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Morphological evaluation of the lipofuscinolytic effect of acetylhomocysteine thiolactone
Arch. Gerontol. Geriatr., 4 (1985) 67-72 Elsevier
67
AGG 00097
Morphological evaluation of the lipofuscinolytic effect of acetylhomocysteine thiolactone Eugenia Aloj Totfiro ~, Francesco Aurelio Pisanti 1, A n n a m a r i a C o n t i n i l l o I a n d Enrico Liberatori 2 I Departments of Biology and General Zoology, First Faculty of Medicine and Surgery, University of Naples, Viale M. Cristina 18, 1-80122 Naples, and 2 Research Department, Roussel MaestrettL Viale Gran Sasso 18, 1-20131 Milan, Italy (Received 1 October 1984; revised version received 14 December 1984; accepted 18 December 1984)
Summary The acetylhomocysteine thiolactone, a free radical scavenger drug able to activate the superoxide dismutase, shows a lytic effect on neuronal lipofuscin. The study was performed in a nervous tissue particularly rich in lipofuscin, the electric lobe of Torpedo marmorata. It is shown that the drug induces the migration of pigment towards the blood vessels and glial cells which remove them. acetylhomocysteine thiolactone; lipofuscin; age pigments; free radicals; Torpedo marmorata
Introduction
Various authors (Feeney-Bourns et al., 1980; Donato, 1981) consider that the production of lipofuscin is a consequence of the peroxidative effect induced by free radicals on membrane lipids. This hypothesis is supported by experimental results of these authors who have used free radical scavenger substances such as centrophenoxine (Glees and Hasan, 1976) and antioxidants as vitamin E (Robison et al., 1979) to reduce the accumulation of lipofuscin. The electric lobe, an anatomotopographical district of the central nervous system of Torpedo marmorata, is particularly rich in lipofuscin (Sterzi, 1909; De Lerma and Ventra, 1956; Aloj Totaro and Pisanti, 1979). Recently, it has been observed in the neurons of this tissue (Aloj Totaro and Pisanti, 1981) that acetylhomocysteine thiolactone, or cythiolone (CYT), inhibits the formation of lipofuscin granulation. These observations were followed by experiments which have clarified the mechanism of action of this drug, which has a free radical scavenger activity (Pisanti et al., 1983; Aloj Totaro et al., 1985b) and a capacity to increase the activity of superoxide dismutase (Pisanti et al., 1985). It seemed interesting to us therefore to complete our 0167-4943/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
68 study on the action of cythiolone on age pigments by demonstrating the qualitative and quantitative modification of pigment granules of electric lobe neurons of Torpedo exposed to the drug.
Materials and Methods
20 Torpedo marmorata of both sexes of 1-2 yr of age, as judged by the comparative evaluation of various growth parameters (Aloj Totaro et al., 1985a), were kept in tanks with closed circulation and fed artificially (Aloj Totaro, 1978). Ten animals were injected daily with 8 mg/kg body weight of acetylhomocysteine thiolactone (Citiolase, Roussel-Maestretti, Milano, Italy) for 30 days, while the remaining ten were treated with a placebo. On the 31st day the animals were anaesthetised in MS 222 (Sandoz) at 0.015%o in sea water and perfused with a solution containing 291 mM NaCI, 3.52 mM Na2SO 4, 3.24 mM KCI, 2.16 mM MgC12, 1.8 mM CaC12 and 450 mM CO(NH2) 2. The perfusion liquid was then replaced with a fixative of 3.5% glutaraldehyde, 2.4% Na-cacodylate and 2% sucrose in 205 mM NaCI, 3.52 mM Na2SO4, 3.24 mM KCI, 2.16 mM MgC12, 1.8 mM CaCI 2 and 450 mM CO(NH2) 2. By dissecting the cranium we then removed the electric lobes (Sterzi, 1909) which were processed for the electron microscope according to standard techniques (Aloj Totaro and Pisanti, 1979). Ultrathin sections (Ultrotome III, LKB), stained with lead citrate and uranyl acetate, were observed with a Philips 400 electron microscope. We took 30 random photographs (6000 × ) on samples selected according to the method proposed by Weibel et al. (1969) for stereological analysis. These photographs were analyzed with a semi-automatic computerized image analyzer (IBAS I, Zeiss).
Results
Neurons from the Torpedo electric lobe have a characteristic shape and appear surrounded by a series of glial cells which, according to the hypothesis proposed by Glees and Hasan (1976) for neurons of higher vertebrates, serve to capture residual debris and to transport these towards the vessel lumen. In the normal condition the electric lobe neurons of adult Torpedo appear rich in lipofuscin granulation generally situated in the perinuclear area (Aloj Totaro and Pisanti, 1979). The pigment granules occupy 2.52 + 0.3% of cytoplasmic area with a mean surface area of 0.58 + 0.2/~m2. In contrast, in neurons of individuals treated with CYT, the granules are less numerous (1.94 + 0.6%, P < 0.01), smaller (0.50 + 0.2 #m 2, P < 0.05) and are found in the peripheral cytoplasm (Fig. 1). Also apparent in Fig. 2 are numerous formations typical of GERL, which confirms the intense phenomenon of phagocytosis and metabolic transformations which are associated with these cells. Figure 2 shows that the lipofuscin granules near the edge of the neuron are probably in the process of being c~iptured by the satellite cells. The presence of both granular and 'finger print' pigment granules indicates that CYT induced a process of lipofuscinol-
69
Fig. 1. Torpedo m. electric lobe neuron. Note GERL formations ( x 4600).
Fig. 2. Treated Torpedo m. electric lobe. Lipofuscin granules (L) have been or are in the process of being captured by a satellite cell (S) ( x 8000). Fig. 3. Treated Torpedo m. Several lipofuscin granules moving from electric lobe neuron toward the glial cell. N: nucleus of the glial cell ( x 8000).
70
Fig. 4. Treated Torpedo m. electric lobe. Lipofuscin granulation near the satellite cell (S) ( x 10000).
ysis, as it has been demonstrated for other drugs such as centrophenoxine (Spoerri and Glees, 1975). This lipofuscinolytic action of CYT is further confirmed in the sequence of Figs. 3-5. In Fig. 3 there are various pigment granules near the cytoplasmic boundary of the neuron. Some of these show a clear tendency of being transported to the nearby satellite cell. In Fig. 4 we show, at higher magnification,
Fig. 5. Treated Torpedo m. electric lobe. A typical glial cell full of lipofuscin granules ( × 6000).
71
various lipofuscin granules which have a prevalently lamellar structure grouped in proximity to a satellite cell. Figure 5 shows the conclusion of the transport of pigment granules induced by CYT demonstrating a satellite glial cell absolutely full of lipofuscin granules.
Discussion
In vitro experiments show that the generation of hydroxyl free radicals by chemical systems results in the cross-linking of bovine serum albumin (Zs.-Nagy and Nagy, 1980); CYT, when introduced in this experimental system, is able to diiminish the extent of cross-linking acting most probably as a free radical scavenger (Pisanti et al., 1983; Aloj Totaro et al., 1985b). CYT injected in vivo in normal adult rats for 30 days significantly (P < 0.001) increased the superoxide dismutase activity, one of the most important of the enzymes of protection against the damaging effects of free radicals (Pisanti et al., 1985). It has also been shown (Aloj Totaro and Pisanti, 1981), and confirmed in the present report, that CYT inhibits the production of lipofuscin granules, shown both as a reduction in the number of granules and in their morphometric parameters. This inhibitory effect of CYT on the production of lipofuscin is shown by a reduction of the cytoplasmic area occupied by the pigment. The observations in this work show, in addition, how the drug induces the migration of pigment granules toward the cell periphery in proximity to the vessels and glial cells. In fact, these latter cells, in animals treated with the drug, appear to be full of lipofuscin granules, ready to send residual material accumulated within them toward the blood vessels which transport the material away from its site of production. The capacity of neurons to free themselves of unwanted material from their cellular circuits seems to be activated by CYT which, in this sense, aids the cellular defense mechanisms. Similar observations have been presented (Spoerri and Glees, 1975) for centrophenoxine, which has a molecular structure very different to that of CYT. Both centrophenoxine and cythiolone have been shown to have free radical scavenger activity (Zs.-Nagy and Semsei, 1984; Aloj Totaro et al., 1985b). Therefore, we believe that neither of these drugs acts directly on lipofuscinolysis. By protecting the membrane components against free radicals that are normally produced during the cell metabolism, these drugs limit their action to the inhibition of production of new pigment granules. In this way the cell system that physiologically assures the removal of residual material of metabolic processes will be made more efficient. One characteristic that makes cythiolone particularly interesting for its effect on age pigments with respect to other free radical scavenger molecules as centrophenoxine and antioxidants as vitamin E is its capacity to activate the superoxide dismutase; in this way aiding its protective effect against free radicals.
References Aloj Totaro, E. (1978): Effects of centrophenoxine on the variation of some behavioural patterns in Torpedo m. Acta Neurol., 34, 332-336.
72 Aloj Totaro, E. and Pisanti, F.A. (1979): Preliminary observations at the electron microscope on the presence of neuronal lipofuscin in Torpedo m. Acta Neurol., 34, 322-331. Aloj Totaro, E. and Pisanti, F.A. (1981): Influence of SH groups in the formation of neuronal lipofuscin in Torpedo m. Acta Neurol., 36, 460-465. Aloj Totaro, E., Pisanti, F.A., Russo, P. and Brunetti, P. (1985a): Evaluation of aging parameters in Torpedo marmorata. Ann. Soc. R. Zool. Belg., in press. Aloj Totaro, E., Pisanti, F.A. and Liberatori, E. (1985b): Possible interrelations of acetyl homocysteine thiolactone in mechanisms of lipofuscinigenesis. Res. Commun. Chem. Pathol. Pharmacol., in press. De Lerma, B. and Ventra, D. (1956): Su un materiale fluorescente individuato nel citoplasma delle cellule gangliari del lobo elettrico di Torpedine. Acta Neurol., 11, 1009-1018. Donato, H. (1981): Lipid peroxidation, cross-linking and diseases. In: Age Pigments, pp. 63-82. Editor: R.S. Sohal. Elsevier/North-Holland, Amsterdam. Feeney-Bourns, L., Berman, E.R. and Rothman, M.S. (1980): Lipofuscin of human retinal pigment epithelium. Am. J. Ophthalmol., 90, 783-791. Glees, P. and Hasan, M. (1976): Lipofuscin in neuronal aging and diseases. In: Normal and Pathological Anatomy, pp. 1-68. Editors: W. Bargmann and W. Doerr. Georg Thieme, Stuttgart. Pisanti, F.A., Fascatore, S., Aloj Totaro, E. and Crisci, A. (1983): Effetti del citiolone sulla dinamica di formazione dei pigmenti di senescenza. G. Gerontol., 31,795-796. Pisanti, F.A., Fascatore, S. and Papaccio, G. (1985): Influence of acetyl homocysteine thiolactone on erythrocyte superoxide dismutase activity. Experientia, in press. Robison, W.A., Kuwabara, T. and Bieri, J.A. (1979): Vitamin E deficiency and the retina. Photoreceptor and pigment epithelial changes. Invest. Ophthalmol. Vis. Sci., 18, 683-689. Spoerri, P.E. and Glees, P. (1975): The mode of lipofuscin removal from hypothalamic neurons. Exp. Gerontol., 10, 225-228. Sterzi, G. (1909): Selacei, anatomia. In: I1 sistema nervoso centrale dei vertebrati, vol. 2, libro 1, pp. 1-415. Editor: G. Sterzi. Draghi, Padova. Weibel, E.R., Staubli, W., Gnagi, H.R. and Hess, F.A. (1969): Correlated morphometric and biochemical studies on the liver cell. J. Cell Biol., 42, 68-91. Zs.-Nagy, I. and Nagy, K. (1980): On the role of cross-linking of cellular proteins in aging. Mech. Ageing Dev., 14, 245-251. Zs.-Nagy, I. and Semsei, I. (1984): Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats: an explanation of its action in terms of the membrane hypothesis of aging. Exp. Gerontol., 19, 171-178.
67
AGG 00097
Morphological evaluation of the lipofuscinolytic effect of acetylhomocysteine thiolactone Eugenia Aloj Totfiro ~, Francesco Aurelio Pisanti 1, A n n a m a r i a C o n t i n i l l o I a n d Enrico Liberatori 2 I Departments of Biology and General Zoology, First Faculty of Medicine and Surgery, University of Naples, Viale M. Cristina 18, 1-80122 Naples, and 2 Research Department, Roussel MaestrettL Viale Gran Sasso 18, 1-20131 Milan, Italy (Received 1 October 1984; revised version received 14 December 1984; accepted 18 December 1984)
Summary The acetylhomocysteine thiolactone, a free radical scavenger drug able to activate the superoxide dismutase, shows a lytic effect on neuronal lipofuscin. The study was performed in a nervous tissue particularly rich in lipofuscin, the electric lobe of Torpedo marmorata. It is shown that the drug induces the migration of pigment towards the blood vessels and glial cells which remove them. acetylhomocysteine thiolactone; lipofuscin; age pigments; free radicals; Torpedo marmorata
Introduction
Various authors (Feeney-Bourns et al., 1980; Donato, 1981) consider that the production of lipofuscin is a consequence of the peroxidative effect induced by free radicals on membrane lipids. This hypothesis is supported by experimental results of these authors who have used free radical scavenger substances such as centrophenoxine (Glees and Hasan, 1976) and antioxidants as vitamin E (Robison et al., 1979) to reduce the accumulation of lipofuscin. The electric lobe, an anatomotopographical district of the central nervous system of Torpedo marmorata, is particularly rich in lipofuscin (Sterzi, 1909; De Lerma and Ventra, 1956; Aloj Totaro and Pisanti, 1979). Recently, it has been observed in the neurons of this tissue (Aloj Totaro and Pisanti, 1981) that acetylhomocysteine thiolactone, or cythiolone (CYT), inhibits the formation of lipofuscin granulation. These observations were followed by experiments which have clarified the mechanism of action of this drug, which has a free radical scavenger activity (Pisanti et al., 1983; Aloj Totaro et al., 1985b) and a capacity to increase the activity of superoxide dismutase (Pisanti et al., 1985). It seemed interesting to us therefore to complete our 0167-4943/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
68 study on the action of cythiolone on age pigments by demonstrating the qualitative and quantitative modification of pigment granules of electric lobe neurons of Torpedo exposed to the drug.
Materials and Methods
20 Torpedo marmorata of both sexes of 1-2 yr of age, as judged by the comparative evaluation of various growth parameters (Aloj Totaro et al., 1985a), were kept in tanks with closed circulation and fed artificially (Aloj Totaro, 1978). Ten animals were injected daily with 8 mg/kg body weight of acetylhomocysteine thiolactone (Citiolase, Roussel-Maestretti, Milano, Italy) for 30 days, while the remaining ten were treated with a placebo. On the 31st day the animals were anaesthetised in MS 222 (Sandoz) at 0.015%o in sea water and perfused with a solution containing 291 mM NaCI, 3.52 mM Na2SO 4, 3.24 mM KCI, 2.16 mM MgC12, 1.8 mM CaC12 and 450 mM CO(NH2) 2. The perfusion liquid was then replaced with a fixative of 3.5% glutaraldehyde, 2.4% Na-cacodylate and 2% sucrose in 205 mM NaCI, 3.52 mM Na2SO4, 3.24 mM KCI, 2.16 mM MgC12, 1.8 mM CaCI 2 and 450 mM CO(NH2) 2. By dissecting the cranium we then removed the electric lobes (Sterzi, 1909) which were processed for the electron microscope according to standard techniques (Aloj Totaro and Pisanti, 1979). Ultrathin sections (Ultrotome III, LKB), stained with lead citrate and uranyl acetate, were observed with a Philips 400 electron microscope. We took 30 random photographs (6000 × ) on samples selected according to the method proposed by Weibel et al. (1969) for stereological analysis. These photographs were analyzed with a semi-automatic computerized image analyzer (IBAS I, Zeiss).
Results
Neurons from the Torpedo electric lobe have a characteristic shape and appear surrounded by a series of glial cells which, according to the hypothesis proposed by Glees and Hasan (1976) for neurons of higher vertebrates, serve to capture residual debris and to transport these towards the vessel lumen. In the normal condition the electric lobe neurons of adult Torpedo appear rich in lipofuscin granulation generally situated in the perinuclear area (Aloj Totaro and Pisanti, 1979). The pigment granules occupy 2.52 + 0.3% of cytoplasmic area with a mean surface area of 0.58 + 0.2/~m2. In contrast, in neurons of individuals treated with CYT, the granules are less numerous (1.94 + 0.6%, P < 0.01), smaller (0.50 + 0.2 #m 2, P < 0.05) and are found in the peripheral cytoplasm (Fig. 1). Also apparent in Fig. 2 are numerous formations typical of GERL, which confirms the intense phenomenon of phagocytosis and metabolic transformations which are associated with these cells. Figure 2 shows that the lipofuscin granules near the edge of the neuron are probably in the process of being c~iptured by the satellite cells. The presence of both granular and 'finger print' pigment granules indicates that CYT induced a process of lipofuscinol-
69
Fig. 1. Torpedo m. electric lobe neuron. Note GERL formations ( x 4600).
Fig. 2. Treated Torpedo m. electric lobe. Lipofuscin granules (L) have been or are in the process of being captured by a satellite cell (S) ( x 8000). Fig. 3. Treated Torpedo m. Several lipofuscin granules moving from electric lobe neuron toward the glial cell. N: nucleus of the glial cell ( x 8000).
70
Fig. 4. Treated Torpedo m. electric lobe. Lipofuscin granulation near the satellite cell (S) ( x 10000).
ysis, as it has been demonstrated for other drugs such as centrophenoxine (Spoerri and Glees, 1975). This lipofuscinolytic action of CYT is further confirmed in the sequence of Figs. 3-5. In Fig. 3 there are various pigment granules near the cytoplasmic boundary of the neuron. Some of these show a clear tendency of being transported to the nearby satellite cell. In Fig. 4 we show, at higher magnification,
Fig. 5. Treated Torpedo m. electric lobe. A typical glial cell full of lipofuscin granules ( × 6000).
71
various lipofuscin granules which have a prevalently lamellar structure grouped in proximity to a satellite cell. Figure 5 shows the conclusion of the transport of pigment granules induced by CYT demonstrating a satellite glial cell absolutely full of lipofuscin granules.
Discussion
In vitro experiments show that the generation of hydroxyl free radicals by chemical systems results in the cross-linking of bovine serum albumin (Zs.-Nagy and Nagy, 1980); CYT, when introduced in this experimental system, is able to diiminish the extent of cross-linking acting most probably as a free radical scavenger (Pisanti et al., 1983; Aloj Totaro et al., 1985b). CYT injected in vivo in normal adult rats for 30 days significantly (P < 0.001) increased the superoxide dismutase activity, one of the most important of the enzymes of protection against the damaging effects of free radicals (Pisanti et al., 1985). It has also been shown (Aloj Totaro and Pisanti, 1981), and confirmed in the present report, that CYT inhibits the production of lipofuscin granules, shown both as a reduction in the number of granules and in their morphometric parameters. This inhibitory effect of CYT on the production of lipofuscin is shown by a reduction of the cytoplasmic area occupied by the pigment. The observations in this work show, in addition, how the drug induces the migration of pigment granules toward the cell periphery in proximity to the vessels and glial cells. In fact, these latter cells, in animals treated with the drug, appear to be full of lipofuscin granules, ready to send residual material accumulated within them toward the blood vessels which transport the material away from its site of production. The capacity of neurons to free themselves of unwanted material from their cellular circuits seems to be activated by CYT which, in this sense, aids the cellular defense mechanisms. Similar observations have been presented (Spoerri and Glees, 1975) for centrophenoxine, which has a molecular structure very different to that of CYT. Both centrophenoxine and cythiolone have been shown to have free radical scavenger activity (Zs.-Nagy and Semsei, 1984; Aloj Totaro et al., 1985b). Therefore, we believe that neither of these drugs acts directly on lipofuscinolysis. By protecting the membrane components against free radicals that are normally produced during the cell metabolism, these drugs limit their action to the inhibition of production of new pigment granules. In this way the cell system that physiologically assures the removal of residual material of metabolic processes will be made more efficient. One characteristic that makes cythiolone particularly interesting for its effect on age pigments with respect to other free radical scavenger molecules as centrophenoxine and antioxidants as vitamin E is its capacity to activate the superoxide dismutase; in this way aiding its protective effect against free radicals.
References Aloj Totaro, E. (1978): Effects of centrophenoxine on the variation of some behavioural patterns in Torpedo m. Acta Neurol., 34, 332-336.
72 Aloj Totaro, E. and Pisanti, F.A. (1979): Preliminary observations at the electron microscope on the presence of neuronal lipofuscin in Torpedo m. Acta Neurol., 34, 322-331. Aloj Totaro, E. and Pisanti, F.A. (1981): Influence of SH groups in the formation of neuronal lipofuscin in Torpedo m. Acta Neurol., 36, 460-465. Aloj Totaro, E., Pisanti, F.A., Russo, P. and Brunetti, P. (1985a): Evaluation of aging parameters in Torpedo marmorata. Ann. Soc. R. Zool. Belg., in press. Aloj Totaro, E., Pisanti, F.A. and Liberatori, E. (1985b): Possible interrelations of acetyl homocysteine thiolactone in mechanisms of lipofuscinigenesis. Res. Commun. Chem. Pathol. Pharmacol., in press. De Lerma, B. and Ventra, D. (1956): Su un materiale fluorescente individuato nel citoplasma delle cellule gangliari del lobo elettrico di Torpedine. Acta Neurol., 11, 1009-1018. Donato, H. (1981): Lipid peroxidation, cross-linking and diseases. In: Age Pigments, pp. 63-82. Editor: R.S. Sohal. Elsevier/North-Holland, Amsterdam. Feeney-Bourns, L., Berman, E.R. and Rothman, M.S. (1980): Lipofuscin of human retinal pigment epithelium. Am. J. Ophthalmol., 90, 783-791. Glees, P. and Hasan, M. (1976): Lipofuscin in neuronal aging and diseases. In: Normal and Pathological Anatomy, pp. 1-68. Editors: W. Bargmann and W. Doerr. Georg Thieme, Stuttgart. Pisanti, F.A., Fascatore, S., Aloj Totaro, E. and Crisci, A. (1983): Effetti del citiolone sulla dinamica di formazione dei pigmenti di senescenza. G. Gerontol., 31,795-796. Pisanti, F.A., Fascatore, S. and Papaccio, G. (1985): Influence of acetyl homocysteine thiolactone on erythrocyte superoxide dismutase activity. Experientia, in press. Robison, W.A., Kuwabara, T. and Bieri, J.A. (1979): Vitamin E deficiency and the retina. Photoreceptor and pigment epithelial changes. Invest. Ophthalmol. Vis. Sci., 18, 683-689. Spoerri, P.E. and Glees, P. (1975): The mode of lipofuscin removal from hypothalamic neurons. Exp. Gerontol., 10, 225-228. Sterzi, G. (1909): Selacei, anatomia. In: I1 sistema nervoso centrale dei vertebrati, vol. 2, libro 1, pp. 1-415. Editor: G. Sterzi. Draghi, Padova. Weibel, E.R., Staubli, W., Gnagi, H.R. and Hess, F.A. (1969): Correlated morphometric and biochemical studies on the liver cell. J. Cell Biol., 42, 68-91. Zs.-Nagy, I. and Nagy, K. (1980): On the role of cross-linking of cellular proteins in aging. Mech. Ageing Dev., 14, 245-251. Zs.-Nagy, I. and Semsei, I. (1984): Centrophenoxine increases the rates of total and mRNA synthesis in the brain cortex of old rats: an explanation of its action in terms of the membrane hypothesis of aging. Exp. Gerontol., 19, 171-178.