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Nuclear and mitochondrial alterations in amebae exposed to ethidium bromide
Printed in Sweden Copyright g 1973 by Academic Press, Inc. All rights of reproduction in any form rescrced
Experimental
NUCLEAR
Cell Research 81
AND MITOCHONDRIAL
(1973) 293-300
ALTERATIONS
EXPOSED TO ETHIDIUM
IN AMEBAE
BROMIDE
CH. J. FLICKINGER Department of Anatomy, School of Medicine, University of Virginia, Charlottesville, Va 22901, USA
SUMMARY Cultures of Amoeba proteus were exposed to ethidium bromide at concentrations ranging between 5 and 100 pg/ml for periods of up to 1 week. Samples of treated and control cells were prepared at intervals for electron microscopy. The main ultrastructural alterations were in nucleoli and in mitochondria. The nucleoli of treated cells increased in density and became spherical with more sharply defined margins than those of normal amebae. Many nucleoli contained electron-lucent regions or nucleolar vacuoles of varying size. The chromatin was unusually condensed in some amebae. Mitochondria developed a central electron-lucent region and accumulated a dense material in the matrix. Some cristae were abnormally dilated. The nuclear alterations occurred at least as early as the mitochondrial changes and were present even in cells exposed to the lower concentrations of inhibitor, in which mitochondrial changes were minimal.
Ethidium bromide is a phenanthridinium compound that leads to the loss of the kinetoplast in trypanosomes [35, 37, 38, 471 and induces cytoplasmic mutations in some yeasts [29, 451. Recently, it has been used in numerous experiments since it selectively inhibits kinetoplast and mitochondrial DNA synthesis [ll, 13, 17, 26, 30, 39, 511and RNA synthesis [21, 28, 521apparently becausedrug molecules intercalate between basesand alter the DNA structure [l, 23, 25 , 30, 31, 40, 501. Consequently, ethidium bromide has effects on biochemical and physiological properties of mitochondria [4, 19, 20, 32, 461 including inhibition of mitochondrial protein synthesis [22, 331, and it causes changes in the mitochondrial ultrastructure [12, 20, 24, 27, 461.In some instances, alterations in nuclear fine structure have also been detected [3, 12, 41, 441. Ethidium bromide is one of several com-
pounds which have been used to study strain differences and the inheritance of resistance to drugs and antibiotics in amebae [14]. Multiplication of amebae is inhibited by ethidium bromide and the related compound dimidium bromide [ 151. Nuclear transplantation experiments [16] have indicated that the cellular site of action of the drug is in the cytoplasm, although interpretation of its action on amebae has been complicated by the previous lack of direct evidence of mitochondrial changes in amebae and the fact that many strains of amebae contain intracellular bacterium-like organisms [42] (also referred to as parasites or symbionts by various authors) which are thought to modify the response of certain amebae to the drug [14, 151. We have been interested in the dynamics of cytoplasmic organelles in amebaeand have used different metabolic inhibitors as tools Exptl Cell Res 81 (1973
294 Ch. J. Flickinger for their study. In the course of these investigations, we observed alterations in the fine structure of both the nucleus and cytoplasm of amebae treated with ethidium bromide. Although several studies on mitochondrial ultrastructure in other cell types treated with ethidium bromide [12, 20, 24, 27, 461 appeared while this work was in progress, our observations on amebae are described briefly in this report for the following reasons. First, although evidence favors the mitochondrion as the main site of action of ethidium bromide, the observations indicate that the nucleus is affected simultaneously, and attention is directed to nucleocytoplasmic interactions in ethidium bromide-treated cells. Second, by providing clear evidence of structural alterations in ameba mitochondria they aid in the interpretation of prior studies on the inheritance of resistance to ethidium bromide and similar drugs in these cells. Third, the results confirm and extend previous accounts of fine structural changes in the mitochondria of other organisms and favorably illustrate the development of mitochondrial lesions in response to different concentrations of the drug.
Table 1. Experiments in which amebae were exposed to ethidium bromide
MATERIALS AND METHODS
RESULTS Nuclei The main nuclear changes were in the multiple irregularly shaped nucleoli (fig. I), which are located at the periphery of the nucleus and have a substructure of intermixed fine fibrils and granules in normal amebae [8]. In the presence of ethidium bromide, the density of the nucleoli increased, and they assumed a spherical shape with a smoother, more sharply demarcated outline than normal (fig. 2). Frequently, electron-lucent regions or nucleolar vacuoles of varying size developed within the substance
Stock cultures of Amoeba proteus were originally obtained from Dr D. M. Prescott, University of Colorado. They were maintained in Prescott’s ameba medium [34] at 21°C with daily feedings of washed Tetrahymena. Samples of amebae were exposed to ethidium bromide (Sigma Chemical Co., St Louis, MO) dissolved in ameba medium at cont. of 5, 15, 30, 50, and 100 yg/ml. Control cells were removed from stock cultures at the same time as the experimental amebae and were placed in normal ameba medium. Both groups of cells were maintained at 21°C in small Petri dishes. In short-term expts lasting a few days the amebae were not fed, but in expts lasting for a week the amebae were fed small numbers of Tetrahymena each day. The length of time that cells were maintained and the intervals at which samples were taken are shown in table 1 in relation to the different ethidium bromide concentrations used. Exptl Cell Res 81 (1973)
fzPt ’ 1 2
Cone. (pug/ml) 5 50 15 30
3
,z
4 5
100 100
6 50 100
Fate of amebae Sample fixed 1 week 80 % dead after 3 days Samples fixed 1, 3, 6 days Samples fixed 1, 3 days Samples fixed 1, 2, 3 days Samples fixed 1, 2 days, all dead by 3 days Sample fixed 1 day Samples fixed 3, 6 h Amebae were exposed to the inhibitor for 1 day, then washed and placed in ameba medium for 1 day 90 % cells survived 75 % cells survived
Samples of both treated and control amebae were fixed for 1 h in Karnovsky’s fixative [18], rinsed overnight in distilled water, and postfixed in 1 % 0~0, in 0.1 M cacodylate buffer. The amebae were dehydrated in a graded series of ethanols followed by propylene oxide, and embedded in Araldite. Cells were collected by centrifugation between the steps. Thin sections showing silver to pale gold interference colors were cut using a Porter-Blum-MT-l ultramicrotome equipped with a diamond knife. The sections were mounted on uncoated copper grids and stained with lead citrate [36]. Electron micrographs were obtained with a Philips EM-300 electron microscope.
Amebae exposed to ethidium bromide
of the nucleoli (fig. 3), lending the appearance of a series of fused rings to sections of nucleoli that contained several large vacuoles (fig. 4). Dense granules appeared within some of the small nucleolar vacuoles and in some instances a granular material was found at the periphery of the nucleoli. In some but not all amebae, the chromatin in the interior of the nucleus was clumped (fig. 2) and the number of RNA-containing helices characteristic of the ameba nucleus [48] declined. The nuclear envelope was not noticeably altered. Mitochondria
Normal ameba mitochondria (fig. 5) have rod-shaped or circular profiles. Tubular cristae are continuous with the inner of the two mitochondrial membranes, A finely granular matrix is distributed uniformly throughout the inner mitochondrial compartment. The density of the matrix often varies from one mitochondrion to another [7]. Bundles of moderately dense parallel filaments are sometimes present in the matrix, possibly varying with the nutritional state of the amebae [2]. In amebae exposed to ethidium bromide, many mitochondria had either a circular profile or a highly irregular outline, while smoothly-contoured rods were less common. The density of the matrix in the center of the mitochondrion declined, leaving an electron-lucent area traversed only by a few fine filaments (fig. 6). As the mitochondrial lesions progressed further, the matrix accumulated irregularly shaped clumps of a dense material (fig. 7), which appeared to have a flocculent texture with granules embedded in it. In some mitochondria, the number of cristae seemed to be reduced, but in others they continued to occupy much of the interior of the mitochondrion. Frequently, one or more cristae were abnormally dilated,
295
forming irregular membrane-bounded clear spaceswithin a mitochondrion (fig. 8). Other cytoplasmic structures
Golgi bodies underwent a slow decline in both size and number after several days treatment with ethidium bromide, although in the first day of treatment with 100 pug/ml of ethidium bromide they tended to be clumped together and a few cells contained Golgi bodies with a greater than normal diameter or an abnormally large number of cisternae. Evidence of an effect on the bacterium-like intracellular organisms [42] was also sought, but we were unable to form any firm conclusions in this regard due to a high degree of variability in the number and appearance of the bacteria in both control and experimental cells, as well as in amebae from stock cultures. Amebae exposed to ethidium bromide for several days to a week displayed circular cisternae of rough endoplasmic reticulum, such as have been noted previously in amebae subjected to different metabolic inhibitors or other deleterious conditions [5, 6, 9, 10, 431. No alterations were detected in the plasma membrane or cytoplasmic matrix. General observations
The alterations in mitochondria, nuclei and other structures occurred with varying rapidity, depending on the ethidium bromide concentration. In cells exposed to 100 pg/ml, both nuclear and mitochondrial lesions were well-developed after only 24 h, while in the presence of 30 or 50 pg/ml alterations required 3 to 6 days to become evident. In cells exposed to 5 or 15 pg/ml for 1 week, nuclear changes were prominent, but mitochondrial alterations did not become as extreme as those in cells treated with the higher concentrations. At these lower concentrations, some mitochondria became Exptl Cell Res 81 (1973)
296
Ch. J. Flickinger
round and acquired a circular dense matrix spot, but we have observed this type of change in response to other treatments [9] and regard this morphology as a non-specific change in ameba mitochondria subjected to a variety of unfavorable conditions. These changes were not detected in control amebae maintained in normal ameba medium.
DISCUSSION Nuclear alterations
The nucleoli were the most greatly altered components in the nuclei of amebae treated with ethidium bromide. In general, nucleoli appear to be particularly sensitive to ethidium bromide, since nucleolar changes have also been detected in mammalian cells in culture [44], trypanosomes [3], sea urchin embryos [12], and onion root tip cells [41] exposed to the drug. The increase in density of nucleoli, formation of nucleolar vacuoles, and clumping of chromatin in amebae were reminiscent of the early changes following treatment with actinomycin D [8]. Although regions of differing texture were noted within some nucleoli in amebae exposed to ethidium bromide, obvious segregation of nucleolar components was not observed, as it was in amebae treated with actinomycin [8] and in some other cells exposed to ethidium bromide [41, 441. In addition, since the granular and fibrillar components of ameba
nucleoli are intermixed with one another [8], it was difficult to ascertain whether the granular component declined [12, 411, but it is possible that the increased density and smooth uniform texture of the nucleoli might be the result of loss of the granular component. The structure of chromatin also appears to be altered by ethidium bromide. Unusual clumping or condensation of chromatin in the interior of the nucleus was observed in ethidium bromide-treated amebae, and similar chromatin alterations have been noted in other cells exposed to the drug [3, 12, 441. Furthermore, abnormal mitoses in sea urchin eggs treated with ethidium bromide have been attributed to defective condensation of chromosomes during prophase [49]. Mitochondrial
changes
Ethidium bromide is known to inhibit both mitochondrial DNA and RNA synthesis (see p. 293). The relation of the ultrastructural changes to these molecular events is uncertain, but formation of central electronlucent areas might result from clumping and condensation of mitochondrial DNA to form the massesof dense matrix material, as in the kinetoplast of trypanosomes [3]. Loss of cristae may also play a role in the generation of electron-lucent areas within mitochondria, but this effect seems less pronounced in amebae than in other cells [12, 20, 24, 461.
Fig. I. In normal ameba nuclei most of the multiple nucleoli are found just inside the nuclear envelope (E). The nucleoli (N) have a fibrillar and granular substructure [8] and an irregular margin. Chromatin (C) has a fibrillar character and is located mainly in the interior of the nucleus. x 20 000. Fig. 2. When amebae are exposed to ethidium bromide, the density of the nucleoli (N) increases and they assume a spherical shape with a smoother more distinct margin than in normal cells. In some cells, chromatin (C) in the interior of the nucleus is condensed into clumps. Nucleus of an ameba treated with ethidium bromide 30 fig/ml for 6 days. x 22 000. Fig. 3. Many round electron-lucent areas, or nucleolar vacuoles (V) of varying size appear within nucleoli of ethidium bromide-treated amebae. Ameba exposed to 100 pg/ml for 1 day. x 23 000. Fig. 4. Nucleolar vacuoles may become very large in cells exposed to ethidium bromide, so that in sections through a nucleolus the image is that of rings of nucleolar material fused to one another. Cell treated with 5 ,ug/ml for 1 week. x 23 000. Exptl Cell Res 81 (1973)
Amebue exposed to ethidium bromide
291
Exptl Cell Res 81 (1973)
298 Ch. J. Flickinger
Fig. 5. Normal ameba mitochondria have rod-shaped or circular profiles. The interior is occupied by tubular cristae and a uniformly distributed matrix. Normal ameba from a stock culture. x 36 000. Fig. 6. In amebae exposed to ethidium bromide, mitochondria develop an electron-lucent region (A) in the central part of the matrix. Mitochondrion from an ameba treated with ethidium bromide 30 pg/ml for 3 days. x 37 000. Fig. 7. In the presence of ethidium bromide, the mitochondrial matrix accumulates clumps of an unusual dense
material (D) with an uneven granular texture. Ameba exposed to ethidium bromide 100 pg/ml for 1 day. x34ooo. Fig. 8. Clear areas (B) bounded by a membrane often appear within mitochondria of ethidium bromide-treated
amebae. They are apparently formed by dilatation of cristae. Ameba treated with lOO,~g/ml for 1 day. x 24 000.
Exptf CelI Res 81 (1973)
Amebae exposed to ethidium bromide Nucleocytoplasmic interactions.
Previous studies showed that’ethidium bromide and the related compound dimidium bromide inhibit the multiplication of Amoeba proteus and Amoeba discoides in a manner that is both strain dependent and related to the concentration of the drug [15]. The fine structural changes described here implicate changes in either the nucleus or the mitochondria or both in this growth inhibition, since alterations in these organelles were most noticeable and occurred at approximately the same time after beginning treatment. Nuclear transfer studies [16], however, have indicated that the site of action of the drug in inhibiting multiplication of amebae is in the cytoplasm, since transfer of nuclei of treated cells to normal cytoplasm results in the formation of some clones, but transfer of normal nuclei to treated cytoplasm fails to produce viable amebae.The ,present study supports the hypothesis [14] that the cytoplasmic site of action of the drug, at least so far as this growth inhibiting effect is concerned, is in the mitochondria. Nevertheless, ethidium bromide also affected the nucleus in an adverse manner. In cells exposed to lower ethidium bromide concentrations (5 and 15 pg/ml), in fact, the nuclear alterations were easily detected while mitochondrial changes were minimal. It is possible that the changes in nuclear fine structure were the result of interaction of ethidium bromide with nuclear DNA. The evidence from other systems that ethidium bromide selectively affects mitochondria [ 11, 13, 17, 21, 28, 30, 521is against this conjecture, however, and suggestsinstead that the nuclear changes followed secondarily as the result of disturbance of mitochondrial metabolism. At low cont. of the drug, derangements in mitochondrial metabolism:may have occurred before structural changesdeveloped,
299
thus accounting for the paucity of mitochondrial alterationsin relation to pronounced nuclear lesions. Alternatively, the presence of nuclear alterations might be attributed to direct effects of the drug upon the nucleus as a result of the use in this study of ethidium bromide concentrations which exceedthose that are selective for mitochondria. The concentrations used here are indeed greater than those commonly used with mammalian cells (e.g. [20-24, 31, 32, 46, 52]), but it should be noted that the nuclear alterations were prominent in amebae exposed to the lower ethidium bromide concentrations (5 and 15 pg/ml), which are within the range of those utilized in previous studies on amebae ([ 15, I6]-dimidium bromide), yeasts [13, 191, some trypanosomes [47], sea urchins [49], and Tetrahymena [27], as well as for ultrastructural studies on several kinds of cells [3, 12, 411. In any event, the presence and time course of the changes in nuclear fine structure suggest that changes in nuclear function occurred in ethidium bromidetreated amebae and that some effects of the drug could be due to these nuclear changes, whether they result from interaction of the drug with the nucleus or are secondary to disturbance of mitochondrial metabolism. The author is indebted for technical assistance to Mrs Mary Kay Trail and Miss Sherry Vinsant. This research was supported by research grants from NSF (GB-32285) and ACS (E-500A).
REFERENCES 1. Bauer, W & Vinograd, J, J mol biol 33 (1968) 141. 2. Daniels, E W & Breyer, E P, Z Zellforsch 91 (1968) 159. 3. Delain, E & Riou, G, Biochem pharmacol 19 (1970) 2521. 4. DeVries, H & Kroon, A M, FEBS letters 7 (1970) 347. 5. Flickinger, C J, J cell biol 37 (1968) 300. 6. - Exptl cell res 53 (1968) 241. 7. - Protoplasma 66 (1968) 139. 8. - J ultrastruct res 23 (1968) 260. Exptl Cell Res 81 (1973)
300 Ch. J. Flickinger 9. - Exptl cell res 68 (1971) 381. 10. - The biology of amoeba (ed K W Jeon) p. 171. Academic Press, New York (1973). 11. Fukuhara, H & Kujawa, C, Biochem biophys res commun 41 (1970) 1002. 12. Geuskens, M, J exptl’zool 178 (1971) 247. 13. Goldring, E S, Grossman, L 1, Krupnick, D, Cryer, D R & Marmur, J, J mol biol 52 (1970) 323. 14. Hawkins, S E, The biology of amoeba (ed K W Jeon) p. 371. Academic Press, New York (1973). 15. Hawkins, S E & Willis, L R, J cell sci 5 (1969) 57. 16. Hawkins, S E&Willis, L R, Nature 222 (1969) 86. 17. Heilporn, V & Limbosch, S, Biochim biophys acta 240 (1971) 94. 18. Karnovsky, M J, J cell biol 27 (1965) 137A. 19. Kellerman, G M, Biggs, D R & Linnane, A W, J cell biol 42 (1969) 378. 20. King, M E, Godman, G C & King, D W, J cell biol 53 (1972) 127. 21. Knight, E, Biochemistry 8 (1969) 5089. 22. Lederman, M & Attardi, G, Biochem biophys res commun 40 (1970) 1492. 23. Leibowitz, R D, J cell biol 51 (1971) 116. 24. Lenk, R & Penman, S, J cell biol 49 (1971) 541. 25. LePecq, J-B & Paoletti, C, J mol biol 27 (1967) 81 -.. 26. Meyer, R R & Simpson, M V, Biochem biophys res commun 34 (1969) 238. 27. Meyer, R, R, Boyd, C R, Rein, D C & Keller, S J, Exptl cell res 70 (1972) 233. 28. Meyer, R R, Probst, G S & Keller, S J, Arch biochem biophys 148 (1972) 425. 29. Nagley, P & Linnane, A W, J mol biol 66 (1972) 181. 30. Nass, M M K, Proc natl acad sci US 67 (1970) lY26.
Exptl
Cell Res 81 (1973)
31. - Exptl cell res 72 (1972) 211. 32. Naum, Y & Pious, D A, Exptl cell res 65 (1971) 335. 33. Perlman, S & Penman, S, Biochem biophys res commun 40 (1970) 941. 34. Prescott, D .M & Carrier, R F, Methods cell physiol 1 (1964) 85. 35. Renger, H C & Wolstenholme, D R, J cell biol 47 (1970) 689. 36. Reynolds, E S, J cell biol 17 (1963) 208. 37. Riou, G C, Compt rend acad sci 265 (1967) 2004. 38. - Ibid 266 (1968) 250. 39. - Biochem pharmacol 19 (1970) 1524. 40. Riou, G C & Delain, E, Proc natl acad sci US 64 (1969) 618. 41. Risuefio, M C, Fernandez-Gomez, M E, de la Torre, C & Gimenez-Martin, G, J ultrastruct res 39 (1972) 163. 42. Roth. L E & Daniels. E W, J bioohvs biochem cytol’9 (1961) 317. . . 43. Sanders, E J, Exptl cell res 61 (1970) 461. 44. Simard, R, Cancer res 26 (1966) 2316. 45. Slonimski. P P. Perrodin. G & Croft. J H. Biothem biophys res commun 30 (1968) 232. ’ 46. Soslau, G & Nass, M M K, J cell biol 51 (1971) 514. 47. Steinert, M, Exptl cell res 55 (1969) 248. 48. Stevens, A R, The control of nuclear activity (ed L Goldstein) u. 189. Academic Press. New York (1967). ’ . 49. Vacauier. V & Brachet. J. Nature 222 (1969) 193. 50. Waring, M J, Nature 219(1968) 1320.‘ ’ 51. Westergaard, 0, Marcker, K A & Keiding, J, Nature 227 (I 970) 708. 52. Zylber, E, Vesco, C & Penman, S, J mol bio144 (1969) 195. Keceived March 23, 1973
Experimental
NUCLEAR
Cell Research 81
AND MITOCHONDRIAL
(1973) 293-300
ALTERATIONS
EXPOSED TO ETHIDIUM
IN AMEBAE
BROMIDE
CH. J. FLICKINGER Department of Anatomy, School of Medicine, University of Virginia, Charlottesville, Va 22901, USA
SUMMARY Cultures of Amoeba proteus were exposed to ethidium bromide at concentrations ranging between 5 and 100 pg/ml for periods of up to 1 week. Samples of treated and control cells were prepared at intervals for electron microscopy. The main ultrastructural alterations were in nucleoli and in mitochondria. The nucleoli of treated cells increased in density and became spherical with more sharply defined margins than those of normal amebae. Many nucleoli contained electron-lucent regions or nucleolar vacuoles of varying size. The chromatin was unusually condensed in some amebae. Mitochondria developed a central electron-lucent region and accumulated a dense material in the matrix. Some cristae were abnormally dilated. The nuclear alterations occurred at least as early as the mitochondrial changes and were present even in cells exposed to the lower concentrations of inhibitor, in which mitochondrial changes were minimal.
Ethidium bromide is a phenanthridinium compound that leads to the loss of the kinetoplast in trypanosomes [35, 37, 38, 471 and induces cytoplasmic mutations in some yeasts [29, 451. Recently, it has been used in numerous experiments since it selectively inhibits kinetoplast and mitochondrial DNA synthesis [ll, 13, 17, 26, 30, 39, 511and RNA synthesis [21, 28, 521apparently becausedrug molecules intercalate between basesand alter the DNA structure [l, 23, 25 , 30, 31, 40, 501. Consequently, ethidium bromide has effects on biochemical and physiological properties of mitochondria [4, 19, 20, 32, 461 including inhibition of mitochondrial protein synthesis [22, 331, and it causes changes in the mitochondrial ultrastructure [12, 20, 24, 27, 461.In some instances, alterations in nuclear fine structure have also been detected [3, 12, 41, 441. Ethidium bromide is one of several com-
pounds which have been used to study strain differences and the inheritance of resistance to drugs and antibiotics in amebae [14]. Multiplication of amebae is inhibited by ethidium bromide and the related compound dimidium bromide [ 151. Nuclear transplantation experiments [16] have indicated that the cellular site of action of the drug is in the cytoplasm, although interpretation of its action on amebae has been complicated by the previous lack of direct evidence of mitochondrial changes in amebae and the fact that many strains of amebae contain intracellular bacterium-like organisms [42] (also referred to as parasites or symbionts by various authors) which are thought to modify the response of certain amebae to the drug [14, 151. We have been interested in the dynamics of cytoplasmic organelles in amebaeand have used different metabolic inhibitors as tools Exptl Cell Res 81 (1973
294 Ch. J. Flickinger for their study. In the course of these investigations, we observed alterations in the fine structure of both the nucleus and cytoplasm of amebae treated with ethidium bromide. Although several studies on mitochondrial ultrastructure in other cell types treated with ethidium bromide [12, 20, 24, 27, 461 appeared while this work was in progress, our observations on amebae are described briefly in this report for the following reasons. First, although evidence favors the mitochondrion as the main site of action of ethidium bromide, the observations indicate that the nucleus is affected simultaneously, and attention is directed to nucleocytoplasmic interactions in ethidium bromide-treated cells. Second, by providing clear evidence of structural alterations in ameba mitochondria they aid in the interpretation of prior studies on the inheritance of resistance to ethidium bromide and similar drugs in these cells. Third, the results confirm and extend previous accounts of fine structural changes in the mitochondria of other organisms and favorably illustrate the development of mitochondrial lesions in response to different concentrations of the drug.
Table 1. Experiments in which amebae were exposed to ethidium bromide
MATERIALS AND METHODS
RESULTS Nuclei The main nuclear changes were in the multiple irregularly shaped nucleoli (fig. I), which are located at the periphery of the nucleus and have a substructure of intermixed fine fibrils and granules in normal amebae [8]. In the presence of ethidium bromide, the density of the nucleoli increased, and they assumed a spherical shape with a smoother, more sharply demarcated outline than normal (fig. 2). Frequently, electron-lucent regions or nucleolar vacuoles of varying size developed within the substance
Stock cultures of Amoeba proteus were originally obtained from Dr D. M. Prescott, University of Colorado. They were maintained in Prescott’s ameba medium [34] at 21°C with daily feedings of washed Tetrahymena. Samples of amebae were exposed to ethidium bromide (Sigma Chemical Co., St Louis, MO) dissolved in ameba medium at cont. of 5, 15, 30, 50, and 100 yg/ml. Control cells were removed from stock cultures at the same time as the experimental amebae and were placed in normal ameba medium. Both groups of cells were maintained at 21°C in small Petri dishes. In short-term expts lasting a few days the amebae were not fed, but in expts lasting for a week the amebae were fed small numbers of Tetrahymena each day. The length of time that cells were maintained and the intervals at which samples were taken are shown in table 1 in relation to the different ethidium bromide concentrations used. Exptl Cell Res 81 (1973)
fzPt ’ 1 2
Cone. (pug/ml) 5 50 15 30
3
,z
4 5
100 100
6 50 100
Fate of amebae Sample fixed 1 week 80 % dead after 3 days Samples fixed 1, 3, 6 days Samples fixed 1, 3 days Samples fixed 1, 2, 3 days Samples fixed 1, 2 days, all dead by 3 days Sample fixed 1 day Samples fixed 3, 6 h Amebae were exposed to the inhibitor for 1 day, then washed and placed in ameba medium for 1 day 90 % cells survived 75 % cells survived
Samples of both treated and control amebae were fixed for 1 h in Karnovsky’s fixative [18], rinsed overnight in distilled water, and postfixed in 1 % 0~0, in 0.1 M cacodylate buffer. The amebae were dehydrated in a graded series of ethanols followed by propylene oxide, and embedded in Araldite. Cells were collected by centrifugation between the steps. Thin sections showing silver to pale gold interference colors were cut using a Porter-Blum-MT-l ultramicrotome equipped with a diamond knife. The sections were mounted on uncoated copper grids and stained with lead citrate [36]. Electron micrographs were obtained with a Philips EM-300 electron microscope.
Amebae exposed to ethidium bromide
of the nucleoli (fig. 3), lending the appearance of a series of fused rings to sections of nucleoli that contained several large vacuoles (fig. 4). Dense granules appeared within some of the small nucleolar vacuoles and in some instances a granular material was found at the periphery of the nucleoli. In some but not all amebae, the chromatin in the interior of the nucleus was clumped (fig. 2) and the number of RNA-containing helices characteristic of the ameba nucleus [48] declined. The nuclear envelope was not noticeably altered. Mitochondria
Normal ameba mitochondria (fig. 5) have rod-shaped or circular profiles. Tubular cristae are continuous with the inner of the two mitochondrial membranes, A finely granular matrix is distributed uniformly throughout the inner mitochondrial compartment. The density of the matrix often varies from one mitochondrion to another [7]. Bundles of moderately dense parallel filaments are sometimes present in the matrix, possibly varying with the nutritional state of the amebae [2]. In amebae exposed to ethidium bromide, many mitochondria had either a circular profile or a highly irregular outline, while smoothly-contoured rods were less common. The density of the matrix in the center of the mitochondrion declined, leaving an electron-lucent area traversed only by a few fine filaments (fig. 6). As the mitochondrial lesions progressed further, the matrix accumulated irregularly shaped clumps of a dense material (fig. 7), which appeared to have a flocculent texture with granules embedded in it. In some mitochondria, the number of cristae seemed to be reduced, but in others they continued to occupy much of the interior of the mitochondrion. Frequently, one or more cristae were abnormally dilated,
295
forming irregular membrane-bounded clear spaceswithin a mitochondrion (fig. 8). Other cytoplasmic structures
Golgi bodies underwent a slow decline in both size and number after several days treatment with ethidium bromide, although in the first day of treatment with 100 pug/ml of ethidium bromide they tended to be clumped together and a few cells contained Golgi bodies with a greater than normal diameter or an abnormally large number of cisternae. Evidence of an effect on the bacterium-like intracellular organisms [42] was also sought, but we were unable to form any firm conclusions in this regard due to a high degree of variability in the number and appearance of the bacteria in both control and experimental cells, as well as in amebae from stock cultures. Amebae exposed to ethidium bromide for several days to a week displayed circular cisternae of rough endoplasmic reticulum, such as have been noted previously in amebae subjected to different metabolic inhibitors or other deleterious conditions [5, 6, 9, 10, 431. No alterations were detected in the plasma membrane or cytoplasmic matrix. General observations
The alterations in mitochondria, nuclei and other structures occurred with varying rapidity, depending on the ethidium bromide concentration. In cells exposed to 100 pg/ml, both nuclear and mitochondrial lesions were well-developed after only 24 h, while in the presence of 30 or 50 pg/ml alterations required 3 to 6 days to become evident. In cells exposed to 5 or 15 pg/ml for 1 week, nuclear changes were prominent, but mitochondrial alterations did not become as extreme as those in cells treated with the higher concentrations. At these lower concentrations, some mitochondria became Exptl Cell Res 81 (1973)
296
Ch. J. Flickinger
round and acquired a circular dense matrix spot, but we have observed this type of change in response to other treatments [9] and regard this morphology as a non-specific change in ameba mitochondria subjected to a variety of unfavorable conditions. These changes were not detected in control amebae maintained in normal ameba medium.
DISCUSSION Nuclear alterations
The nucleoli were the most greatly altered components in the nuclei of amebae treated with ethidium bromide. In general, nucleoli appear to be particularly sensitive to ethidium bromide, since nucleolar changes have also been detected in mammalian cells in culture [44], trypanosomes [3], sea urchin embryos [12], and onion root tip cells [41] exposed to the drug. The increase in density of nucleoli, formation of nucleolar vacuoles, and clumping of chromatin in amebae were reminiscent of the early changes following treatment with actinomycin D [8]. Although regions of differing texture were noted within some nucleoli in amebae exposed to ethidium bromide, obvious segregation of nucleolar components was not observed, as it was in amebae treated with actinomycin [8] and in some other cells exposed to ethidium bromide [41, 441. In addition, since the granular and fibrillar components of ameba
nucleoli are intermixed with one another [8], it was difficult to ascertain whether the granular component declined [12, 411, but it is possible that the increased density and smooth uniform texture of the nucleoli might be the result of loss of the granular component. The structure of chromatin also appears to be altered by ethidium bromide. Unusual clumping or condensation of chromatin in the interior of the nucleus was observed in ethidium bromide-treated amebae, and similar chromatin alterations have been noted in other cells exposed to the drug [3, 12, 441. Furthermore, abnormal mitoses in sea urchin eggs treated with ethidium bromide have been attributed to defective condensation of chromosomes during prophase [49]. Mitochondrial
changes
Ethidium bromide is known to inhibit both mitochondrial DNA and RNA synthesis (see p. 293). The relation of the ultrastructural changes to these molecular events is uncertain, but formation of central electronlucent areas might result from clumping and condensation of mitochondrial DNA to form the massesof dense matrix material, as in the kinetoplast of trypanosomes [3]. Loss of cristae may also play a role in the generation of electron-lucent areas within mitochondria, but this effect seems less pronounced in amebae than in other cells [12, 20, 24, 461.
Fig. I. In normal ameba nuclei most of the multiple nucleoli are found just inside the nuclear envelope (E). The nucleoli (N) have a fibrillar and granular substructure [8] and an irregular margin. Chromatin (C) has a fibrillar character and is located mainly in the interior of the nucleus. x 20 000. Fig. 2. When amebae are exposed to ethidium bromide, the density of the nucleoli (N) increases and they assume a spherical shape with a smoother more distinct margin than in normal cells. In some cells, chromatin (C) in the interior of the nucleus is condensed into clumps. Nucleus of an ameba treated with ethidium bromide 30 fig/ml for 6 days. x 22 000. Fig. 3. Many round electron-lucent areas, or nucleolar vacuoles (V) of varying size appear within nucleoli of ethidium bromide-treated amebae. Ameba exposed to 100 pg/ml for 1 day. x 23 000. Fig. 4. Nucleolar vacuoles may become very large in cells exposed to ethidium bromide, so that in sections through a nucleolus the image is that of rings of nucleolar material fused to one another. Cell treated with 5 ,ug/ml for 1 week. x 23 000. Exptl Cell Res 81 (1973)
Amebue exposed to ethidium bromide
291
Exptl Cell Res 81 (1973)
298 Ch. J. Flickinger
Fig. 5. Normal ameba mitochondria have rod-shaped or circular profiles. The interior is occupied by tubular cristae and a uniformly distributed matrix. Normal ameba from a stock culture. x 36 000. Fig. 6. In amebae exposed to ethidium bromide, mitochondria develop an electron-lucent region (A) in the central part of the matrix. Mitochondrion from an ameba treated with ethidium bromide 30 pg/ml for 3 days. x 37 000. Fig. 7. In the presence of ethidium bromide, the mitochondrial matrix accumulates clumps of an unusual dense
material (D) with an uneven granular texture. Ameba exposed to ethidium bromide 100 pg/ml for 1 day. x34ooo. Fig. 8. Clear areas (B) bounded by a membrane often appear within mitochondria of ethidium bromide-treated
amebae. They are apparently formed by dilatation of cristae. Ameba treated with lOO,~g/ml for 1 day. x 24 000.
Exptf CelI Res 81 (1973)
Amebae exposed to ethidium bromide Nucleocytoplasmic interactions.
Previous studies showed that’ethidium bromide and the related compound dimidium bromide inhibit the multiplication of Amoeba proteus and Amoeba discoides in a manner that is both strain dependent and related to the concentration of the drug [15]. The fine structural changes described here implicate changes in either the nucleus or the mitochondria or both in this growth inhibition, since alterations in these organelles were most noticeable and occurred at approximately the same time after beginning treatment. Nuclear transfer studies [16], however, have indicated that the site of action of the drug in inhibiting multiplication of amebae is in the cytoplasm, since transfer of nuclei of treated cells to normal cytoplasm results in the formation of some clones, but transfer of normal nuclei to treated cytoplasm fails to produce viable amebae.The ,present study supports the hypothesis [14] that the cytoplasmic site of action of the drug, at least so far as this growth inhibiting effect is concerned, is in the mitochondria. Nevertheless, ethidium bromide also affected the nucleus in an adverse manner. In cells exposed to lower ethidium bromide concentrations (5 and 15 pg/ml), in fact, the nuclear alterations were easily detected while mitochondrial changes were minimal. It is possible that the changes in nuclear fine structure were the result of interaction of ethidium bromide with nuclear DNA. The evidence from other systems that ethidium bromide selectively affects mitochondria [ 11, 13, 17, 21, 28, 30, 521is against this conjecture, however, and suggestsinstead that the nuclear changes followed secondarily as the result of disturbance of mitochondrial metabolism. At low cont. of the drug, derangements in mitochondrial metabolism:may have occurred before structural changesdeveloped,
299
thus accounting for the paucity of mitochondrial alterationsin relation to pronounced nuclear lesions. Alternatively, the presence of nuclear alterations might be attributed to direct effects of the drug upon the nucleus as a result of the use in this study of ethidium bromide concentrations which exceedthose that are selective for mitochondria. The concentrations used here are indeed greater than those commonly used with mammalian cells (e.g. [20-24, 31, 32, 46, 52]), but it should be noted that the nuclear alterations were prominent in amebae exposed to the lower ethidium bromide concentrations (5 and 15 pg/ml), which are within the range of those utilized in previous studies on amebae ([ 15, I6]-dimidium bromide), yeasts [13, 191, some trypanosomes [47], sea urchins [49], and Tetrahymena [27], as well as for ultrastructural studies on several kinds of cells [3, 12, 411. In any event, the presence and time course of the changes in nuclear fine structure suggest that changes in nuclear function occurred in ethidium bromidetreated amebae and that some effects of the drug could be due to these nuclear changes, whether they result from interaction of the drug with the nucleus or are secondary to disturbance of mitochondrial metabolism. The author is indebted for technical assistance to Mrs Mary Kay Trail and Miss Sherry Vinsant. This research was supported by research grants from NSF (GB-32285) and ACS (E-500A).
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