- Email: [email protected]
Dithizone-induced changes in the rat lateral prostate
EXPERIMENTAL
AND
MOLECULAR
Dithizone-Induced
Changes G.
Department
of Pathology,
2, SO3-514
PATHOLOGY
University
in the
RANDOLPH
Rat
Lateral
Prostate1
SCHRODT
of Louisville
Received
(1963)
November
School
of Medicine,
Louisville,
Kentucky
1, 1962
Biochemical and fine structural differences have been demonstrated among the lobes of the rat prostate complex (Brandes et al., 1961; Gunn et al., 1956, 1957; Miller et al., 1961; Schrodt, 1961). A most interesting difference is the high level of zinc in the lateral lobe as compared with the others (Dixon et al., 1951; Fisher et al., 1955; Gunn et al., 1956; Wetterdal, 1958). When dithizone (diphenylthiocarbazone) , a metal chelating agent, is administered to rats, there is extensive damage of the lateral lobe epithelium and only slight change in the dorsal lobe. No changeshave been noted in the epithelia of the other lobes. After a single injection of dithizone there is necrosis of most of the epithelial cells. Several weeks later the gland has returned to an almost normal appearance. These sequential changes in the lateral lobe epithelium have been described by Logothetopoulos, using light and dark-field microscopy. In order to understand more fully the morphologic changeswhich occur following dithizone, the lateral lobe epithelium of the rat prostate has been studied with the electron microscope. MATERIALS
AND METHODS
Male CFN rats (Carworth Farms, New City, New York), weighing between 280 and 320 gm, were used. These animals were fed a routine diet and given water ad Zibitum. They were segregatedfrom females. Each rat was given a single dose of dithizone intravenously, approximately 50 mg per kilogram body weight. The dithizone was prepared according to the method of Philips et al. Five hundred mg of dithizone is dissolved by the addition of 7.5 ml of 1.00 M NaOH. To this solution 27.75 ml of 0.2 N HCl are added with constant stirring. The mixture is transferred to a volumetric flask, brought to 50 ml with water, and filtered. Zinc-free water is used throughout. The rats were sacrificed in pairs after 20 minutes, 1, 3, 6, 9, and 24 hours, and after 2, 7, 14, 20, 48, and 110 days. Single rats were sacrificed after 35 minutes, 45 minutes, 2% hours, and 3 days. Under ether anesthesia, the tips of the lateral lobes were excised and placed immediately in cold 1% sucrose osmium. One millimeter pieces were minced with razor blades and placed in fresh fixative for 1 hour at 4°C. Following alcohol dehydration, the tissue was embedded in 8: 1 butyl-methyl methacrylate using 1% benzoyl peroxide as catalyst, or in Epon according to the method of Luft, using 3 parts mixture A to one part mixture B (Luft, 1961). 1 Supportedby NIH Grant No. C-5676; presented of the Prostate” held at the Airlie House, Warranton, so3
in part at the “Workshop Va., October l-3, 1962.
on the
Biology
504
G. RANDOLPH
SCHRODT
Methacrylate was polymerized under ultraviolet light. Sections were cut with glass knives on a Porter-Blum microtome and placed on carbon-supported collodion-covered grids. The sections were stained in lead hydroxide. Methacrylate sections were sandwiched with another layer of carbon. A Siemens Elmiskop I was used. RESULTS The epithelium of the lateral lobe of the rat prostate is simple columnar and arranged in acini. The cells are characterized by abundant microvilli, a prominent Golgi zone containing electron-dense granules in vacuoles, and flattened cisternae of the endoplasmic reticulum. The earliest changes in the lateral lobe epithelium following dithizone were focal. After 20 minutes a thin layer of electron-dense material covered the microvilli (Fig. 1). At 1 hour there was a loss of microvilli from some epithelial cells with disruption of the plasma membrane. The secretory granules in the Golgi zone were decreased in number. Further changes were noted at 2% hours. In addition to the deposit of electrondense material on the apical surface of the epithelial cells, there was beginning separation of the cells from the basement membrane. Some clumping of nuclear chromatin was apparent. The Golgi zone vesicles were dilated, and the dense granules of this zone were smaller and fewer. Pronounced changes were presented in the lateral prostate 3 hours after dithizone. The apical cytoplasm of some epithelial cells had formed blebs. These blebs of cytoplasm contained scattered granules and a few large membrane-lined vacuoles. Some of the vacuoles appeared to represent dilated cisternae of rough endoplasmic reticulum. There was focal smudging of the plasma membranes, with some disruption of the membranes (Fig. 2). Similar relatively organelle-free areas were present in the basilar cytoplasm. Some cells had separated from the underlying stroma, with deposition of electron-dense material at the sites of separation. This dense material was amorphous or else appeared to be composed of clumps of disrupted membranes (Fig. 3). The nuclear material showed condensation, especially at the nuclear membrane. The mitochondria were intact in general, though there was some loss of the usual orientation of the cristae and increased density of the mitochondrial matrix. The underlying stroma was unaltered. The basement membrane could not always be distinguished from the electron-dense layer deposited at the site of epithelial cell separation. Where seen, it appeared unaltered or occasionally discontinuous. Despite these marked changes, some lateral lobe epithelial cells appeared normal (Fig. 4). At 6 hours most of the epithelial cells had separated from the basement membrane and lay free in the lumina of the acini either singly or in sheets. Cell membranes were lost from many cells. Mitochondria were still recognizable, but appeared altered. Nuclear material was arranged in distinct clumps (Fig. 5). Neutrophils were present in the stroma and lumina at 9 hours. There was further disruption of the epithelial cells, so that by 1 day, only cell debris (membranes and vacuoles) were seen in some areas. Three days after the dithizone injection regeneration of lateral lobe epithelium was quite evident. Some of the acini were lined by a single layer of epithelium;
DITHIZONE
EFFECT
ON
RAT
PROSTATE
505
FIG. 1. Micrograph showing the supranuclear portion of an epithelial cell of the rat lateral prostate. The animal was sacrificed 20 minutes after an injection of di’thizone. A layer of electrondense material covers the microvilli (mv). The mitochondria (m) and endoplasmic reticulum (er) appear unchanged. A large secretory granule (g) is present in the apical cytoplasm. X 44,000.
506
G. RANDOLPH
SCHRODT
FIG. 2. Lateral prostate epithelium 3 hours following a single injection of dithizone. The apical cytoplasm of one cell has formed blebs (b), some of which lie free in the lumen of the acinus (L). The arrows indicate the disruption and the smudgy appearance of the plasma membranes. X 31,000.
DITHIZONE
EFFECT
ON
RAT
PROSTATE
507
FIG. 3. The basilar cytoplasm of the epithelium 3 hours after dithizone. The cytoplasm is light A few dilated cisternae of the endoplasmic reticulum (er) are seen. The basement amd granular. nembrane (BM), reticulum fibrils of the stroma, and a stromal cell (S) are intact. X 58,000.
508
FIG. 4. dithizone. membrane
G. RANDOLPH
SCHRODT
Photomicrograph showing two epithelial cells of the lateral prostate 3 hours following Cell A is unaltered; cell B shows loss of microvilli and some disruption of its plasma (arrow). X 35,000.
DITHIZONE
EFFECT
ON
RAT
PROSTATE
509
FIG. 5. Necrotic cells 6 hours after dithizone. The cells are separated from the basement membrane (BM) and are breaking apart. Nuclear material (N) shows marked clumping. At the right lower portion of the photomicrograph is an intact capillary (c) containing an erythrocyte. x 21,000.
510
FIG. 6. The :mv), a small :ellular debris.
G. RANDOLPH
SCHRODT
apical portion of two epithelial cells 1 week Golgi zone (G), and mitochondria (m) are x 35,000.
following dithizone. noted. The lumen
(L)
Short microvilli still contains
DITHIZONE
EFFECT
ON
RAT
511
PROSTATE
FIG. 7. Epithelial cells of the lateral prostate 3 weeks following dithizone. “normal.” Nuclei (N), mitochondria Cm), endoplasmic reticulum (er) , basement ar.d a etromal cell (s) are seen. X 20,000.
The cells membrane
appear (bm),
512
G. RANDOLPH
SCHRODT
others were lined by epithelium two or three cells thick. The lumina were still filled with necrotic cells. Many of the nuclei contained one or several large nucleoli. At 1 week most acini were lined by a single layer of epithelium. The cells, however, often appeared immature. The microvilli were fewer in number and shorter than in the normal adult epithelium. The Golgi zones were distinct but small. Cisternae of the endoplasmic reticulum were inconspicuous (Fig. 6). The lateral lobe epithelium at 3, 7, and 16 weeks appeared identical to that of control animals (Fig. 7). DISCUSSION The zinc level of the rat lateral prostate is 2.3 mg per gram dry weight. Radioactive zinc-65 studies and histochemical studies suggest that most of it is concentrated in the apical and basilar portions of the epithelial cytoplasm. Although some of the zinc is bound to enzymes, the exact location and chemical combination of most of it is unknown. The pronounced structural changes in the prostate following dithizone administration have been studied by a few investigators at the light microscopic level. Logothelopoulos (1961), in a study of the rat lateral prostate, showed that zinc dithizonate granules were located in the apical and basilar portions of the cytoplasm. Mackenzie et al. (1962), in their study of the dog prostate, demonstrated that zinc was lost from the prostate following dithizone administration, Our studies show that the earliest and most pronounced changes in the rat lateral prostate occur in the microvilli, Golgi zone, and cisternae of the endoplasmic reticulum. These organelles are prominent in the apical and/or basilar areas of the epithelial cells. Despite these findings, which suggest that zinc is chelated with dithizone in &JO and that the most pronounced structural changes occur in loci of high zinc concentration, it does not necessarily follow that chelation of zinc produces the epithelial necrosis. One objection is that dithizone chelates metals other than zinc. Inactivation of other metallo-enzymes containing copper, manganese, or iron could produce the structural changes. An enlightening discussion of this problem has been given by Phillips ( 196 I ) . If we make the assumption that chelation of zinc by dithizone does produce a lesion of the rat lateral prostate, two intermediate and simultaneous steps have occurred: (1) Zinc has been removed from certain zinc-containing compounds; (2) zinc dithizonate has been produced. Either or both could produce cell damage. In support of the first is the findings of Philips et al. that metal chelators other than dithizone produce prostatic lesions. (It should be mentioned, however, that Logothelopoulos found no lesions in the rat prostate following administration of o-aminobenzoic acid, 8-hydroxyquinoline, and 2-quinoline-carboxylic acid.) In support of the second point is the work of Mackenzie, who found dithizoneinduced lesions in the dog prostate only when the zinc level was above 0.74 mg per gram dry weight. This finding suggests that at a certain level zinc dithizonate is cytotoxic. Whatever the exact mechanism of the dithizone-induced lesion of the rat lateral
DITHIZONE
EFFECT
ON
RAT
513
PROSTATE
prostate, our findings show early change in two organelles where one might expect high levels of zinc, viz., the Golgi zone and the endoplasmic reticulum. This is highly probable since the prostatic secretion is zinc rich, and these two organelles are regarded as important in the synthesis and release of secretory products. The alterations in the cell membranes, including the microvilli, are more difficult to interpret. There is experimental evidence suggesting that some divalent ions are important in maintaining cell membrane integrity. Coman (1959) described changes in the plasma membrane of liver parenchymal cells following perfusion with Versene, and attributed the changes to a loss of calcium and magnesium. Leeson and Kalant ( 1961)) in their study of the effect of Versene on rat liver, also suggested that calcium, magnesium, and other divalent ions may be important in cell integrity. To our knowledge there is no report on the presence or absence of zinc in the cell membrane. We think it entirely possible that this divalent ion is important in maintaining the cell membrane integrity of rat lateral prostate epithelium. To follow up this hypothesis, we are planning to study the prostate epithelium by means of electron microscopic radioautography using radioactive zinc SUMMARY The destructive changes in the epithelium of the rat lateral prostate following dithizone have been studied with the electron microscope. Focal changes are seen within the first hour and marked changes are noted within 3 hours. The possible mechanisms of these changes are briefly discussed. It is suggested that the Golgi granules, endoplasmic reticulum, and plasma membranes, especially the microvilli, may be zinc-rich organelles. ACKNOWLEDGMENT The author
wishes
to thank
Mr.
Charles
Foreman
for his excellent
technical
assistance.
REFERENCES D., and GROTK, D. P. (1961). The fine structure of the rat prostatic complex. Exptl. Res. 23. 159-175. COMAN, DALE REX (1959). Cellular adhesiveness in relation to the invasiveness of cancer. Cancer Res. 14, 519-521. DIXON, R. H., and WHITFIELD, J. F. (1959). The histochemical localization of zinc in the dorsolateral prostate of the rat. J. Hislochenz. Cytochem. 7, 262-266. FISHER, N. I., TIKKAM, A. D., and MAWSON, C. A. (1955). Zinc, carbonic anhydrase, and phosphatase in the prostatic glands of the rat. Can. J. Biockem. Physiol. 33, 181-190. GUNN, SAMUCL A., and GOULD, THELMA CLARK (1956). Difference between dorsal and lateral components of dorsolateral prosta’te of rats in Znc” uptake. Proc. Sm. Erptl. Biol. Med. 92, 17-20. GUNN, SAMUEL A., and GOULD, THELMA CLARK (1957). A correlative anatomical and functional study of the dorsolateral prostate of the rat. dnat. Record 128, 41-53. LEESON, T. S., and KALANT, H. (1961). Effects of in vivo decalcification on ultrastructure of adult rat liver. J. Biophys. Biochem. Cytol. 10, 93-104. LOGOTHETOPOULOS, JOHN (1961). Intravital chelation of zinc in the prostate of the rat. (Histochemical and Histologic Observations.) Am. J. Pathol. 37, 357-375. LUFT, JOHN H. (1961). Improvements in epoxy resin embedding methods. J. Biopkys. Biochem. Cytol. 9, 409-414. MACKENZIE, A. RANALD, HALL, THEODORE, and WHITMORE, WILLET F., JR. (1962). The relation between the zinc content of the canine prostate and the histiologic effects of dithizone. J. Ural. 37, 923-929. MILLAR, M. J., VINCENT, N. R., and MAWSON, C. A. (1961). An autoradiographic study of the distribution of Zincas rn . rat tissues. J. Histockem. Cytockem. 9, 111.116. BRANDES,
Cell
514 PHILIPS,
G. RANDOLPH FREDERICK
S., ASKARA,
communication. PHILIPS, FREDERICK S. diphenylthiocarbazone.
AUGUSTA
SCHRODT
B., SCHWARTZ,
HERBERT
S., and
STERNBERG,
STEPHEN
S.
Personal
(1961). Relations between zinc content and the selective cytoxicity of Federalion Proc. 20, (3, Part II) 129.131. SCHRODT, G. KAND~LPH (1961). The fine structure of the lateral lobe of the rat prostate gland. J. Ultrastruct. Res. 5, 485-496. WETTF,RDAL, B. (1958). Experimental studies on radioactive zinc in the male reproductive organs of the rat. dcta Radiol. Suppl. 156, l-83.
AND
MOLECULAR
Dithizone-Induced
Changes G.
Department
of Pathology,
2, SO3-514
PATHOLOGY
University
in the
RANDOLPH
Rat
Lateral
Prostate1
SCHRODT
of Louisville
Received
(1963)
November
School
of Medicine,
Louisville,
Kentucky
1, 1962
Biochemical and fine structural differences have been demonstrated among the lobes of the rat prostate complex (Brandes et al., 1961; Gunn et al., 1956, 1957; Miller et al., 1961; Schrodt, 1961). A most interesting difference is the high level of zinc in the lateral lobe as compared with the others (Dixon et al., 1951; Fisher et al., 1955; Gunn et al., 1956; Wetterdal, 1958). When dithizone (diphenylthiocarbazone) , a metal chelating agent, is administered to rats, there is extensive damage of the lateral lobe epithelium and only slight change in the dorsal lobe. No changeshave been noted in the epithelia of the other lobes. After a single injection of dithizone there is necrosis of most of the epithelial cells. Several weeks later the gland has returned to an almost normal appearance. These sequential changes in the lateral lobe epithelium have been described by Logothetopoulos, using light and dark-field microscopy. In order to understand more fully the morphologic changeswhich occur following dithizone, the lateral lobe epithelium of the rat prostate has been studied with the electron microscope. MATERIALS
AND METHODS
Male CFN rats (Carworth Farms, New City, New York), weighing between 280 and 320 gm, were used. These animals were fed a routine diet and given water ad Zibitum. They were segregatedfrom females. Each rat was given a single dose of dithizone intravenously, approximately 50 mg per kilogram body weight. The dithizone was prepared according to the method of Philips et al. Five hundred mg of dithizone is dissolved by the addition of 7.5 ml of 1.00 M NaOH. To this solution 27.75 ml of 0.2 N HCl are added with constant stirring. The mixture is transferred to a volumetric flask, brought to 50 ml with water, and filtered. Zinc-free water is used throughout. The rats were sacrificed in pairs after 20 minutes, 1, 3, 6, 9, and 24 hours, and after 2, 7, 14, 20, 48, and 110 days. Single rats were sacrificed after 35 minutes, 45 minutes, 2% hours, and 3 days. Under ether anesthesia, the tips of the lateral lobes were excised and placed immediately in cold 1% sucrose osmium. One millimeter pieces were minced with razor blades and placed in fresh fixative for 1 hour at 4°C. Following alcohol dehydration, the tissue was embedded in 8: 1 butyl-methyl methacrylate using 1% benzoyl peroxide as catalyst, or in Epon according to the method of Luft, using 3 parts mixture A to one part mixture B (Luft, 1961). 1 Supportedby NIH Grant No. C-5676; presented of the Prostate” held at the Airlie House, Warranton, so3
in part at the “Workshop Va., October l-3, 1962.
on the
Biology
504
G. RANDOLPH
SCHRODT
Methacrylate was polymerized under ultraviolet light. Sections were cut with glass knives on a Porter-Blum microtome and placed on carbon-supported collodion-covered grids. The sections were stained in lead hydroxide. Methacrylate sections were sandwiched with another layer of carbon. A Siemens Elmiskop I was used. RESULTS The epithelium of the lateral lobe of the rat prostate is simple columnar and arranged in acini. The cells are characterized by abundant microvilli, a prominent Golgi zone containing electron-dense granules in vacuoles, and flattened cisternae of the endoplasmic reticulum. The earliest changes in the lateral lobe epithelium following dithizone were focal. After 20 minutes a thin layer of electron-dense material covered the microvilli (Fig. 1). At 1 hour there was a loss of microvilli from some epithelial cells with disruption of the plasma membrane. The secretory granules in the Golgi zone were decreased in number. Further changes were noted at 2% hours. In addition to the deposit of electrondense material on the apical surface of the epithelial cells, there was beginning separation of the cells from the basement membrane. Some clumping of nuclear chromatin was apparent. The Golgi zone vesicles were dilated, and the dense granules of this zone were smaller and fewer. Pronounced changes were presented in the lateral prostate 3 hours after dithizone. The apical cytoplasm of some epithelial cells had formed blebs. These blebs of cytoplasm contained scattered granules and a few large membrane-lined vacuoles. Some of the vacuoles appeared to represent dilated cisternae of rough endoplasmic reticulum. There was focal smudging of the plasma membranes, with some disruption of the membranes (Fig. 2). Similar relatively organelle-free areas were present in the basilar cytoplasm. Some cells had separated from the underlying stroma, with deposition of electron-dense material at the sites of separation. This dense material was amorphous or else appeared to be composed of clumps of disrupted membranes (Fig. 3). The nuclear material showed condensation, especially at the nuclear membrane. The mitochondria were intact in general, though there was some loss of the usual orientation of the cristae and increased density of the mitochondrial matrix. The underlying stroma was unaltered. The basement membrane could not always be distinguished from the electron-dense layer deposited at the site of epithelial cell separation. Where seen, it appeared unaltered or occasionally discontinuous. Despite these marked changes, some lateral lobe epithelial cells appeared normal (Fig. 4). At 6 hours most of the epithelial cells had separated from the basement membrane and lay free in the lumina of the acini either singly or in sheets. Cell membranes were lost from many cells. Mitochondria were still recognizable, but appeared altered. Nuclear material was arranged in distinct clumps (Fig. 5). Neutrophils were present in the stroma and lumina at 9 hours. There was further disruption of the epithelial cells, so that by 1 day, only cell debris (membranes and vacuoles) were seen in some areas. Three days after the dithizone injection regeneration of lateral lobe epithelium was quite evident. Some of the acini were lined by a single layer of epithelium;
DITHIZONE
EFFECT
ON
RAT
PROSTATE
505
FIG. 1. Micrograph showing the supranuclear portion of an epithelial cell of the rat lateral prostate. The animal was sacrificed 20 minutes after an injection of di’thizone. A layer of electrondense material covers the microvilli (mv). The mitochondria (m) and endoplasmic reticulum (er) appear unchanged. A large secretory granule (g) is present in the apical cytoplasm. X 44,000.
506
G. RANDOLPH
SCHRODT
FIG. 2. Lateral prostate epithelium 3 hours following a single injection of dithizone. The apical cytoplasm of one cell has formed blebs (b), some of which lie free in the lumen of the acinus (L). The arrows indicate the disruption and the smudgy appearance of the plasma membranes. X 31,000.
DITHIZONE
EFFECT
ON
RAT
PROSTATE
507
FIG. 3. The basilar cytoplasm of the epithelium 3 hours after dithizone. The cytoplasm is light A few dilated cisternae of the endoplasmic reticulum (er) are seen. The basement amd granular. nembrane (BM), reticulum fibrils of the stroma, and a stromal cell (S) are intact. X 58,000.
508
FIG. 4. dithizone. membrane
G. RANDOLPH
SCHRODT
Photomicrograph showing two epithelial cells of the lateral prostate 3 hours following Cell A is unaltered; cell B shows loss of microvilli and some disruption of its plasma (arrow). X 35,000.
DITHIZONE
EFFECT
ON
RAT
PROSTATE
509
FIG. 5. Necrotic cells 6 hours after dithizone. The cells are separated from the basement membrane (BM) and are breaking apart. Nuclear material (N) shows marked clumping. At the right lower portion of the photomicrograph is an intact capillary (c) containing an erythrocyte. x 21,000.
510
FIG. 6. The :mv), a small :ellular debris.
G. RANDOLPH
SCHRODT
apical portion of two epithelial cells 1 week Golgi zone (G), and mitochondria (m) are x 35,000.
following dithizone. noted. The lumen
(L)
Short microvilli still contains
DITHIZONE
EFFECT
ON
RAT
511
PROSTATE
FIG. 7. Epithelial cells of the lateral prostate 3 weeks following dithizone. “normal.” Nuclei (N), mitochondria Cm), endoplasmic reticulum (er) , basement ar.d a etromal cell (s) are seen. X 20,000.
The cells membrane
appear (bm),
512
G. RANDOLPH
SCHRODT
others were lined by epithelium two or three cells thick. The lumina were still filled with necrotic cells. Many of the nuclei contained one or several large nucleoli. At 1 week most acini were lined by a single layer of epithelium. The cells, however, often appeared immature. The microvilli were fewer in number and shorter than in the normal adult epithelium. The Golgi zones were distinct but small. Cisternae of the endoplasmic reticulum were inconspicuous (Fig. 6). The lateral lobe epithelium at 3, 7, and 16 weeks appeared identical to that of control animals (Fig. 7). DISCUSSION The zinc level of the rat lateral prostate is 2.3 mg per gram dry weight. Radioactive zinc-65 studies and histochemical studies suggest that most of it is concentrated in the apical and basilar portions of the epithelial cytoplasm. Although some of the zinc is bound to enzymes, the exact location and chemical combination of most of it is unknown. The pronounced structural changes in the prostate following dithizone administration have been studied by a few investigators at the light microscopic level. Logothelopoulos (1961), in a study of the rat lateral prostate, showed that zinc dithizonate granules were located in the apical and basilar portions of the cytoplasm. Mackenzie et al. (1962), in their study of the dog prostate, demonstrated that zinc was lost from the prostate following dithizone administration, Our studies show that the earliest and most pronounced changes in the rat lateral prostate occur in the microvilli, Golgi zone, and cisternae of the endoplasmic reticulum. These organelles are prominent in the apical and/or basilar areas of the epithelial cells. Despite these findings, which suggest that zinc is chelated with dithizone in &JO and that the most pronounced structural changes occur in loci of high zinc concentration, it does not necessarily follow that chelation of zinc produces the epithelial necrosis. One objection is that dithizone chelates metals other than zinc. Inactivation of other metallo-enzymes containing copper, manganese, or iron could produce the structural changes. An enlightening discussion of this problem has been given by Phillips ( 196 I ) . If we make the assumption that chelation of zinc by dithizone does produce a lesion of the rat lateral prostate, two intermediate and simultaneous steps have occurred: (1) Zinc has been removed from certain zinc-containing compounds; (2) zinc dithizonate has been produced. Either or both could produce cell damage. In support of the first is the findings of Philips et al. that metal chelators other than dithizone produce prostatic lesions. (It should be mentioned, however, that Logothelopoulos found no lesions in the rat prostate following administration of o-aminobenzoic acid, 8-hydroxyquinoline, and 2-quinoline-carboxylic acid.) In support of the second point is the work of Mackenzie, who found dithizoneinduced lesions in the dog prostate only when the zinc level was above 0.74 mg per gram dry weight. This finding suggests that at a certain level zinc dithizonate is cytotoxic. Whatever the exact mechanism of the dithizone-induced lesion of the rat lateral
DITHIZONE
EFFECT
ON
RAT
513
PROSTATE
prostate, our findings show early change in two organelles where one might expect high levels of zinc, viz., the Golgi zone and the endoplasmic reticulum. This is highly probable since the prostatic secretion is zinc rich, and these two organelles are regarded as important in the synthesis and release of secretory products. The alterations in the cell membranes, including the microvilli, are more difficult to interpret. There is experimental evidence suggesting that some divalent ions are important in maintaining cell membrane integrity. Coman (1959) described changes in the plasma membrane of liver parenchymal cells following perfusion with Versene, and attributed the changes to a loss of calcium and magnesium. Leeson and Kalant ( 1961)) in their study of the effect of Versene on rat liver, also suggested that calcium, magnesium, and other divalent ions may be important in cell integrity. To our knowledge there is no report on the presence or absence of zinc in the cell membrane. We think it entirely possible that this divalent ion is important in maintaining the cell membrane integrity of rat lateral prostate epithelium. To follow up this hypothesis, we are planning to study the prostate epithelium by means of electron microscopic radioautography using radioactive zinc SUMMARY The destructive changes in the epithelium of the rat lateral prostate following dithizone have been studied with the electron microscope. Focal changes are seen within the first hour and marked changes are noted within 3 hours. The possible mechanisms of these changes are briefly discussed. It is suggested that the Golgi granules, endoplasmic reticulum, and plasma membranes, especially the microvilli, may be zinc-rich organelles. ACKNOWLEDGMENT The author
wishes
to thank
Mr.
Charles
Foreman
for his excellent
technical
assistance.
REFERENCES D., and GROTK, D. P. (1961). The fine structure of the rat prostatic complex. Exptl. Res. 23. 159-175. COMAN, DALE REX (1959). Cellular adhesiveness in relation to the invasiveness of cancer. Cancer Res. 14, 519-521. DIXON, R. H., and WHITFIELD, J. F. (1959). The histochemical localization of zinc in the dorsolateral prostate of the rat. J. Hislochenz. Cytochem. 7, 262-266. FISHER, N. I., TIKKAM, A. D., and MAWSON, C. A. (1955). Zinc, carbonic anhydrase, and phosphatase in the prostatic glands of the rat. Can. J. Biockem. Physiol. 33, 181-190. GUNN, SAMUCL A., and GOULD, THELMA CLARK (1956). Difference between dorsal and lateral components of dorsolateral prosta’te of rats in Znc” uptake. Proc. Sm. Erptl. Biol. Med. 92, 17-20. GUNN, SAMUEL A., and GOULD, THELMA CLARK (1957). A correlative anatomical and functional study of the dorsolateral prostate of the rat. dnat. Record 128, 41-53. LEESON, T. S., and KALANT, H. (1961). Effects of in vivo decalcification on ultrastructure of adult rat liver. J. Biophys. Biochem. Cytol. 10, 93-104. LOGOTHETOPOULOS, JOHN (1961). Intravital chelation of zinc in the prostate of the rat. (Histochemical and Histologic Observations.) Am. J. Pathol. 37, 357-375. LUFT, JOHN H. (1961). Improvements in epoxy resin embedding methods. J. Biopkys. Biochem. Cytol. 9, 409-414. MACKENZIE, A. RANALD, HALL, THEODORE, and WHITMORE, WILLET F., JR. (1962). The relation between the zinc content of the canine prostate and the histiologic effects of dithizone. J. Ural. 37, 923-929. MILLAR, M. J., VINCENT, N. R., and MAWSON, C. A. (1961). An autoradiographic study of the distribution of Zincas rn . rat tissues. J. Histockem. Cytockem. 9, 111.116. BRANDES,
Cell
514 PHILIPS,
G. RANDOLPH FREDERICK
S., ASKARA,
communication. PHILIPS, FREDERICK S. diphenylthiocarbazone.
AUGUSTA
SCHRODT
B., SCHWARTZ,
HERBERT
S., and
STERNBERG,
STEPHEN
S.
Personal
(1961). Relations between zinc content and the selective cytoxicity of Federalion Proc. 20, (3, Part II) 129.131. SCHRODT, G. KAND~LPH (1961). The fine structure of the lateral lobe of the rat prostate gland. J. Ultrastruct. Res. 5, 485-496. WETTF,RDAL, B. (1958). Experimental studies on radioactive zinc in the male reproductive organs of the rat. dcta Radiol. Suppl. 156, l-83.