Summary
Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slightly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.
Similar content being viewed by others
References
Annison, E.F., Hill, K.J., Lindsay, D.B., Peters, R.A.: Fluroacetate poisoning in sheep. J. comp. Pathol. & Ther. 70, 145–155 (1960)
Bahr, G.F., Phil, E., Engler, W., Mikel, U.: Mitochondria of rat liver. Variations in gross conformation and size. Cytobiologie 2, 163–187 (1970)
Barasa, A., Godina, G., Buffa, P., Pasquali-Ronchetti, I.: Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo. I. The effect of fluoroacetate: A study by phasecontrast microscopy and time-lapse cinemicrography. Z. Zellforsch. 138, 187–210 (1973)
Bickis, I.J., Quastel, J.H.: Effects of metabolic inhibitors on energy metabolism of Ehrlich ascites carcinoma. Nature (Lond.) 205, 44–46 (1965)
Bovis, R., Kasten, F.H., Okigaki, T.: Electron microscopic study of the toxic effect of sodium fluoroacetate on rat myocardial cultures. Exp. Cell Res. 43, 611–621 (1966)
Bowman, R.H.: Inhibition of citrate metabolism by sodium fluoroacetate in the perfused rat heart and the effect on phosphofructokinase activity and glucose utilization. Biochem. J. 93, 13c-15c (1964)
Buffa, P.: Behaviour of easily hydroly sable P in heart muscle during fluoroacetate poisoning. Chem. and Ind., p. 1080 (1952)
Buffa, P., Azzone, G.F., Carafoli, E., Muscatello, U.: Modificazioni biochimiche mitocondriali e tessutali in relazione ad alcune ipo-ed ipertermie sperimentali del ratio. Sperimentale 110, 79–107 (1960a)
Buffa, P., Carafoli, E., Muscatello, U.: Differenze metaboliche tra diaframma e muscolo grande dorsale del ratio rivelate dall'intossicazione da fluoroacetato. Rend. Accad. Naz. Lincei 28, Ser. VIII, 929–936 (1960b)
Buffa, P., Godina, G., Barasa, A.: Mitochondrial changes induced by fluoroacetate in living cells. 5th Internat. Congr. Biochem., Moscow, 1961. Abstracts of Communications, 9.4. 1509
Buffa, P., Guarriero-Bobyleva, V., Costa-Tiozzo, R.: Metabolic effects of fluoroacetate poisoning in animals. Fluoride 6, 224–245 (1973)
Buffa, P., Guarriera-Bobyleva, V., Muscatello, U., Pasquali-Ronchetti, I.: Conformational changes of mitochondria associated with uncoupling of oxidative phosphorylation in vivo and in vitro. Nature (Lond.) 226, 272–274 (1970)
Buffa, P., Guarriera-Bobyleva, V., Pasquali-Ronchetti, I.: Biochemical effects of fluoroacetate poisoning in rat liver. In: Carbon-Fluorine Compounds. Chemistry, biochemistry and biological activities (K. Elliott and J. Birch, eds.), pp. 303–330. Amsterdam: Elsevier. Excerpta Medica. North-Holland 1972
Buffa, P., Muscatello, U., Godina, G., Barasa, A.: Mitochondrial modifications associated with the biochemical lesion caused by fluoroacetate. 3rd Europ. Regional Conf. Electr. Micr., Prague, 1964a, pp. 125–126
Buffa, P., Muscatello, U., Godina, G., Barasa, A.: Modification de la structure submicroscopique des mitochondries des cellules en culture sous l'action du fluoroacetate. Bull. Assoc. Anatomistes XLIX Reunion, Madrid, 1964b, pp. 352–356
Buffa, P., Pasquali-Ronchetti, I.: Ultrastructural modulations of mitochondria correlated to biochemical activity in vivo. 7th Internat. Congr. Electr. Micr., Grenoble, 1970. Vol. III, pp. 147–148
Buffa, P., Pasquali-Ronchetti, I., Coppi, G.: Effects of uncoupling of oxidative phosphorylation on the ultrastructure of mitochondria in vivo. Atti VI. Congr. Ital. Microsc. Elettr., Siena, 1967, pp. 7–11
Buffa, P., Peters, R.A.: Formation of citrate in vivo induced by fluoroacetate poisoning. Nature (Lond.) 163, 914 (1949a)
Buffa, P., Peters, R.A.: The in vivo formation of citrate induced by fluoroacetate and its significance. J. Physiol. (Lond.) 110, 488–500 (1949b)
Carey, N.H., Greville, G.D.: Mitochondria from embryonic tissues of the chick. I. Preparation, characterization and some enzymic properties. Biochem. J. 71, 159–166 (1959a)
Carey, N.H., Greville, G.D.: Mitochondria from embryonic tissues of the chick. 2. Metabolic activities. Biochem. J. 71, 166–176 (1959b)
Chenoweth, M.B.: Monofluoroacetatic acid and related compounds. J. Pharmacol. exp. Ther., Part. II 97, 383–424 (1949)
Corsi, A., Granata, A.L.: Differential toxicity of fluoroacetate to heart, kidney, and brain mitochondria of the living rat. Biochem. Pharmacol. 16, 1083–1089 (1967)
Cox, R.W., Horne, R.W.: Accurate calibration of the magnification of the A.E.I. E.M. 6B electron microscope using catalase crystals. 4th Europ. Regional Conf. Electr. Micr., Rome, 1968, Vol. I, pp. 579–580
Danes, B.S., Paul, J.: Environmental factors influencing respiration of strain L cells. Exp. Cell Res. 24, 344–355 (1961)
DuBois, K.P., Cochran, K.W., Doull, J.: Inhibition of citric acid synthesis in vivo by X-irradiation. Proc. Soc. exp. Biol. (N.Y.) 76, 422–427 (1951)
Fairhurst, A.S., Smith, R.E., Gál, E.M.: The effects of fluoro-compounds on oxidative phosphorylation. I. Effects on P/O ratios with fumarate and with reduced diphosphopyridine nucleotide. Biochem. Pharmacol. 1, 273–279 (1959a)
Fairhurst, A.S., Smith, R.E., Gál, E.M.: The effects of fluoro-compounds on oxidative phosphorylation. II. Effects on the breakdown of nucleotides by liver mitochondria. Biochem. Pharmacol. 1, 280–287 (1959b)
Gál. E.M., Peters, R.A., Wakelin, R.A.: Some effects of synthetic fluoro-compounds on the metabolism of acetate and citrate. Biochem. J. 64, 161–168 (1956)
Guarriera-Bobyleva, V., Buffa, P.: The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase. Biochem. J. 113, 853–860 (1969)
Guarriera-Bobyleva, V., Buffa, P.: Alternative pathway of citrate oxidation in the liver of the rat poisoned with fluoroacetate. Abstracts 10th FEBS Meeting, Paris, 1975, No. 1445
Hackenbrock, C.R.: Ultrastructural bases for metabolically linked mechanical activity in mitochondria I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J. Cell Biol. 30, 269–297 (1966)
Hackenbrock, C.R.: Ultrastructural bases for metabolically linked mechanical activity in mitochondria. II. Electron transport-linked ultrastructural transformations in mitochondria. J. Cell Biol. 37, 345369 (1968)
Hackenbrock, C.R., Renn, T.G., Gamble, J.L., Weinbach, E.C., Lemasters, J.J.: Ultrastructural transformation in the mitochondrion: its relationship to the energy state of the mitochondrion and the energy state of the cell. In: Energy transduction in respiration and phosphorylation (E. Quagliariello, S. Papa and C.S. Rossi, eds.), pp. 285–305. Bari: Adriatica Editrice 1971
Judah, J.D., Rees, K.R.: The activation of fatty acid oxidation by kidney and liver mitochondria. Biochem. J. 55, 664–668 (1953)
Koenig, H.: Acute axonal dystrophy caused by fluorocitrate: The role of mitochondrial swelling. Science 164, 310–312 (1969)
Lehninger, A.L.: Water uptake and extrusion by mitochondria in relation to oxidative phosphorylation. Physiol. Rev. 42, 467–517 (1962)
Lehninger, A.L.: The mitochondrion. Molecular basis of structure and function, p. 10. New York: Benjamin Inc. 1964
Liébecq, C., Liébecq-Hutter, S.: Effet du fluoroacétate et des rayon X sur la teneur en acide citrique du fois de la souris. Experientia (Basel) 14, 216–217 (1958)
Lindebaum, A., White, R.M., Schubert, J.: Citrate formation in vivo induced by non-lethal doses of fluoroacetate. J. biol. Chem. 190, 585–593 (1951)
Lowenstein, J.M.: Citrate and the conversion of carbohydrate into fat. In: The metabolic roles of citrate (T.W. Goodwin, ed.), pp. 61–86. London: Academic Press 1968
Max, S.R., Purvis, J.L.: Energy-linked incorporation of citrate into rat liver mitochondria. Biochem. biophys. Res. Commun. 21, 587–594 (1965)
McDowell, E.M.: Light and electron microscopic studies of the rat kidney after administration of inhibitors of the citric acid cycle in vivo: I. Effects of sodium fluoroacetate on the proximal convoluted tubule. Amer. J. Path. 66, 513–542 (1972)
Muscatello, U., Buffa, P., Barasa, A., Godina, G.: Modificazioni substrutturali nei mitocondri di cellule coltivate in vitro, sottoposte all'azione del fluoroacetato. Atti Soc. Ital. Anat., Monit. Zool. Ital. 72, Suppl., 86 (1964)
Muscatello, U., Guarriera-Bobyleva, V., Buffa, P.: Configurational changes in isolated rat liver mitochondria as revealed by negative staining. I. Modifications caused by osmotic and other factors. J. Ultrastruct. Res. 40, 215–234 (1972a)
Muscatello, U., Guarriera-Bobyleva, V., Buffa, P.: Configurational changes in isolated liver mitochondria as revealed by negative staining. II. Modifications caused by changes in respiratory states. J. Ultrastruct. Res. 40, 235–260 (1972b)
Muscatello, U., Pasquali-Ronchetti, I.: Structural asymmetry of mitochondrial inner membrane. J. submicr. Cytol. 4, 211–214 (1972)
Muscatello, U., Pasquali-Ronchetti, I., Barasa, A.: An electron microscopic study of myoblasts from chick embryo heart cultured in vitro. J. Ultrastruct. Res. 23, 44–59 (1968)
Ord, M.C., Stocken, L.A.: Sex differences in effect of radiation and fluoroacetate poisoning on liver metabolism. Proc. Soc. exp. Biol. (N.Y.) 83, 695–697 (1953)
Packer, L.: Relation of structure to energy coupling in rat liver mitochondria. Fed Proc. 29, 1533–1540 (1970)
Paradisi, F., Galanti, B., Mancini, A.: ATP levels in normal and diphteria toxin treated cell cultures. Path. Microbiol. 32, 24–28 (1968)
Parsons, D.F., Williams, G.R., Thompson, W., Wilson, D., Chance, B.: Improvements in the procedure for purification of mitochondrial outer and inner membrane. Comparison of the outer membrane with smooth endoplasmic reticulum. In: Mitochondrial structure and compartmentation (E. Quagliariello, S. Papa, E.C. Slater and J.M. Tager, eds.), pp. 29–70. Bari: Adriatica Editrice 1967
Pasquali-Ronchetti, I., Buffa, P.: Correlazione fra ultrastruttura e biochimica respiratoria dei mitocondri in vivo. Atti VII. Congr. Ital. Microsc. Elettr., Modena, 1969, pp. 113–115
Pasquali-Ronchetti, I., Muscatello, U.: Studio sui metodi di preparazione per l'esame al microscopic elettronico di mioblasti di cuore embrionale di polio coltivati in vitro. Sperimentale 116, 93–147 (1966)
Peters, R.A.: Lethal synthesis. Proc. roy. Soc. B 139, 143–170 (1952)
Peters, R.A.: Biochemical lesions and lethal synthesis, pp. 88–130. Oxford: Pergamon Press 1963
Peters, R.A., Wakelin, R.W., Buffa, P., Thomas, L.C.: Biochemistry of fluoroacetate poisoning. The isolation and some properties of the fluorotricarboxylic acid inhibitor of citrate metabolism. Proc. roy. Soc. B 140, 497–507 (1953)
Potter, V.R., Bush, H.: Citric acid content of normal and tumor tissue in vivo following injection of fluoroacetate. Cancer Res. 10, 353–356 (1950)
Pucci, I.: Mitochondrial changes induced by fluoroacetate in chick embryo myocardium in vivo. Exp. Cell Res. 35, 412–414 (1964)
Pucher, G.W., Sherman, C.C., Vickery, H.B.: A method to determine small amounts of citric acid in biological material. J. biol. Chem. 113, 235–245 (1936)
Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–213 (1963)
Ruggiero, G., Paradisi, F., Giusti, G.: Mitochondrial swelling induced by diphtheria toxin. The behaviour of citrate in intoxicated cells. Enzym. Biol. Clin. 9, 168–174 (1968)
Seno, S., Otsumi, K., Yokomura, E.: Swelling and contraction of mitochondria related to metabolism. In: Intracellular membranous structure (S. Seno and E.V. Cowdry, eds.), pp. 155–166. Okayama: Japan Society for Cell Biology 1965
Slater, E.C., Ter Welle, H.F.: Applications of oligomycin and related inhibitors in bioenergetics. In: Inhibitors. Tools in cell research (Th. Bücher and H. Sies, eds.), pp. 258–278. Berlin: Springer 1969
Strehler, B.L., McElroy, W.D.: Assay of adenosine triphosphate. In: Methods in enzymology, Vol. III (S.P. Colowick and M.O. Kaplan, eds.), pp. 871–873. New York: Academic Press 1957
Ward, P.F.V., Peters, R.A.: The chemical and biochemical properties of fluorocitric acid. Biochem. J. 78, 661–668 (1961)
Williams, C.H., Vail, W.J., Harris, R.A., Caldwell, M., Green, D.E.: Conformational basis of energy transduction in membrane systems. VIII. Configurational changes of mitochondria in situ and in vitro. Bioenergetics 1, 147–180 (1970)
Author information
Authors and Affiliations
Additional information
The present study is dedicated to Prof. Otto Bucher on occasion of his 65th birthday
This work was supported by grants of the Consiglio Nazionale delle Ricerche to both Institutes
Rights and permissions
About this article
Cite this article
Buffa, P., Pasquali-Ronchetti, I., Barasa, A. et al. Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo. Cell Tissue Res. 183, 1–23 (1977). https://doi.org/10.1007/BF00219988
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00219988