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Fine structural alterations of Yoshida ascites hepatoma cells following treatment with L-erythro-α,β-dihydroxybutyraldehyde
Europ. ,7. CancerVol. 3, pp. 103-109. Pergamon Press 1967. Printed in Great Britain
Fine Structural Alterations of Yoshida Ascites Hepatoma cells following treatment with L-Erythro,p-Dihydroxybutyraldehyde MARIA BASSI Centro di Microscopia Elettronica, Politecni¢o di Milano, and Istituto di Patologia Generale, Universit~ di Milano, Milano, Italy
INTRODUCTION
commercial rat pellets and received tap water. Approximately 107 cells of Yoshida hepatoma (AH-130) were inoculated intraperitoneally and maintained in ascitic form by intraperitoneal transplantation. Five days after the injection, they were subjected to the following treatments [11]: - - t e n animals were subjected to a 4 hr intraperitoneal infusion with 10 ml of a 0-14 M solution of DIBA, made isotonic with NaC1. This solution was injected by means of a low flow infusion apparatus. - - t e n animals were subjected to a 4 hr intraperitoneal infusion with 10 ml of saline. For electron microscopy, 0.3 ml of ascitic fluid were drawn with a syringe from the peritoneal cavity of the animals at different time intervals: just before the treatment, at the end of it, and respectively 2, 4, 6, 24 and 48 hr after the end of it. The samples were put immediately into icechilled centrifuge tubes containing 6 ml of 2" 5% glutaraldehyde (Eastman Organic) Chemicals) in 0.15 M phosphate buffer, p H 7.3, and fixed for 15 min at + 4 ° C . Then, by centrifuging and resuspending the samples at each step of the procedure, they were washed in phosphate buffer, post-fixed in phosphate-
SEVERAL researches have shown that a n u m b e r of aliphatic aldehydes act as carcinostatic agents [1-6], probably on account of their capacity of inhibiting amino acid incorporation into protein [7-10]. One of these aldehydes, L-erythro-a, B-dihydroxybutyraldehyde (DIBA) was shown to possess a marked carcinostatic and carcinolytic activity at concentrations which are non-toxic to the tumourbearing animals, and was therefore selected for further studies. [11]. Microscopical observations on Yoshida ascites hepatoma cells treated with DIBA have shown that this substance induces marked nuclear alterations, leading to nuclear rhexis and cell death [12]. It seemed therefore interesting to study the morphology of these cells with the electron microscope, in an attempt to discover early modifications in the cell structure and to follow their progressive evolution towards cell death.
METHODS Twenty Wistar albino rats of either sex, weighing about 170 g, were used in these experiments. T h e y were fed "ad libitum" on a diet of 103
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buffered 1% OsO 4, dehydrated in ethanol, and the final pellet was embedded in Araldite [13]. When in absolute ethanol, the samples were impregnated with uranyl acetate in semisaturated solution. Ultrathin sections were obtained with an LKB Ultrotome, mounted on grids coated with carbon-stabilized celloidin film, stained with lead citrate [14], and examined in a Siemens Elmiskop 1 A at an accelerating voltage of 80 kV. Micrographs were taken at magnifications of 5000-30 000and enlarged photographically as necessary.
RESULTS Untreated cells The ultrastructure of Yoshida ascites hepatoma cells has already been described by Bairati [15]. However, since the fixative used in the present study differs from those used in previous works, additional informations on the sub-microscopic structures have been recorded and will be reviewed. At low magnification, the cells appear either isolated or assembled in groups of two or three (Fig. 1) ; they are roundish, with irregular contours due to the presence of a number of cytoplasmic projections (microvilli). At the site of contact between two cells, the adjacent plasma membranes fuse to form a junctional complex, where only two of the usual junctional structures are recognizable, i.e. the zonulae occludens and adhaerens [16]. The cytoplasm is dense and contains a large number of ribosomes, mitochondria, flattened profiles of the endoplasmic reticulum, a well developed Golgi apparatus, dense bodies of undetermined nature (possibly lysosomes), and bundles of filaments. At higher magnification (Fig. 2), the ribosomes appear often aggregated in irregular clusters or associated in coiled structures. The maximum observed length of these structures is about 2500 A (Fig. 2, inset). The endoplasmic reticulum, composed of flattened sacs, has a random disposition in the cytoplasm and shows long segments devoid of ribosomes. In these regions free particles, larger than ribosomes, are visible, and might be tentatively interpreted as glycogen particles. The Golgi complex is much developed, and is composed of several stacks of flattened sacs and a great number of small vesicles. Mitochondria have irregular, roundish or elongated shapes and a clear matrix; their internal membranes have variable lengths and are arranged either transversally or along the longer axis of the mitochondrion. Dense matrix granules are inconspicuous.
In the cytoplasm are often seen filaments, about 60 A in diameter and of indeterminate length. They occur in small, variously oriented bundles, which thicken around the nucleus and often seem to wrap it completely (Fig. 3). The cytoplasm surrounding the fibrous whorls is sometimes particularly rich in clustered ribosomes. The nucleus (Fig. 4) is large and often deeply lobated; chromatin is sometimes finely dis, persed, sometimes more prominent adjacent to the nuclear membrane. Perichromatin granules are often visible. The nucleolus is generally voluminous, of reticular appearance. DIBA-treated cells The sequence of alterations induced in Yoshida ascites hepatoma cells by DIBA treatment can be traced as follows : at first, formation of long cytoplasmic extrusions at the periphery of the cells, and appearance of a strongly electron-opaque substance in the Golgi vesicles ; next, mitochondrial alterations, with increase in matrix density and dilatation of the intracristal spaces, leading to mitochondrial disruption; then nuclear changes, with chromatin clumping and final nuclear rhexis. The first morphological alterations are the formation of long cytoplasmic extrusions (Fig. 5) and the appearance of a strongly electronopaque substance in the Golgi complex (Figs. 6 and 7). The cytoplasmic extrusions appear at the end of the perfusion, and tend to disappear after 2 hours. Later, they are no longer visible. They contain hyaloplasm, sometimes also ribosomes, but never other cell organelles. The free extremities of such extrusions tend to fuse and form large, electron-lucid vacuoles at the periphery of the cells. The electron-opaque substance appears in a few cells at the end of the treatment, and is present in all the cells 2 hr later. It begins to fill only a few Golgi vesicles (Fig. 6), then it extends to many more elements (Fig. 7). This substance has a great affinity for osmium tetroxide and is homogeneous. At this stage, in a few cells the number of coiled polysomes seems increased (Fig. 6); however, since this finding is not at all constant it will not be discussed here. Then the aspect of mitochondria changes: their matrix becomes very dense and the intracristal spaces dilate (Fig. 7). Glutaraldehyde is known to induce profound alterations of the mitochondrial matrix. However, the constant appearance of mitochondrial alterations in DIBA-treated cells and their absence in pairedfixed controls rule out the possibility that the
Keys to symbols E R = endoplasmic retieulum F - - filaments G -- Golgi complex L = lysosomes
M = mitochondria N -- nucleus n -- nucleolus
All figures are o f Yoshida ascit,es hepatoma cells.
Fig. 1. Two untreated cells in close contact. The cell membranes form a number o f microvilli, and at the sttes o f contact they fuse to form junctional complexes. The cytoplasm contains irregularly shaped mitochondria, a f e w flattened profiles o f the endoplasmic reticulum, a well developed Golgi apparatus, many ribosomes and bundles o f filaments. In the lower cell the filaments contact two lysosome-like structures. The nuclei show finely dispersed chromatin. 15,000×.
(to face p. 104)
Fig. 2. Untreated cell. The mitochondria vary in size and shape, and show a clear matrix and variously oriented "cristae." A number of ribosomes associated in coiled structures is visible in the cytoplasm (arrows). Many segments of the endoplasmic reticulum are devoid of ribosomes. Close to them free particles, larger than ribosomes, are visible, and might represent glycogen particles (double arrow). 30,000 x Inset : a coiled polysome, measuring about 2,500 .'\ in length. 80,000 .:.
Fig. 3. Untreated cell. This section cuts the nucleus at one pole, and shows that it is completely wrapped by a thick bundle of filaments. The surrounding cytoplasm is particularly rich in clustered ribosomes. 30,000 × . Fig. 4. Untreated cell. The nucleus is lobated, with chromatin somewhat more prominent adjacent to the nuclear membrane. The nncleolus is large, of reticular appearance. One perichromatin granule is visible (arrow). 2 5 , 0 0 0 × .
Fig. 5. Treated cell, at the end of the treatment. The cytoplasm forms long extrusions, some of which contain only hyaloplasm. These extrusions tend to fuse their free extremities, thus forming large vacuoles at the cell periphery. The aspect of the cell is otherwise normal. 2 0 , 0 0 0 × . Fig. 6. Treated cell, 2 hr after the end of the treatment. A few elements of the Golgi complex contain an electron-opaque substance. Mitochondria retain their normal appearance. One centriole and a great number of polysomes are visible. 2 0 , 0 0 0 ?..: .
Fig. 7. Treated cell, 2 hr after the end of the treatment. Many Golgi elements are filled with an electronopaque substance, and the mitochondria show denser matrix and clearer intracristal spaces. Thin bundles of filaments are visible in the Golgi region. Coiled polysomes are visible in the cytoplasm (arrows). 30,000 × .
Fig. 8. Treated cell, 4 hr after the end o f the treatment. ,'Vlitochondria contain star-shaped empty spaces ; the mitochondrial matrix clumps in dark bands at their periphery. The space between outer and inner membranes remains unchanged, and there is no obvious variation in mitochondrial size. Arrow points to a mitochondrion that is presumably undergoing fragmentation. 44,000 × . Inset : a mitochondrion that has lost its outer membrane and resembles a loose skein. 44,000 x . Fig. 9.
Treated cell, 4 hr after the end o]" the treatment. Chromatin clamps at one pole o f the nucleus. zllitochondria have the usual altered appearance. 15,000 ~ .
Fig. 10. Treated ceil, 4 hr after the end o f the treatment. Chromatin is clumped at the periphery o f the nucleus; interchromatin granules tend to aggregate in groups in the nucleoplasm. The nucleolus is more compact, and its R N A granules are somewhat more prominent. 22,000 ×. Fig. 11. Treated cell, 4 hr after the end o f the treatment. The nuclear membrane encircles the single chromatin masses and ruptures, so that the nuclear sap mixes with the cytoplasmic content. A large nucleolus is still present. 22,000x.
Fig. 12. Treated cell, 6 hr after the end of the treatment. The cytoplasm contains only nuclear remnants and free ribosomes. Note the strong osmiophilia of the fragments of the nuclear membrane, ll/Iitochondria have totally disappeared. A4icrovi[[i have al~o disappeared, and the cell membrane is distended. 15,000/. Fig. 13.
Treated cell, 6 hr after the end of the treatment. Broad vacuoIation oJ"the cytoplasm, where a few altered mitochondria are still visible. 25,000 ~'~.
Fig. 14.
Cell treated with saline, 4 hr after the end of the treatment. No alterations are visible, and the cell retains its normal aspect 30,000 × .
Fine Structural Alterations of Yoshida Ascites Hepatoma Cells observed changes m a y be due to the action of the fixative. The mitochondrial changes come second to the appearance of the dark substance in the Golgi elements: they begin 2 hr after the end of the treatment and are present in all the cells 4 hours after the end of it. With the progressing of the lesion, a dramatic reorganization of the foldings of the inner mitochondrial membrane is seen, with formation of large, star-shaped empty spaces inside the mitochondria, and clumping of the matrix in dark bands at their periphery (Fig. 8). The outer membrane keeps its normal appearance, and there is no obvious decrease in total mitochondrial volume; moreover, the space between outer and inner membranes remains unchanged at the periphery of the mitochondria, notwithstanding the diminution of the whole membraneb o u n d matrical mass. Later, the mitochondrial external membrane dissolves, and the mitochondria assume the aspect of loose skeins; then they break up into fragments, which gradually disappear, leaving large cytoplasmic areas completely devoid of these organelles (Fig. 12). After the cytoplasmic alterations, also nuclear alterations appear. First chromatin clumps at the periphery of the nucleus (Fig. 9), then the interchromatin granules become separate and tend to aggregate in groups in the nucleoplasm (Fig. 10). The nucleolus does not seem markedly affected: it becomes more compact and its R N A granules become more prominent (Figs. 10 and 11). Later, the nuclear membrane encircles the single chromatin masses and then it ruptures (Fig. 11), so that the nuclear sap mixes with the cytoplasmic content, and the interchromatin granules and nucleolus become free in the cytoplasm. Often, at this stage, the nuclear membrane is more osmiophilic than normal. Nuclear alterations begin in a few cells as soon as 2 hr after the end of the treatment, and affect nearly all cells 4 hr after the end of the treatment. When nuclear alterations begin, polyribosomes disappear from the cytoplasm, while free ribosomes remain always in great number. At this stage, many cells show broad cytoplasmic vacuolation, which extends from the cell border to the Golgi region (Fig. 13). The cell membrane becomes distended, microvilli disappear, and the cells are transformed into roundish bodies full of ribosomes, with no other organelles except a few nuclear remnants (Fig. 12). The cells present in the samples taken 6 hr after the end of the treatment are invariably all so deeply damaged, as to render any possibility of recovery extremely unlikely. Some cells, however, seem to escape the action of the drug.
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In fact, very few cells are present in the ascitic fluid both 24 and 48 hr after the end of the treatment, but they have a perfectly normal appearance. Control cells, i.e. cells treated with saline, keep their normal aspect throughout the whole experiment (Fig. 14).
DISCUSSION For a satisfactory discussion of the observed ultrastructural changes, good information on the concomitant biochemical changes would be necessary. The available data demonstrate an inhibition of protein synthesis [7-10], but since biochemical information is on the whole scanty, morphological interpretation is tentative. However, it seems of interest to have succeeded in tracing a chronological sequence of morphological lesions: first the Golgi complex is affected, second the mitochondria, and third the nucleus. This sequence might provide a basis to direct future biochemical research. The modifications induced in the cell surface, such as the formation of long cytoplasmic extrusions, are probably not specific, since they have been observed also in Ehrlich ascites tumour cells following treatment with a number of different substances [17]. They might be due to variations in the surface tension at the b o u n d a r y between cell membrane and surrounding medium. Occasional and aspecific seems also to the formation of large vacuoles at the cell periphery, by fusion of the extrusions' ends; it seems in no way related to an increased pinocytosis [18], since the number of pinocytotic vesicles present in the inner cytoplasmic regions is not increased. All the other alterations seem to be peculiar to the action of DIBA. The nature of the substance that fills the Golgi elements remains obscure: whether it derives from an accumulation of lipids, from a strong condensation of the usual Golgi content, or from the deposition of a new substance elaborated in the Golgi complex under the action of DIBA, can not be decided at present. Anyway, even if it cannot be ascertained what kind of reactions take place in the Golgi complex under the action of DIBA, it can be affirmed that this organelle is the first target hit by the drug. The degenerative changes undergone by mitochondria do not seem to resemble any of the mitochondrial alterations found in different pathological conditions [19]. Generally, an increase of the matrix density was found either in enlarged mitochondria following storage of foreign materials [20], or in shrunken mito.
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chondria, as a consequence of the concentration of their content [21-23]. In our case, the mitochondrial volume does not seem to change. To the increase of matrix density corresponds a diminution of the density of the intracristal spaces, as if there was a passage of substance from the intra- to the intermembranous compartments, or, more likely, a passage of water in the opposite direction. The aspect of these mitochondria is partially comparable to that of mitochondria after active removal of Ca ++ and Mg~ [24] or of ADP-activated phosphorylating mitochondria [25], where extrusion of water is known to take place [26, 27]. The mitochondrial changes following treatment with D I B A might be due to a simple redistribution of ions or metabolites within the mitochondrial compartments, or to a damage of the inner membrane, with changes in its molecular components and loss of capacity to regulate water efflux. It would be interesting to know whether these changes are due to a direct action of DIBA, or if this action is mediated by the substance(s) elaborated by the Golgi complex. The fact that mitochondrial alterations always begin after the appearance of the electron-opaque substance in the Golgi elements can be tentatively taken as a support to the latter view; however, one can not rule out the possibility that a longer time must elapse before the damage induced by DIBA in the mitochondria becomes patent. At variance with what seems to happen in most other cells, both in normal and pathological conditions [28-33], the process of mitochondrial disruption seems to take place by simple fragmentation and subsequent dissolution of the fragments in the cytoplasm, without formation of autophagic vacuoles. In the nucleus, chromatin appears deeply affected, while the nucleolus retains a relatively
normal appearance. This might represent the morphological basis of a derangement in D N A and R N A synthesis at the chromatin level [34-37], while nucleolar R N A remains unchanged. Subsequent alterations, such as rupture of the nuclear membrane and nuclear fragmentation, are most likely the consequence of chromatin damage. On the other hand, nuclear alterations might not be directly related to the action of the drug, but might represent only a stage in cell death, brought about by the impairment or suppression of mitochondrial function. The disappearance of polyribosomes from the cytoplasm is a consequence of the derangement of nuclear R N A synthesis and may explain the diminution of cytoplasmic protein synthesis. The final picture of swollen, rounded cells with marked cytoplasmic vacuolation is the usual aspect of cells undergoing necrosis [3840]. It is not possible to establish why the cells present in the ascitic fluid both 24 and 48 hr after the end of the treatment are apparently undamaged. It seems that the lesion is of the "all-or-none" type, since no recovering cell was seen at whatsoever time. One may guess that the surviving cells are descendants either of genetically resistant cells or of cells that during the treatment were hidden in some abdominal recess, and were therefore not reached by a sufficient amount of the drug.
Acknowledgements- The author thanks Prof. E. Ciaranfi for suggesting this research, Drs. A. Perin and A. Arnaboldi for treating the animals, and Miss N. Barbieri for valuable technical assistance. This research was supported in part by a grant of the C.N.R. National Centre of Experimental Medicine.
RESUME
Vingt rats de souche Wistar, porteurs de l'Mpatome ascitique de l/'oshida, onl gtg soumis une infusion intrapgritongale, avec une solution d'aldghyde L-grylhro-~, ~-dihydroxybutyrique. Pour l'examen des cellules tumorales au microscope glectronique, on a prglevg des gchantillons de liquide ascitique avant el ~ latin du traitement, ainsi que 2, 4, 6, 24 et 48 heures apr~s latin de celui-ci. Les premieres allgrations des cellules tumorales apparaissent ~ latin du trailement ; elles se manifestent par la formation de longues excroissances cytoplasmiques ~ la pgriphgrie des cellules et par l' apparition d'une substance tr~s dense g, l'int~rieur des vgsicules de Golgi. Apr~s ces prem#res altgrations, on constate que la matrice mitochondriale devient tr~s dense el que l'espace intracristal se dilate; ensuile le membrane mitochondriale extgrieure dispara~t et les mitochondries sefragmentent. Ces altgrations sont tr~s gvidentes 2 el 4 heures apr~s latin du traitement. Enfin, on observe des altgrations nuclgaires : la chromatine se condense ~ la pgriphgrie du noyau, tandis que les granules interchromatiniens se groupent dans le nuclgoplasme.
Fine Structural Alterations of Yoshida Ascites Hepatoma Cells Ensuite, la membrane nuclgaire se fragmente et le contenu du noyau se disperse dans le cytoplasme. Les cellules prgsentent alors les caractgristiques de la ngcrose. Les cellules prgsentes dans le liquide ascitique, 24 ou 48 heures aprks latin du traitement, sont tr~speu nombreuses et apparemment normales. S UMMAdR Y Yoshida ascites hepatoma-bearing Wistar rats were subjected to peritoneal infusion with a solution of L-erythro- et, ~-dihydroxybutyraldehyde. For the examination of the tumour cells, samples of the asciticfluid were taken at thefoUowing intervals : just before and immediately after the treatment, and then respectively 2, 4, 6, 24 and 48 hr after the end of it. Thetirst changes in Yoshida ascites hepatoma cells appear at the end of the treatment. They are: formation of long cytoplasmic extrusions at the periphery of the cells and appearance of an electron-opaque substance in the Golgi vesicles. Subsequent changes are: condensation of the mitochondrial matrix and dilatation of the intracristal spaces, followed by disruption of the mitochondrial outer membrane and fragmentation of the mitochondria. These changes are most evident both 2 and 4 hr after the end of the treatment. Then nuclear alterations follow, with chromatin clumping, separation of the interchromatin granules, rupture of the nuclear membrane and nuclearfragmentation. Finally, the cells undergo necrosis. The cells present in the asciticfluid respectively 24 and 48 hr after the end of the treatment are veryfew and have a normal aspect. ZUSAMMENFASSUNG
Zwanzig Wistar albino Ratten, Triiger von Yoshida-Ascites-Hepatom, wurden einer intraperitonealen Infusion mit einer Lgsung von L-Erythro- a, ~-dihydroxybutyraldehyd unterworfen. Fiir die elektronenmikroskopwidsche Untersuchung der HepatomzeUen wurden Proben der Ascites-Fliissigkeit vor und am Ende der Infusion entnommen, wei auch 2, 4, 6, 24 und 48 Stunden nach dem Ende der Infusion. Die ersten Veriinderungen der Hepatomzellen erscheinen am Ende der Infusion: sie bestehen in der Bildung langer Cytoplasma-Ausstiilpungen an der Zellperipherie und im Auftreten einer sehr dunklen Substanz in den Golgi-Blgschen. Nach diesen ersten Ver gnderungen wurde beobachtet, dass die mitochondriale Matrix sehr dunkel wird und die Intramembranriiume sich ausdehnen. Dann verschwindet die Aussenmembran der Mitochondrien, und die Mitochondrien zersplittern. Diese Ver~nderungen sind sehr deutlich 2 und 4 Stunden nach dem Ende der Infusion. Schliesslich wurden Kernveriinderungen beobachtet : das Chromatin kondensiert sich an der Kernperipherie, und die interchromatinen Granulationen gruppieren sich im Kernplasma. 1)arauf zerreisst die Kernmenbran und der Kerninhalt tritt in das Cytoplasma ein. Die Zellen zeigen dann die charakteristischen Merkmale der Nekrose. In den 24 und 48 Stunden nach dem Ende der Infusion entnommener Proben sind die Zellen sp~rlich und vollkommen normal. REFERENCES I. A. FURST, B. L. FREEDLANDER, F. A. FRENCH, H. GROSS and D. DEMSHER, ~Ethoxy-a-ketobutyraldehyde (kethoxal) as a carcinostatic agent in mouse tumours. Proc. Am. Ass. Cancer Res. 2, 204 (1957). 2. F.A. FRENCH and B. L. FREEDLANDER, Carcinostaticaction ofpolycarbonyl compounds and their derivatives. I. 3-Ethoxy-2-ketobutyraldehyde and related compounds. Cancer Res. 18, 172 (1958).
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Maria Bassi 4. H.G. PETERING,H. H. BUSKIRKand G. E. UNDERWOOD,The anti-tumour activity of 2-keto-3-ethoxybutyraldehyde bis(thio-semicarbazone) and related compounds. Cancer Res. 24, 357 (1964). 5. G. BRUNS,W. JUNGSTANDand H. KN~LL, Zur Wirkung des DL-Glycerinaldehyds aufdas Ehrlich-Ascites-Carcinom der weissen Maus. Naturwissenschaften 51~ 560 (1964). 6. E. CIARANFI,L. LORETI, A. BORGHETTIand G. G. Guidotti, Studies on the antitumour activity of aliphatic aldehydes--II. Effect on survival of Yoshida ascites hepatoma bearing rats. Europ. J. Cancer 1, 147 (1965). 7. E. CIARANFIand A. FONNESU,Sui rapporti fra metabolismo energetico e incorporazione di aminoacidi nelle proteine delle cellule di tumori-ascite. Atti Accad. Lincei, Rend. 32, 835 (1962). 8. G. G. GUIDOTTI, La gliceraldeide, un nuovo inibitore delle sintesi proteiche. Tumori 49, 303 (1963). 9. G.G. GUIDOTTI,A. FONNESUand E. CIARANFI,Inhibition of amino acid incorporation into protein ofYoshida ascites hepatoma cells by glyceraldehyde. CancerRes. 24, 900 (1964). 10. C.G. GUIDOTTI,L. LORETIand E. CIARANFI,Studies on the anti-tumour activity of aliphatic aldehydes--I. The mechanism of inhibition of amino acid incorporation into protein ofYoshida ascites hepatoma cells. Europ. J. Cancer 1, 23 (1965). 11. E. CIARANFI,personal communication. 12. A. PERIN, personal communication. 13. J . H . LUFT, Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytolg, 409 (1961). 14. E.S. REYNOLDS,The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. cell Biol. 17, 208 (1953). 15. A. BAiRATi, Jr., Submicroscopic structure of Yoshida ascites hepatoma. CancerRes. 21, 989 (1961). 16. M . G . FARQUIIARand G. E. PALADE,Junctional complexes in various epithelia. J. cellBiol. 17, 375 (1963). 17. M. RATZENHOFER and J. ZANGGER, Cytotoxische Effekte an Krebszellen. Verb. dtsch. Ges. Path. 443, 120 (1964). 18. H. HOLIER, Pinocytosis. Int. Rev. Cytol. 8, 481 (1959). 19. CH. ROUILLER,Physiological and pathological changes in mitochondrial morphology. Int. Rev. Cytol. 9, 227 (1960). 20. H. GANSLERand CH. ROUILLER,Modifications physiologiques et pathologiques du condriome. Schweiz. Z" allg. Path. 19, 217 (1956). 21. H. DAVID, Submikroskopische Ortho- und Pathomorphologie der Leber. Akademie Verlag, Berlin (1964). 22. M. WACHSTEINand M. BESEN, Electron microscopy of renal coagulative necrosis due to DL-serine, with special reference to mitochondrial pycnosis. Am. J. Pathol. 44, 383 (1964). 23. T. SUZUKI and F. K. MosworI, Electron microscopic studies of acute tubular necrosis. Early changes in proximal convoluted tubules of the rat kidney following subcutaneous injection of glycerin. Lab. Invest. 15, 1225 (1966). 24. C.C. HARRISand CH. LEONE,Some effects of EDTA and tetraphenylboron on the ultrastructure of mitochondria in mouse liver cells. J. cell Biol. 28, 405 (1966). 25. C . e . HACKENBROCK,Ultrastructural bases for metabolically linked mechanical activity in mitochondria. J. cell Biol. 30, 269 (1966). 26. C.A. PRICE, A. FONNESUand R. E. DAVIES, Movements of water and ions in mitochondria. Biochem. J. 64, 754 (1956). 27. A.L. LEHNINOER,Reversal ofthyroxine-inducedswelling ofrat liver mitochondria by adenosine triphosphate. J. biol. Chem. 234, 2187 (1959). 28. T. P. ASH~ORD and K. R. PORTER, Cytoplasmic components in hepatic cell lysosomes. J. cell. Biol. 12, 198 (1962). 29. A. B. NOVIKOFFand E. ESSNER, Cytolysomes and mitochondrial degeneration. J. cell Biol. 15, 140 (1962). 30. H. MOE and O. BEHNKE,Cytoplasmic bodies containing mitochondria, ribosomes, and rough surfaced endoplasmic membranes in the epithelium of the small intestine of newborn rats. J. cell Biol. 13~ 168 (1962). 31. Z. HRUBAN,H. SWIFT,F. W. DUNNand D. E. LEwis, Effect ofl3-3-furylalanine on the ultrastructure of the hepatocytes and pancreatic acinar cells. Lab. invest. 14, 70 (1965).
Fine Structural Alterations of Yoshida Ascites Hepatoma Cells 32. J . L . E . ERICSSONand W. H. GLINSMANN,Observations on the subcellular organization of hepatic parenchymal cells. I. Golgi apparatus, cytosomes and cytosegresomes in normal cells. Lab. Invest. 15, 750 (1966). 33. W . H . GLINSMANNandJ. L. E. ERICSSON,Observations on the subcellular organization of hepatic parenchymal cells. II. Evolution of reversible alterations induced by hypoxia. Lab. Invest. 15, 762 (1966). 34. E. D. HAY and J. P. REVEL, The fine structure of the DNA component of the nucleus. An electron microscopy study utilizing autoradiography to localize DNA synthesis. J. cell Biol. 16, 29 (1963). 35. J. L. SIRLIN,Facts and speculations on the function of nuclear components. In: The Cell Nucleus, pp. 35-48. J. S. MITCHELL(Editor), Academic Press, New York (1960). 36. J. H. RHo and J. BONNER,The site of ribonucleic acid synthesis in the isolated nucleus. Proc. nat. Acad. Sci. (U.S.A.) 47, 1611 (1961). 37. S. KARASAKI, Electron microscopic examination of the sites of nuclear RNA synthesis during amphibian embryogenesis. J. call Biol. 26~ 937 (I 965). 38. B.F. TRUMP,P.J. GOLDBLATTand R. E. STOWELL,An electron microscopy study of early cytoplasmic alterations in hepatic parenchymal cells of mouse liver during necrosis "in vitro" (autolysis). Lab. Invest. 11~ 986 (1963). 39. M. BASSI and A. BERNELLI-ZAzzERA,Ultrastructural cytoplasmic changes of liver cells after reversible and irreversible ischemia. Expt. Molec. Path. 3, 332 (1964). 40. G. MAJNO,Death of liver tissue. A review of cell death, necrosis and autolysis. In: The Liver Morphology, Biochemistry, Physiology, pp. 267-313. Ch. ROUILLER (Editor), Academic Press, New York (1964).
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Fine Structural Alterations of Yoshida Ascites Hepatoma cells following treatment with L-Erythro,p-Dihydroxybutyraldehyde MARIA BASSI Centro di Microscopia Elettronica, Politecni¢o di Milano, and Istituto di Patologia Generale, Universit~ di Milano, Milano, Italy
INTRODUCTION
commercial rat pellets and received tap water. Approximately 107 cells of Yoshida hepatoma (AH-130) were inoculated intraperitoneally and maintained in ascitic form by intraperitoneal transplantation. Five days after the injection, they were subjected to the following treatments [11]: - - t e n animals were subjected to a 4 hr intraperitoneal infusion with 10 ml of a 0-14 M solution of DIBA, made isotonic with NaC1. This solution was injected by means of a low flow infusion apparatus. - - t e n animals were subjected to a 4 hr intraperitoneal infusion with 10 ml of saline. For electron microscopy, 0.3 ml of ascitic fluid were drawn with a syringe from the peritoneal cavity of the animals at different time intervals: just before the treatment, at the end of it, and respectively 2, 4, 6, 24 and 48 hr after the end of it. The samples were put immediately into icechilled centrifuge tubes containing 6 ml of 2" 5% glutaraldehyde (Eastman Organic) Chemicals) in 0.15 M phosphate buffer, p H 7.3, and fixed for 15 min at + 4 ° C . Then, by centrifuging and resuspending the samples at each step of the procedure, they were washed in phosphate buffer, post-fixed in phosphate-
SEVERAL researches have shown that a n u m b e r of aliphatic aldehydes act as carcinostatic agents [1-6], probably on account of their capacity of inhibiting amino acid incorporation into protein [7-10]. One of these aldehydes, L-erythro-a, B-dihydroxybutyraldehyde (DIBA) was shown to possess a marked carcinostatic and carcinolytic activity at concentrations which are non-toxic to the tumourbearing animals, and was therefore selected for further studies. [11]. Microscopical observations on Yoshida ascites hepatoma cells treated with DIBA have shown that this substance induces marked nuclear alterations, leading to nuclear rhexis and cell death [12]. It seemed therefore interesting to study the morphology of these cells with the electron microscope, in an attempt to discover early modifications in the cell structure and to follow their progressive evolution towards cell death.
METHODS Twenty Wistar albino rats of either sex, weighing about 170 g, were used in these experiments. T h e y were fed "ad libitum" on a diet of 103
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buffered 1% OsO 4, dehydrated in ethanol, and the final pellet was embedded in Araldite [13]. When in absolute ethanol, the samples were impregnated with uranyl acetate in semisaturated solution. Ultrathin sections were obtained with an LKB Ultrotome, mounted on grids coated with carbon-stabilized celloidin film, stained with lead citrate [14], and examined in a Siemens Elmiskop 1 A at an accelerating voltage of 80 kV. Micrographs were taken at magnifications of 5000-30 000and enlarged photographically as necessary.
RESULTS Untreated cells The ultrastructure of Yoshida ascites hepatoma cells has already been described by Bairati [15]. However, since the fixative used in the present study differs from those used in previous works, additional informations on the sub-microscopic structures have been recorded and will be reviewed. At low magnification, the cells appear either isolated or assembled in groups of two or three (Fig. 1) ; they are roundish, with irregular contours due to the presence of a number of cytoplasmic projections (microvilli). At the site of contact between two cells, the adjacent plasma membranes fuse to form a junctional complex, where only two of the usual junctional structures are recognizable, i.e. the zonulae occludens and adhaerens [16]. The cytoplasm is dense and contains a large number of ribosomes, mitochondria, flattened profiles of the endoplasmic reticulum, a well developed Golgi apparatus, dense bodies of undetermined nature (possibly lysosomes), and bundles of filaments. At higher magnification (Fig. 2), the ribosomes appear often aggregated in irregular clusters or associated in coiled structures. The maximum observed length of these structures is about 2500 A (Fig. 2, inset). The endoplasmic reticulum, composed of flattened sacs, has a random disposition in the cytoplasm and shows long segments devoid of ribosomes. In these regions free particles, larger than ribosomes, are visible, and might be tentatively interpreted as glycogen particles. The Golgi complex is much developed, and is composed of several stacks of flattened sacs and a great number of small vesicles. Mitochondria have irregular, roundish or elongated shapes and a clear matrix; their internal membranes have variable lengths and are arranged either transversally or along the longer axis of the mitochondrion. Dense matrix granules are inconspicuous.
In the cytoplasm are often seen filaments, about 60 A in diameter and of indeterminate length. They occur in small, variously oriented bundles, which thicken around the nucleus and often seem to wrap it completely (Fig. 3). The cytoplasm surrounding the fibrous whorls is sometimes particularly rich in clustered ribosomes. The nucleus (Fig. 4) is large and often deeply lobated; chromatin is sometimes finely dis, persed, sometimes more prominent adjacent to the nuclear membrane. Perichromatin granules are often visible. The nucleolus is generally voluminous, of reticular appearance. DIBA-treated cells The sequence of alterations induced in Yoshida ascites hepatoma cells by DIBA treatment can be traced as follows : at first, formation of long cytoplasmic extrusions at the periphery of the cells, and appearance of a strongly electron-opaque substance in the Golgi vesicles ; next, mitochondrial alterations, with increase in matrix density and dilatation of the intracristal spaces, leading to mitochondrial disruption; then nuclear changes, with chromatin clumping and final nuclear rhexis. The first morphological alterations are the formation of long cytoplasmic extrusions (Fig. 5) and the appearance of a strongly electronopaque substance in the Golgi complex (Figs. 6 and 7). The cytoplasmic extrusions appear at the end of the perfusion, and tend to disappear after 2 hours. Later, they are no longer visible. They contain hyaloplasm, sometimes also ribosomes, but never other cell organelles. The free extremities of such extrusions tend to fuse and form large, electron-lucid vacuoles at the periphery of the cells. The electron-opaque substance appears in a few cells at the end of the treatment, and is present in all the cells 2 hr later. It begins to fill only a few Golgi vesicles (Fig. 6), then it extends to many more elements (Fig. 7). This substance has a great affinity for osmium tetroxide and is homogeneous. At this stage, in a few cells the number of coiled polysomes seems increased (Fig. 6); however, since this finding is not at all constant it will not be discussed here. Then the aspect of mitochondria changes: their matrix becomes very dense and the intracristal spaces dilate (Fig. 7). Glutaraldehyde is known to induce profound alterations of the mitochondrial matrix. However, the constant appearance of mitochondrial alterations in DIBA-treated cells and their absence in pairedfixed controls rule out the possibility that the
Keys to symbols E R = endoplasmic retieulum F - - filaments G -- Golgi complex L = lysosomes
M = mitochondria N -- nucleus n -- nucleolus
All figures are o f Yoshida ascit,es hepatoma cells.
Fig. 1. Two untreated cells in close contact. The cell membranes form a number o f microvilli, and at the sttes o f contact they fuse to form junctional complexes. The cytoplasm contains irregularly shaped mitochondria, a f e w flattened profiles o f the endoplasmic reticulum, a well developed Golgi apparatus, many ribosomes and bundles o f filaments. In the lower cell the filaments contact two lysosome-like structures. The nuclei show finely dispersed chromatin. 15,000×.
(to face p. 104)
Fig. 2. Untreated cell. The mitochondria vary in size and shape, and show a clear matrix and variously oriented "cristae." A number of ribosomes associated in coiled structures is visible in the cytoplasm (arrows). Many segments of the endoplasmic reticulum are devoid of ribosomes. Close to them free particles, larger than ribosomes, are visible, and might represent glycogen particles (double arrow). 30,000 x Inset : a coiled polysome, measuring about 2,500 .'\ in length. 80,000 .:.
Fig. 3. Untreated cell. This section cuts the nucleus at one pole, and shows that it is completely wrapped by a thick bundle of filaments. The surrounding cytoplasm is particularly rich in clustered ribosomes. 30,000 × . Fig. 4. Untreated cell. The nucleus is lobated, with chromatin somewhat more prominent adjacent to the nuclear membrane. The nncleolus is large, of reticular appearance. One perichromatin granule is visible (arrow). 2 5 , 0 0 0 × .
Fig. 5. Treated cell, at the end of the treatment. The cytoplasm forms long extrusions, some of which contain only hyaloplasm. These extrusions tend to fuse their free extremities, thus forming large vacuoles at the cell periphery. The aspect of the cell is otherwise normal. 2 0 , 0 0 0 × . Fig. 6. Treated cell, 2 hr after the end of the treatment. A few elements of the Golgi complex contain an electron-opaque substance. Mitochondria retain their normal appearance. One centriole and a great number of polysomes are visible. 2 0 , 0 0 0 ?..: .
Fig. 7. Treated cell, 2 hr after the end of the treatment. Many Golgi elements are filled with an electronopaque substance, and the mitochondria show denser matrix and clearer intracristal spaces. Thin bundles of filaments are visible in the Golgi region. Coiled polysomes are visible in the cytoplasm (arrows). 30,000 × .
Fig. 8. Treated cell, 4 hr after the end o f the treatment. ,'Vlitochondria contain star-shaped empty spaces ; the mitochondrial matrix clumps in dark bands at their periphery. The space between outer and inner membranes remains unchanged, and there is no obvious variation in mitochondrial size. Arrow points to a mitochondrion that is presumably undergoing fragmentation. 44,000 × . Inset : a mitochondrion that has lost its outer membrane and resembles a loose skein. 44,000 x . Fig. 9.
Treated cell, 4 hr after the end o]" the treatment. Chromatin clamps at one pole o f the nucleus. zllitochondria have the usual altered appearance. 15,000 ~ .
Fig. 10. Treated ceil, 4 hr after the end o f the treatment. Chromatin is clumped at the periphery o f the nucleus; interchromatin granules tend to aggregate in groups in the nucleoplasm. The nucleolus is more compact, and its R N A granules are somewhat more prominent. 22,000 ×. Fig. 11. Treated cell, 4 hr after the end o f the treatment. The nuclear membrane encircles the single chromatin masses and ruptures, so that the nuclear sap mixes with the cytoplasmic content. A large nucleolus is still present. 22,000x.
Fig. 12. Treated cell, 6 hr after the end of the treatment. The cytoplasm contains only nuclear remnants and free ribosomes. Note the strong osmiophilia of the fragments of the nuclear membrane, ll/Iitochondria have totally disappeared. A4icrovi[[i have al~o disappeared, and the cell membrane is distended. 15,000/. Fig. 13.
Treated cell, 6 hr after the end of the treatment. Broad vacuoIation oJ"the cytoplasm, where a few altered mitochondria are still visible. 25,000 ~'~.
Fig. 14.
Cell treated with saline, 4 hr after the end of the treatment. No alterations are visible, and the cell retains its normal aspect 30,000 × .
Fine Structural Alterations of Yoshida Ascites Hepatoma Cells observed changes m a y be due to the action of the fixative. The mitochondrial changes come second to the appearance of the dark substance in the Golgi elements: they begin 2 hr after the end of the treatment and are present in all the cells 4 hours after the end of it. With the progressing of the lesion, a dramatic reorganization of the foldings of the inner mitochondrial membrane is seen, with formation of large, star-shaped empty spaces inside the mitochondria, and clumping of the matrix in dark bands at their periphery (Fig. 8). The outer membrane keeps its normal appearance, and there is no obvious decrease in total mitochondrial volume; moreover, the space between outer and inner membranes remains unchanged at the periphery of the mitochondria, notwithstanding the diminution of the whole membraneb o u n d matrical mass. Later, the mitochondrial external membrane dissolves, and the mitochondria assume the aspect of loose skeins; then they break up into fragments, which gradually disappear, leaving large cytoplasmic areas completely devoid of these organelles (Fig. 12). After the cytoplasmic alterations, also nuclear alterations appear. First chromatin clumps at the periphery of the nucleus (Fig. 9), then the interchromatin granules become separate and tend to aggregate in groups in the nucleoplasm (Fig. 10). The nucleolus does not seem markedly affected: it becomes more compact and its R N A granules become more prominent (Figs. 10 and 11). Later, the nuclear membrane encircles the single chromatin masses and then it ruptures (Fig. 11), so that the nuclear sap mixes with the cytoplasmic content, and the interchromatin granules and nucleolus become free in the cytoplasm. Often, at this stage, the nuclear membrane is more osmiophilic than normal. Nuclear alterations begin in a few cells as soon as 2 hr after the end of the treatment, and affect nearly all cells 4 hr after the end of the treatment. When nuclear alterations begin, polyribosomes disappear from the cytoplasm, while free ribosomes remain always in great number. At this stage, many cells show broad cytoplasmic vacuolation, which extends from the cell border to the Golgi region (Fig. 13). The cell membrane becomes distended, microvilli disappear, and the cells are transformed into roundish bodies full of ribosomes, with no other organelles except a few nuclear remnants (Fig. 12). The cells present in the samples taken 6 hr after the end of the treatment are invariably all so deeply damaged, as to render any possibility of recovery extremely unlikely. Some cells, however, seem to escape the action of the drug.
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In fact, very few cells are present in the ascitic fluid both 24 and 48 hr after the end of the treatment, but they have a perfectly normal appearance. Control cells, i.e. cells treated with saline, keep their normal aspect throughout the whole experiment (Fig. 14).
DISCUSSION For a satisfactory discussion of the observed ultrastructural changes, good information on the concomitant biochemical changes would be necessary. The available data demonstrate an inhibition of protein synthesis [7-10], but since biochemical information is on the whole scanty, morphological interpretation is tentative. However, it seems of interest to have succeeded in tracing a chronological sequence of morphological lesions: first the Golgi complex is affected, second the mitochondria, and third the nucleus. This sequence might provide a basis to direct future biochemical research. The modifications induced in the cell surface, such as the formation of long cytoplasmic extrusions, are probably not specific, since they have been observed also in Ehrlich ascites tumour cells following treatment with a number of different substances [17]. They might be due to variations in the surface tension at the b o u n d a r y between cell membrane and surrounding medium. Occasional and aspecific seems also to the formation of large vacuoles at the cell periphery, by fusion of the extrusions' ends; it seems in no way related to an increased pinocytosis [18], since the number of pinocytotic vesicles present in the inner cytoplasmic regions is not increased. All the other alterations seem to be peculiar to the action of DIBA. The nature of the substance that fills the Golgi elements remains obscure: whether it derives from an accumulation of lipids, from a strong condensation of the usual Golgi content, or from the deposition of a new substance elaborated in the Golgi complex under the action of DIBA, can not be decided at present. Anyway, even if it cannot be ascertained what kind of reactions take place in the Golgi complex under the action of DIBA, it can be affirmed that this organelle is the first target hit by the drug. The degenerative changes undergone by mitochondria do not seem to resemble any of the mitochondrial alterations found in different pathological conditions [19]. Generally, an increase of the matrix density was found either in enlarged mitochondria following storage of foreign materials [20], or in shrunken mito.
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chondria, as a consequence of the concentration of their content [21-23]. In our case, the mitochondrial volume does not seem to change. To the increase of matrix density corresponds a diminution of the density of the intracristal spaces, as if there was a passage of substance from the intra- to the intermembranous compartments, or, more likely, a passage of water in the opposite direction. The aspect of these mitochondria is partially comparable to that of mitochondria after active removal of Ca ++ and Mg~ [24] or of ADP-activated phosphorylating mitochondria [25], where extrusion of water is known to take place [26, 27]. The mitochondrial changes following treatment with D I B A might be due to a simple redistribution of ions or metabolites within the mitochondrial compartments, or to a damage of the inner membrane, with changes in its molecular components and loss of capacity to regulate water efflux. It would be interesting to know whether these changes are due to a direct action of DIBA, or if this action is mediated by the substance(s) elaborated by the Golgi complex. The fact that mitochondrial alterations always begin after the appearance of the electron-opaque substance in the Golgi elements can be tentatively taken as a support to the latter view; however, one can not rule out the possibility that a longer time must elapse before the damage induced by DIBA in the mitochondria becomes patent. At variance with what seems to happen in most other cells, both in normal and pathological conditions [28-33], the process of mitochondrial disruption seems to take place by simple fragmentation and subsequent dissolution of the fragments in the cytoplasm, without formation of autophagic vacuoles. In the nucleus, chromatin appears deeply affected, while the nucleolus retains a relatively
normal appearance. This might represent the morphological basis of a derangement in D N A and R N A synthesis at the chromatin level [34-37], while nucleolar R N A remains unchanged. Subsequent alterations, such as rupture of the nuclear membrane and nuclear fragmentation, are most likely the consequence of chromatin damage. On the other hand, nuclear alterations might not be directly related to the action of the drug, but might represent only a stage in cell death, brought about by the impairment or suppression of mitochondrial function. The disappearance of polyribosomes from the cytoplasm is a consequence of the derangement of nuclear R N A synthesis and may explain the diminution of cytoplasmic protein synthesis. The final picture of swollen, rounded cells with marked cytoplasmic vacuolation is the usual aspect of cells undergoing necrosis [3840]. It is not possible to establish why the cells present in the ascitic fluid both 24 and 48 hr after the end of the treatment are apparently undamaged. It seems that the lesion is of the "all-or-none" type, since no recovering cell was seen at whatsoever time. One may guess that the surviving cells are descendants either of genetically resistant cells or of cells that during the treatment were hidden in some abdominal recess, and were therefore not reached by a sufficient amount of the drug.
Acknowledgements- The author thanks Prof. E. Ciaranfi for suggesting this research, Drs. A. Perin and A. Arnaboldi for treating the animals, and Miss N. Barbieri for valuable technical assistance. This research was supported in part by a grant of the C.N.R. National Centre of Experimental Medicine.
RESUME
Vingt rats de souche Wistar, porteurs de l'Mpatome ascitique de l/'oshida, onl gtg soumis une infusion intrapgritongale, avec une solution d'aldghyde L-grylhro-~, ~-dihydroxybutyrique. Pour l'examen des cellules tumorales au microscope glectronique, on a prglevg des gchantillons de liquide ascitique avant el ~ latin du traitement, ainsi que 2, 4, 6, 24 et 48 heures apr~s latin de celui-ci. Les premieres allgrations des cellules tumorales apparaissent ~ latin du trailement ; elles se manifestent par la formation de longues excroissances cytoplasmiques ~ la pgriphgrie des cellules et par l' apparition d'une substance tr~s dense g, l'int~rieur des vgsicules de Golgi. Apr~s ces prem#res altgrations, on constate que la matrice mitochondriale devient tr~s dense el que l'espace intracristal se dilate; ensuile le membrane mitochondriale extgrieure dispara~t et les mitochondries sefragmentent. Ces altgrations sont tr~s gvidentes 2 el 4 heures apr~s latin du traitement. Enfin, on observe des altgrations nuclgaires : la chromatine se condense ~ la pgriphgrie du noyau, tandis que les granules interchromatiniens se groupent dans le nuclgoplasme.
Fine Structural Alterations of Yoshida Ascites Hepatoma Cells Ensuite, la membrane nuclgaire se fragmente et le contenu du noyau se disperse dans le cytoplasme. Les cellules prgsentent alors les caractgristiques de la ngcrose. Les cellules prgsentes dans le liquide ascitique, 24 ou 48 heures aprks latin du traitement, sont tr~speu nombreuses et apparemment normales. S UMMAdR Y Yoshida ascites hepatoma-bearing Wistar rats were subjected to peritoneal infusion with a solution of L-erythro- et, ~-dihydroxybutyraldehyde. For the examination of the tumour cells, samples of the asciticfluid were taken at thefoUowing intervals : just before and immediately after the treatment, and then respectively 2, 4, 6, 24 and 48 hr after the end of it. Thetirst changes in Yoshida ascites hepatoma cells appear at the end of the treatment. They are: formation of long cytoplasmic extrusions at the periphery of the cells and appearance of an electron-opaque substance in the Golgi vesicles. Subsequent changes are: condensation of the mitochondrial matrix and dilatation of the intracristal spaces, followed by disruption of the mitochondrial outer membrane and fragmentation of the mitochondria. These changes are most evident both 2 and 4 hr after the end of the treatment. Then nuclear alterations follow, with chromatin clumping, separation of the interchromatin granules, rupture of the nuclear membrane and nuclearfragmentation. Finally, the cells undergo necrosis. The cells present in the asciticfluid respectively 24 and 48 hr after the end of the treatment are veryfew and have a normal aspect. ZUSAMMENFASSUNG
Zwanzig Wistar albino Ratten, Triiger von Yoshida-Ascites-Hepatom, wurden einer intraperitonealen Infusion mit einer Lgsung von L-Erythro- a, ~-dihydroxybutyraldehyd unterworfen. Fiir die elektronenmikroskopwidsche Untersuchung der HepatomzeUen wurden Proben der Ascites-Fliissigkeit vor und am Ende der Infusion entnommen, wei auch 2, 4, 6, 24 und 48 Stunden nach dem Ende der Infusion. Die ersten Veriinderungen der Hepatomzellen erscheinen am Ende der Infusion: sie bestehen in der Bildung langer Cytoplasma-Ausstiilpungen an der Zellperipherie und im Auftreten einer sehr dunklen Substanz in den Golgi-Blgschen. Nach diesen ersten Ver gnderungen wurde beobachtet, dass die mitochondriale Matrix sehr dunkel wird und die Intramembranriiume sich ausdehnen. Dann verschwindet die Aussenmembran der Mitochondrien, und die Mitochondrien zersplittern. Diese Ver~nderungen sind sehr deutlich 2 und 4 Stunden nach dem Ende der Infusion. Schliesslich wurden Kernveriinderungen beobachtet : das Chromatin kondensiert sich an der Kernperipherie, und die interchromatinen Granulationen gruppieren sich im Kernplasma. 1)arauf zerreisst die Kernmenbran und der Kerninhalt tritt in das Cytoplasma ein. Die Zellen zeigen dann die charakteristischen Merkmale der Nekrose. In den 24 und 48 Stunden nach dem Ende der Infusion entnommener Proben sind die Zellen sp~rlich und vollkommen normal. REFERENCES I. A. FURST, B. L. FREEDLANDER, F. A. FRENCH, H. GROSS and D. DEMSHER, ~Ethoxy-a-ketobutyraldehyde (kethoxal) as a carcinostatic agent in mouse tumours. Proc. Am. Ass. Cancer Res. 2, 204 (1957). 2. F.A. FRENCH and B. L. FREEDLANDER, Carcinostaticaction ofpolycarbonyl compounds and their derivatives. I. 3-Ethoxy-2-ketobutyraldehyde and related compounds. Cancer Res. 18, 172 (1958).
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