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Localization of gold in the anterior pituitary gland of rats exposed to sodium aurothiomalate
EXPERIMENTAL
AND
MOLECULAR
Localization
of Anatomy
41, 74-80
(1984)
of Gold in the Anterior Pituitary Gland of Rats Exposed to Sodium Aurothiomalate
BJARNE Institut
PATHOLOGY
MIZILLER-MADSENANDOLETHORLACIUS-USSING
B and Rheumatism
Received
December
Unit.
University
of Aarhus,
14, 1983, and in revised
form
DK-8000 February
Aarhus
C, Denmark
9, 1984
The ultrastructural localization of gold in the anterior pituitary of rats injected intraperitoneally with sodium aurothiomalate has been demonstrated using a histochemical technique that visualizes minute traces of gold. Gold was located intracellularly within lysosome-like bodies of marginal layer cells, follicular cells, and thyrotrophs. Gold was not seen within secretion granules. The possible mechanism by which gold is taken up into the cells is discussed. INTRODUCTION Gold has been used in the treatment of rheumatoid arthritis since the 1920s but it was not until comparatively recently that the beneficial effect of the drug was fully documented (Empire Rheumatism Council, 1960, 1961; American Rheumatism Association, 1973). The mechanism(s) of its action and toxicity, however, remain(s) obscure. Toxic effects of gold compounds are usually observed at areas of penetration, in the organ or tissue of elimination, and at points where active cellular turnover occurs. Heavy metals are known to influence the endocrine system at several levels including hormone secretion, hormone activation, and binding to target tissue (Henkin, 1976). The aim of this study was to visualize trace amounts of gold in cells of the anterior pituitary gland of rats treated with sodium aurothiomalate (SAT). This was achieved at both light and electron microscope levels by using a histochemical technique based on physical development of trace elements (Danscher 1981a). MATERIALS AND METHODS Twelve Wistar rats of both sexes weighing 230 g each were injected intraperitoneally with 8 mg SAT (Myocrisin) four times in the course of 2 weeks. Controls were injected with 0.9% NaCl instead of SAT. Four days after the last injection, the animals were anesthetized with sodium pentobarbital and killed by transcardial perfusion at 120 mm Hg for 12 min with 3% glutaraldehyde in Verona1 buffer, pH 7.4. The anterior pituitary gland was excised, bisected, dehydrated, and embedded in Epon. Sections (3pm) were placed under UV light before silver intensification by physical development (Danscher, 1981a). The sections were counterstained with 1% toluidine blue before light microscope examination. For ultrathin sections, the sections were reembedded upon Epon blocks, cut with a diamond knife, and stained with both lead citrate and uranyl acetate before examination in the electron microscope. RESULTS Gold deposits were found selectively in the cytoplasm of marginal layer cells, follicular cells, and thyrotrophs. For a given cell type, no differences were found 74 0014-4800/84 Copyright All rights
$3.00
D 1984 by Academic Press, Inc. of reproduction in any form reserved.
GOLD IN THE ANTERIOR
.PITUITARY
GLAND
75
FIG. 1. Epon section (3 pm) showing silver-intensified gold deposits (arrows) in cells of the anterior pituitary. PD = pars distalis; PI = pars intermedia. x 880.
between cells containing gold and those not containing gold, nor were there visible differences between cells taken from the two sexes. In the light microscope, silver-amplified gold particles were especially numerous in the region between the pars intermedia and pars distalis (Fig. I). Ultrastructurally, the gold deposits were located in lysosome-like bodies within the cytoplasm (Figs. 2-5). The deposits were of different shapes and sizes and were often superimposed upon one another. Gold was not found in secretion granules or in extracellular sites. Anterior pituitary gland cells from control animals never contained silver-amplified particles. Silver-intensified gold particles were seen in marginal layer cells (Fig. 2) which were identified as nongranular cells lining a relatively narrow intercellular space between pars distalis and pars intermedia. This space either was tilled with a colloid-like substance or was empty, but never contained any gold deposits. Gold was located in lysosome-like bodies, whereas other parts of the cells, for example, junctional complexes, microvilli, or cilia, were devoid of deposits. Gold deposits were consistently found in the follicular cells (Fig. 3), which were identified as nongranular stellate cells by their large cytoplasmic processes which protuded into a follicular cavity. In secretory cells significant amounts of gold deposits were found in the lysosome-like bodies of thyrotrophs (Figs. 4, 5), which were characterized by their angular shape and their 150- to 200-nm secretory granules. DISCUSSION Discussion of the method. The process of physical development requires controlled experiments since substances other than gold can act as catalysts for silver reduction. Silver itself and the sulfides and selenides of mercury, zinc, and other
76
MOLLER-MADSEN
AND THORLACIUS-USING
FIGS. 2-5. Electron micrographs showing localization of gold in lysosome-like bodies. FIG. 2. Marginal layer cell (MLC). x 15,400. Framed area x 35,800.
heavy metals can also act as catalysts for the reaction (Timm, 1958; Danscher and Schroder, 1979; Danscher, 1981b, c). The controls in this study were devoid of silver-amplified deposits. Discussion of the results. The present study has demonstrated gold accumulations in nonsecretory and secretory cells of the anterior pituitary gland of rats
GOLD
IN
THE
ANTERIOR
FIG. 3. Follicular
PITUITARY
cell (FC).
GLAND
77
x 17,300.
exposed to sodium aurothiomalate, particularly in the region between the pars intermedia and pars distalis where marginal layer cells are located. Gold was found only intracellularly and was always located in lysosome-like bodies identical to the aurosomes defined by Oryschack and Ghadially (1974). Identical accumulations of gold have been described in other tissues and cells such as placenta and mouse peritoneal macrophages (Moller-Madsen and Danscher, 1983; Moller-Madsen et al., 1984). Gold deposits within follicular, nongranulated, or nonsecretory cells support the results published by Yamashita (1969), Dingemans and Feltkamp (1972), Fukuda (1973), Farquhar er al. (1975), and Liwska (1978). They propose that the follicular cells constitute a scavenger system which is responsible for the disposal of cells and cell debris. The mechanism by which the follicular cells take up the gold salt has not been resolved in this study. However, the localization in lysosome-like bodies might indicate that gold is taken up by either phagocytosis or pinocytosis, but a transport through the cellular membrane by amphoteric molecules cannot be excluded. Gold accumulation in marginal layer cells may be explained by their intimate relationship with follicular cells. Both cell types are thought to have the same developmental origin, that is, from the anterior wall of Rathke’s pouch (Yoshimura et al., 1977; Fremont and Ferrand, 1980). The mechanism by which gold enters the lysosome-like bodies of the secretory cells remains unknown. Secretory cells of the anterior pituitary have not been shown to be capable of phagocytosis. However, Farquhar (1969, 1971) has described a process termed crinophagia whereby secretion granules are taken up and disposed of by lysosomes. In our study, gold particles were found exclusively in lysosome-like bodies. It is possible, however, that the gold particles may have
78
M@LLER-MADSEN
FIG. 4. Thyrotroph
AND THORLACIUS-USSING
(T). X 15,000. Framed area x90.000. Corticotroph
(C) is devoid of gold.
been initially located in secretion granules, primarily bound to their contents, and then secondarily transferred by crinophagia to the lysosome-like structures where they have been visualized. Another possibility is an uptake through amphoteric molecules. With our present knowledge it is difficult to specify the interrelationships be-
GOLD IN THE ANTERIOR
FIG. 5. Thyrotroph
PITUITARY
GLAND
79
(T). x 14,500. Framed area x 31,000.
tween a trace meal and the function of the endocrine system. However, the accumulation of gold in lysosome-like bodies in the secretory cells of the anterior pituitary gland, as described here, indicates that gold may influence the incorporation and destruction of excess granules and thereby play a role in the regulation of the secretory process. This hypothesis is supported by the findings of gold deposits in ovary and adrenal gland (Danscher, 1981a). ACKNOWLEDGMENTS The authors gratefully acknowledge the inspiration and help given by Dr. G. Danscher and the skillful technical assistance of Mrs. Eva Ann-Louise Petersen, Mrs. Karin Wiedemann, Mr. Albert Meier, and Mr. Thorkild A. Nielsen. This study was supported by the Danish Rheumatism Association Grant 233-458.
REFERENCES American Rheumatism Association (The Cooperating Clinics Committee) cilc (1973). A controlled trial of gold salt therapy in rheumatoid arthritis. Arthrifis Rheum. 16, 353-358. DANSCHER, G. (1981a). Localization of gold in biological tissue. A photochemical method for light 71, 81-88. and electron microscopy. Histochemistry DANSCHER, G. (1981b). Light and electron microscopic localization of silver in biological tissue. Histochemistry 71, 177-186. DANSCHER, G. (1981~). Histochemical demonstration of heavy metals. A revised version of sulphide 71, I-16. silver method suitable for both light and electron microscopy. Histochemistry DANSCHER, G., and SCHRODER,H. (1979). Histochemical demonstration of mercury induced changes in rat neurons. Histochemistry 60, 1-7. DINGEMANS, K. P., and FELTKAMP. C. A. (1972). Nongranulated cells in the mouse adenohypophysis. Z. Zellforsch.
124, 387-405.
EMPIRE RHEUMATISM COUNCIL (RESEARCH SUBCOMMITTEE) (1960). thritis: Report of a multicentre controlled trial. Ann. Rheum. Dis. EMPIRE RHEUMATISM COUNCIL (RESEARCH SUBCOMMITTEE) (1961). thritis: Final report of a multicentre controlled trial. Ann. Rheum.
Gold therapy in rheumatoid ar19, 95-l 19. Gold therapy in rheumatoid arDis. 20, 315-334.
80
M@LLER-MADSEN
AND THORLACIUS-USSING
M. G. (1969). Lysosome function in regulating secretion: Disposal of secretory granules in cells of the anterior pituitary gland. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 462-482, North-Holland, Amsterdam. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Mem. Sot. Endocrinol. 19, 79- 122. FARQUHAR, M. Cl., SHUTALSKY, E. H., and HOPKINS, C. R. (1975). Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In “The Anterior Pituitary” (A. Tixier-Vidal and M. G. Farquhar, eds.), pp. 83-135. Academic Press, New York. FREMONT, P. H., and FERRAND, R. (1980). The differentiation of follicular-like cells from the epithelium of Rathke’s pouch grown in vitro. Annt. Embryo/. 160, 275-284. FUKUDA, T. (1973). Agranular stellate cells (so-called follicular cells) in human fetal and adult adenohypophysis and in pituitary adenoma. Virchows Arch. Abt. A 359, 19-30. HENKIN, R. I. (1976). Trace metals in endocrinology. In “Medical Clinics, North America,” Vol. 60, No. 4, pp. 779-797. LIWSKA, J. (1978). Investigation of ultrastructure of the adenohypophysis in the domestic pig (Sus serota domestica). Part II: “Dark cells” in the pars anterior. Folk Hisrochem. Cytochem. 16, 315-322. MILLER-MADSEN, B., and DANSCHER, G. (1983). Transplacental transport of gold in rats exposedto sodium aurothiomalate. Exp. Mol. Pathol. 39, 327-33 1. MILLER-MADSEN, B., MOGENSEN, S. C., and DANSCHER, G. (1984). Ultrastructural localization of gold in macrophages and mast cells exposed to aurothio-glucose. Exp. Mol. Pathol. 40, 148-154. ORYSCHAK, A. F., and GHADIALLY, E N. (1974). Aurosomes in rabbit articular cartilage. Virchows Arch. B. 17, 159-168. TIMM, F. (1958). Zur Histochemie der Schwermetalle das Sulfid-Silber-Verfahren. Dtsch. Z. ges. ger. Med. 46, 706-711. YAMASHITA, K. (1969). Electron microscopic observations on the postnatal development of the anterior pituitary of the mouse. In “Gunma Symposia on Endocrinology,” Vol. 6, pp. 177-194. YOSHIMURA, F., Son, T., and KIGUCHI, Y. (1977). Relationship between the follicular cells and marginal layer cells of the anterior pituitary. Endocrinol. Japan 24, 301-305. FARQUHAR,
AND
MOLECULAR
Localization
of Anatomy
41, 74-80
(1984)
of Gold in the Anterior Pituitary Gland of Rats Exposed to Sodium Aurothiomalate
BJARNE Institut
PATHOLOGY
MIZILLER-MADSENANDOLETHORLACIUS-USSING
B and Rheumatism
Received
December
Unit.
University
of Aarhus,
14, 1983, and in revised
form
DK-8000 February
Aarhus
C, Denmark
9, 1984
The ultrastructural localization of gold in the anterior pituitary of rats injected intraperitoneally with sodium aurothiomalate has been demonstrated using a histochemical technique that visualizes minute traces of gold. Gold was located intracellularly within lysosome-like bodies of marginal layer cells, follicular cells, and thyrotrophs. Gold was not seen within secretion granules. The possible mechanism by which gold is taken up into the cells is discussed. INTRODUCTION Gold has been used in the treatment of rheumatoid arthritis since the 1920s but it was not until comparatively recently that the beneficial effect of the drug was fully documented (Empire Rheumatism Council, 1960, 1961; American Rheumatism Association, 1973). The mechanism(s) of its action and toxicity, however, remain(s) obscure. Toxic effects of gold compounds are usually observed at areas of penetration, in the organ or tissue of elimination, and at points where active cellular turnover occurs. Heavy metals are known to influence the endocrine system at several levels including hormone secretion, hormone activation, and binding to target tissue (Henkin, 1976). The aim of this study was to visualize trace amounts of gold in cells of the anterior pituitary gland of rats treated with sodium aurothiomalate (SAT). This was achieved at both light and electron microscope levels by using a histochemical technique based on physical development of trace elements (Danscher 1981a). MATERIALS AND METHODS Twelve Wistar rats of both sexes weighing 230 g each were injected intraperitoneally with 8 mg SAT (Myocrisin) four times in the course of 2 weeks. Controls were injected with 0.9% NaCl instead of SAT. Four days after the last injection, the animals were anesthetized with sodium pentobarbital and killed by transcardial perfusion at 120 mm Hg for 12 min with 3% glutaraldehyde in Verona1 buffer, pH 7.4. The anterior pituitary gland was excised, bisected, dehydrated, and embedded in Epon. Sections (3pm) were placed under UV light before silver intensification by physical development (Danscher, 1981a). The sections were counterstained with 1% toluidine blue before light microscope examination. For ultrathin sections, the sections were reembedded upon Epon blocks, cut with a diamond knife, and stained with both lead citrate and uranyl acetate before examination in the electron microscope. RESULTS Gold deposits were found selectively in the cytoplasm of marginal layer cells, follicular cells, and thyrotrophs. For a given cell type, no differences were found 74 0014-4800/84 Copyright All rights
$3.00
D 1984 by Academic Press, Inc. of reproduction in any form reserved.
GOLD IN THE ANTERIOR
.PITUITARY
GLAND
75
FIG. 1. Epon section (3 pm) showing silver-intensified gold deposits (arrows) in cells of the anterior pituitary. PD = pars distalis; PI = pars intermedia. x 880.
between cells containing gold and those not containing gold, nor were there visible differences between cells taken from the two sexes. In the light microscope, silver-amplified gold particles were especially numerous in the region between the pars intermedia and pars distalis (Fig. I). Ultrastructurally, the gold deposits were located in lysosome-like bodies within the cytoplasm (Figs. 2-5). The deposits were of different shapes and sizes and were often superimposed upon one another. Gold was not found in secretion granules or in extracellular sites. Anterior pituitary gland cells from control animals never contained silver-amplified particles. Silver-intensified gold particles were seen in marginal layer cells (Fig. 2) which were identified as nongranular cells lining a relatively narrow intercellular space between pars distalis and pars intermedia. This space either was tilled with a colloid-like substance or was empty, but never contained any gold deposits. Gold was located in lysosome-like bodies, whereas other parts of the cells, for example, junctional complexes, microvilli, or cilia, were devoid of deposits. Gold deposits were consistently found in the follicular cells (Fig. 3), which were identified as nongranular stellate cells by their large cytoplasmic processes which protuded into a follicular cavity. In secretory cells significant amounts of gold deposits were found in the lysosome-like bodies of thyrotrophs (Figs. 4, 5), which were characterized by their angular shape and their 150- to 200-nm secretory granules. DISCUSSION Discussion of the method. The process of physical development requires controlled experiments since substances other than gold can act as catalysts for silver reduction. Silver itself and the sulfides and selenides of mercury, zinc, and other
76
MOLLER-MADSEN
AND THORLACIUS-USING
FIGS. 2-5. Electron micrographs showing localization of gold in lysosome-like bodies. FIG. 2. Marginal layer cell (MLC). x 15,400. Framed area x 35,800.
heavy metals can also act as catalysts for the reaction (Timm, 1958; Danscher and Schroder, 1979; Danscher, 1981b, c). The controls in this study were devoid of silver-amplified deposits. Discussion of the results. The present study has demonstrated gold accumulations in nonsecretory and secretory cells of the anterior pituitary gland of rats
GOLD
IN
THE
ANTERIOR
FIG. 3. Follicular
PITUITARY
cell (FC).
GLAND
77
x 17,300.
exposed to sodium aurothiomalate, particularly in the region between the pars intermedia and pars distalis where marginal layer cells are located. Gold was found only intracellularly and was always located in lysosome-like bodies identical to the aurosomes defined by Oryschack and Ghadially (1974). Identical accumulations of gold have been described in other tissues and cells such as placenta and mouse peritoneal macrophages (Moller-Madsen and Danscher, 1983; Moller-Madsen et al., 1984). Gold deposits within follicular, nongranulated, or nonsecretory cells support the results published by Yamashita (1969), Dingemans and Feltkamp (1972), Fukuda (1973), Farquhar er al. (1975), and Liwska (1978). They propose that the follicular cells constitute a scavenger system which is responsible for the disposal of cells and cell debris. The mechanism by which the follicular cells take up the gold salt has not been resolved in this study. However, the localization in lysosome-like bodies might indicate that gold is taken up by either phagocytosis or pinocytosis, but a transport through the cellular membrane by amphoteric molecules cannot be excluded. Gold accumulation in marginal layer cells may be explained by their intimate relationship with follicular cells. Both cell types are thought to have the same developmental origin, that is, from the anterior wall of Rathke’s pouch (Yoshimura et al., 1977; Fremont and Ferrand, 1980). The mechanism by which gold enters the lysosome-like bodies of the secretory cells remains unknown. Secretory cells of the anterior pituitary have not been shown to be capable of phagocytosis. However, Farquhar (1969, 1971) has described a process termed crinophagia whereby secretion granules are taken up and disposed of by lysosomes. In our study, gold particles were found exclusively in lysosome-like bodies. It is possible, however, that the gold particles may have
78
M@LLER-MADSEN
FIG. 4. Thyrotroph
AND THORLACIUS-USSING
(T). X 15,000. Framed area x90.000. Corticotroph
(C) is devoid of gold.
been initially located in secretion granules, primarily bound to their contents, and then secondarily transferred by crinophagia to the lysosome-like structures where they have been visualized. Another possibility is an uptake through amphoteric molecules. With our present knowledge it is difficult to specify the interrelationships be-
GOLD IN THE ANTERIOR
FIG. 5. Thyrotroph
PITUITARY
GLAND
79
(T). x 14,500. Framed area x 31,000.
tween a trace meal and the function of the endocrine system. However, the accumulation of gold in lysosome-like bodies in the secretory cells of the anterior pituitary gland, as described here, indicates that gold may influence the incorporation and destruction of excess granules and thereby play a role in the regulation of the secretory process. This hypothesis is supported by the findings of gold deposits in ovary and adrenal gland (Danscher, 1981a). ACKNOWLEDGMENTS The authors gratefully acknowledge the inspiration and help given by Dr. G. Danscher and the skillful technical assistance of Mrs. Eva Ann-Louise Petersen, Mrs. Karin Wiedemann, Mr. Albert Meier, and Mr. Thorkild A. Nielsen. This study was supported by the Danish Rheumatism Association Grant 233-458.
REFERENCES American Rheumatism Association (The Cooperating Clinics Committee) cilc (1973). A controlled trial of gold salt therapy in rheumatoid arthritis. Arthrifis Rheum. 16, 353-358. DANSCHER, G. (1981a). Localization of gold in biological tissue. A photochemical method for light 71, 81-88. and electron microscopy. Histochemistry DANSCHER, G. (1981b). Light and electron microscopic localization of silver in biological tissue. Histochemistry 71, 177-186. DANSCHER, G. (1981~). Histochemical demonstration of heavy metals. A revised version of sulphide 71, I-16. silver method suitable for both light and electron microscopy. Histochemistry DANSCHER, G., and SCHRODER,H. (1979). Histochemical demonstration of mercury induced changes in rat neurons. Histochemistry 60, 1-7. DINGEMANS, K. P., and FELTKAMP. C. A. (1972). Nongranulated cells in the mouse adenohypophysis. Z. Zellforsch.
124, 387-405.
EMPIRE RHEUMATISM COUNCIL (RESEARCH SUBCOMMITTEE) (1960). thritis: Report of a multicentre controlled trial. Ann. Rheum. Dis. EMPIRE RHEUMATISM COUNCIL (RESEARCH SUBCOMMITTEE) (1961). thritis: Final report of a multicentre controlled trial. Ann. Rheum.
Gold therapy in rheumatoid ar19, 95-l 19. Gold therapy in rheumatoid arDis. 20, 315-334.
80
M@LLER-MADSEN
AND THORLACIUS-USSING
M. G. (1969). Lysosome function in regulating secretion: Disposal of secretory granules in cells of the anterior pituitary gland. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 462-482, North-Holland, Amsterdam. FARQUHAR, M. G. (1971). Processing of secretory products by cells of the anterior pituitary gland. Mem. Sot. Endocrinol. 19, 79- 122. FARQUHAR, M. Cl., SHUTALSKY, E. H., and HOPKINS, C. R. (1975). Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In “The Anterior Pituitary” (A. Tixier-Vidal and M. G. Farquhar, eds.), pp. 83-135. Academic Press, New York. FREMONT, P. H., and FERRAND, R. (1980). The differentiation of follicular-like cells from the epithelium of Rathke’s pouch grown in vitro. Annt. Embryo/. 160, 275-284. FUKUDA, T. (1973). Agranular stellate cells (so-called follicular cells) in human fetal and adult adenohypophysis and in pituitary adenoma. Virchows Arch. Abt. A 359, 19-30. HENKIN, R. I. (1976). Trace metals in endocrinology. In “Medical Clinics, North America,” Vol. 60, No. 4, pp. 779-797. LIWSKA, J. (1978). Investigation of ultrastructure of the adenohypophysis in the domestic pig (Sus serota domestica). Part II: “Dark cells” in the pars anterior. Folk Hisrochem. Cytochem. 16, 315-322. MILLER-MADSEN, B., and DANSCHER, G. (1983). Transplacental transport of gold in rats exposedto sodium aurothiomalate. Exp. Mol. Pathol. 39, 327-33 1. MILLER-MADSEN, B., MOGENSEN, S. C., and DANSCHER, G. (1984). Ultrastructural localization of gold in macrophages and mast cells exposed to aurothio-glucose. Exp. Mol. Pathol. 40, 148-154. ORYSCHAK, A. F., and GHADIALLY, E N. (1974). Aurosomes in rabbit articular cartilage. Virchows Arch. B. 17, 159-168. TIMM, F. (1958). Zur Histochemie der Schwermetalle das Sulfid-Silber-Verfahren. Dtsch. Z. ges. ger. Med. 46, 706-711. YAMASHITA, K. (1969). Electron microscopic observations on the postnatal development of the anterior pituitary of the mouse. In “Gunma Symposia on Endocrinology,” Vol. 6, pp. 177-194. YOSHIMURA, F., Son, T., and KIGUCHI, Y. (1977). Relationship between the follicular cells and marginal layer cells of the anterior pituitary. Endocrinol. Japan 24, 301-305. FARQUHAR,