr HD, Wiethõlter H, Pfeiffer J. Muscle wasting in chronic alcoholics: comparative histochemical and biochemical studies. J Neurol Neurosurg Psychiatry, 1983;46:248-254.
20. Lin JH, Duffy JL, Roginsky MS. Microcirculation in diabetes mellitus. Human Pathol 1975;6:77-96.
21. Martin FC, Slavin G, Levi AJ. Alcoholic muscle disease. Brit.Med.Bull, 1982;38:53-56.
22. Martin JB, Craig J W, Eckel RE, Munger J. Hypokalemic myopathy in chronic alcoholism. Neurology, 1971 ;21:1160-1168.
23. Martinez AJ, Hooshmand H, Fans AA. Acute alcoholic myopathy. Enzyme histochemistry and electron microscopic findings. J Neurol Sci, 1973;20:245-252.
24. Munsat TL, Neustein H, Higgins J, Chui L. Experimental acute alcoholic myopathy. Neurology, 1973;23:407.
25. National Academy of Science. Nutrient requirements of laboratory animals. Ed3. Washington: 1978.
26. Nonaka I, Satoyoshi E. Myotonic disorders. In: Mastaglia FL, Walton J. (eds). Skeletal muscle pathology. Ed2. Edinburgh: Churchill Livingstone, !992;3I9-342.
27. Organizacion Panamericana de la Salud. Manual para técnicos en animates de laboratório. Buenos Aires, Centro Panamericano de Zoonosis, 1974;75-1I0.
28. Perkoff GT, Hardy P, Velez-Garcia E. Reversible acute muscular syndrome in chronic alcoholism. N Engl J Med, 1966;274:1277-1285.
29. Perkoff GT, Dioso MM, Bleisch V, Klinkerfuss G. A spectrum of myopathy associated with alcoholism: I. Clinical and laboratory features. Ann.Intern.Med, 1967;67:481-492.
30. Perkoff GT. Alcoholic myopathy. Ann Rev Med, 1971;22:125-132.
31. Porta EA, Gomez-Dumm CLA. A new experimental approach in the study of chronic alcoholism: I. Effects of high alcohol intake in rats fed a commercial laboratory diet. Lab Invest, 1968,18:352-364.
32. Rodgers DA, McClean GE. 0, Mouse strain differences in preference for various concentrations of alcohol. Q J Stud Alcohol, 1962;23:26-33.
33. Rossouw JE, Keeton RG, Hewlett RH. Chronic proximal muscular weakness in alcoholics. S Afr Med J, 1976;50:2095-2098.
34. Rubin E, Katz AM, Lieber CS, Stein EP, Puszkin S. Muscle damage produced by chronic alcohol consumption. Amer J Pathol, 1976;83:499-515.
35. Rubin E. Alcoholic myopathy in heart and skeletal muscle. New Engl J Med, 1979;301:28-33.
36. Sewry CA. Ultrastructural changes in diseased muscle. In: Dubowitz V. Muscle biopsy: a practical approach. Ed2. London: BaillièreTindalI, 1985:129-183.
37. Song SK, Rubin E. Ethanol produces muscle damage in human volunteers. Science, 1972; 175:327-328.
38. Trounce I, Byrne E, Dennett X. Biochemical and morphological studies of skeletal muscle in experimental chronic alcoholic myopathy. Acta Med Scand, 1990;82:386-391.
39. Urbano-Marquez A, Estruch R, Navarro-Lopez F, Grau JM, Mont L, Rubin E. The effects of alcoholism on skeletal and cardiac muscle. N Engl J Med, 1989;320:409-414.
40. Yoshitoshi M, Ishihara T, Tsugane T, Shinohara Y. The effect of sex hormones on tubular aggregates in normal mouse skeletal muscles (Abstr). Rinsho-Shinkeigaku 1991;31:974-980.

Datas de Publicação

Publicação nesta coleção
06 Dez 2010
Data do Fascículo
Jun 1996

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. Baruah J.K, Washington M, Kinder D. Ethanol induced skeletal muscle degeneration: role of calcium. Exp Pathol 1988;33:207-212.

[2] 2. Bosshardt DK, Paul WJ, O'Doherty K, Barnes R.H. Caloric restriction and protein metabolism in the growing mouse. J Nutr 1948;36:773-782.

[3] 3. Canolty NL, Koong LJ. Utilization of energy for maintenance and for fat lean gains by mice selected for rapid postweaning growth rate. J Nutr 1976;106:1202-1208.

[4] 4. Chui LA, Neustein H, Munsat TL. Tubular aggregates in subclinical alcoholic myopathy. Neurology 1975;25:405-412.

[5] 5. Coiro JRR, Brunner A Jr. Comportamento do polylite 8001 com aditivos plastificantes. An Acad Bras Ciênc 1973;45:679-680.

[6] 6. Cullen MJ, Johnson MA, Mastaglia FL. Pathological reactions of skeletal muscle.In: Mastaglia FL, Walton J. (eds). Skeletal muscle pathology Ed 2. Edinburgh: Churchill Livingstone, 1992;! 23-184.

[7] 7. Del Villar Negrio A, Merino Angulo J, Rivera Pomar JM, Aguirre Errasti C. Tubular aggregates in skeletal muscle of chronic alcoholic patients. Acta Neuropathol (Berl) 1982;56:250-254.

[8] 8. Del Villar Negro A, Merino Angulo J, Rivera Pomar JM. Skeletal muscle changes in chronic alcoholic patients: a conventional, histochemical, ultrastructural and morphometric study. Acta Neurol Scand 1984;70:185-196.

[9] 9. Ekbom K, Hed R, Kirstein L, Astrom K-E.:Muscular affections in chronic alcoholism. Arch Neurol 1964;10:449-458.

[10] 10. Engel WK, Bishop DW, Cunnigham GG. Tubular aggregates in type II muscle fibers:ultrastructural and histochemical correlation. J UltrastrRes, 1970;31:507-525.

[11] 11. Ferraz ML, Gabbai AA, Oliveira ASB, Ferrari APJ, Miszputen SJ, Ferreira Neto A, Castelo Filho A, Schimdt B. Estudo histoquímico do músculo esquelético no alcoolismo crônico. Arq Neuropsiquiatr 1989,47:139-149.

[12] 12. Fisher ER, Puntereri AJ, Jung Y, Corredor DG, Danowski TS. Alcoholism and other concomitants of mitochondrial inclusion in skeletal muscle. Amer J Med Sci, 1971;261:85-99.

[13] 13. Freitas MR, Marques HA, Guedes e Silva JB. Associação de miopatia e miocardiopatia alcoólicas: estudo clinico, eletroneuromiográflco e histopatológico do músculo esquelético em 10 casos de miocardiopatia alcoólica. Arq Neuropsiquiatr 1988;46:339-350.

[14] 14. Hand A, Slavin G, Mair W, Sowter C, Ward P, Webb J, Levi J. Fibre type changes in striated muscle of alcoholics. J Clin Pathol, 1981;34:991-995.

[15] 15. Humphrey CD, Pittman FE. Azure II: basic fuchsin stain for epoxy-embedded tissue sections. Stain Technol 1974;49:9-14.

[16] 16. Klinkerfuss G, Bleisch V, Dioso MM, Perkoff GT. A spectrum of myopathy associated with alcoholism. II-light and electron microscopic observations. Ann Intern Med 1967;67:493-510.

[17] 17. Knapka JJ, Smith KP, Judge FJ. Effect of crude fat and crude protein on reproduction and weanling growth in four strains of inbred mice. J Nutr 1977;107:61-69.

[18] 18. Kunel RW, Peshonk A, Lane J, Alexander E. The MRL +/+ mouse: a new model of tubular aggregates which are gender and age-related. Acta Neuropathol 1989;78:615-620.

[19] 19. Langohr HD, Wiethõlter H, Pfeiffer J. Muscle wasting in chronic alcoholics: comparative histochemical and biochemical studies. J Neurol Neurosurg Psychiatry, 1983;46:248-254.

[20] 20. Lin JH, Duffy JL, Roginsky MS. Microcirculation in diabetes mellitus. Human Pathol 1975;6:77-96.

[21] 21. Martin FC, Slavin G, Levi AJ. Alcoholic muscle disease. Brit.Med.Bull, 1982;38:53-56.

[22] 22. Martin JB, Craig J W, Eckel RE, Munger J. Hypokalemic myopathy in chronic alcoholism. Neurology, 1971 ;21:1160-1168.

[23] 23. Martinez AJ, Hooshmand H, Fans AA. Acute alcoholic myopathy. Enzyme histochemistry and electron microscopic findings. J Neurol Sci, 1973;20:245-252.

[24] 24. Munsat TL, Neustein H, Higgins J, Chui L. Experimental acute alcoholic myopathy. Neurology, 1973;23:407.

[25] 25. National Academy of Science. Nutrient requirements of laboratory animals. Ed3. Washington: 1978.

[26] 26. Nonaka I, Satoyoshi E. Myotonic disorders. In: Mastaglia FL, Walton J. (eds). Skeletal muscle pathology. Ed2. Edinburgh: Churchill Livingstone, !992;3I9-342.

[27] 27. Organizacion Panamericana de la Salud. Manual para técnicos en animates de laboratório. Buenos Aires, Centro Panamericano de Zoonosis, 1974;75-1I0.

[28] 28. Perkoff GT, Hardy P, Velez-Garcia E. Reversible acute muscular syndrome in chronic alcoholism. N Engl J Med, 1966;274:1277-1285.

[29] 29. Perkoff GT, Dioso MM, Bleisch V, Klinkerfuss G. A spectrum of myopathy associated with alcoholism: I. Clinical and laboratory features. Ann.Intern.Med, 1967;67:481-492.

[30] 30. Perkoff GT. Alcoholic myopathy. Ann Rev Med, 1971;22:125-132.

[31] 31. Porta EA, Gomez-Dumm CLA. A new experimental approach in the study of chronic alcoholism: I. Effects of high alcohol intake in rats fed a commercial laboratory diet. Lab Invest, 1968,18:352-364.

[32] 32. Rodgers DA, McClean GE. 0, Mouse strain differences in preference for various concentrations of alcohol. Q J Stud Alcohol, 1962;23:26-33.

[33] 33. Rossouw JE, Keeton RG, Hewlett RH. Chronic proximal muscular weakness in alcoholics. S Afr Med J, 1976;50:2095-2098.

[34] 34. Rubin E, Katz AM, Lieber CS, Stein EP, Puszkin S. Muscle damage produced by chronic alcohol consumption. Amer J Pathol, 1976;83:499-515.

[35] 35. Rubin E. Alcoholic myopathy in heart and skeletal muscle. New Engl J Med, 1979;301:28-33.

[36] 36. Sewry CA. Ultrastructural changes in diseased muscle. In: Dubowitz V. Muscle biopsy: a practical approach. Ed2. London: BaillièreTindalI, 1985:129-183.

[37] 37. Song SK, Rubin E. Ethanol produces muscle damage in human volunteers. Science, 1972; 175:327-328.

[38] 38. Trounce I, Byrne E, Dennett X. Biochemical and morphological studies of skeletal muscle in experimental chronic alcoholic myopathy. Acta Med Scand, 1990;82:386-391.

[39] 39. Urbano-Marquez A, Estruch R, Navarro-Lopez F, Grau JM, Mont L, Rubin E. The effects of alcoholism on skeletal and cardiac muscle. N Engl J Med, 1989;320:409-414.

[40] 40. Yoshitoshi M, Ishihara T, Tsugane T, Shinohara Y. The effect of sex hormones on tubular aggregates in normal mouse skeletal muscles (Abstr). Rinsho-Shinkeigaku 1991;31:974-980.