Ultrastructural hepatic changes following the administration of benzylideneyohimbol

Ultrastructural Administration FELIX

Hepatic Changes Following of Benzylideneyohimboll,2,3

the

A. DE LA IGLESIA, JUAN C. SOSA-LUCERO,AND GEORGE LUMB

The Warner-Lambert

Research Institute of Canada, Sheridan Park, Ontario, and the Department of Pathology, University of Toronto, Toronto, Ontario, Canada Received January 20, I969

Ultrastructural Hepatic Changes Following the Administration of Benzylideneyohimbol.DELA IGLESIA,FELIX A., SOSA-LUCERO,JUAN C.,and LUMB, GEORGE (1970). Toxicol. Appf. Pharmacof. 16,239-255.The administration of low levels of benzylideneyohimbol, a potential nonsteroid, anti-inflammatory agent,together with a nutritionally adequatediet to rats and dogs,doesnot induce light microscopicor biochemicalchangesin the liver; however, several ultrastructural modifications are found after administrationof the drug for 4,8, and 16weeksto rats and for 12weeksto dogs. In rats receiving 30 mg/kg of drug daily, mitochondrial changes appearedat 4 weeksand were present also at 8 and 16 weeks.Bizarreshapedorganelleswerefound to be exclusivelylocatedin the central areasof the liver lobule in this species.Hepatocytesfrom dogsreceiving 60 mg/kg alsoevidencedpeculiar mitochondrial forms, someof which are frequently observedin humanhepatic injury. In dogsthesemitochondrial aberrations were located in the peripheralzonesof the liver lobule. Other cytoplasmic changes, including smooth endoplasmicreticulum hypertrophy, were observedin both species.It is possiblethat the morphological alterations found representbasic compensatorymechanismsaimed to support more fundamental cellular processes rather than the preservation of organelle architecture. There are several morphological responsesof liver cells to injurious agents in which mitochondria participate by undergoing variations in structure. These include increase in size (Svoboda and Higginson, 1964) swelling with or without the apparent loss of matrix (Trump et al., 1965),increasein the number of disconformity of cristae (Wilson and Leduc, 1963), lossofaggregation of densegranules(Herman and Bensch, 1967),and appearance of inclusion bodies (Minick et al., 1965) or paracrystalline filaments (Ericsson et al., 1966). Changes affecting mitochondria, of possibletoxic origin, may 1 These studies have been partially supported by the National Research Council of Canada, Grant Toxicity 867. 2 Preliminary results were presented at the 65th Annual Meeting of the American Association of Pathologists and Bacteriologists, Chicago, Illinois, March l-3, 1968, and at the Annual Meeting of the European Society for the Study of Drug Toxicity, Oxford, England, April 8-10, 1968. 3 The series of experiments described here were carried out within the Guiding Principles for the Care of Laboratory Animals, prepared by the Committee on Animal Care of the Canadian Federation of Biological Societies. 239

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have important biochemical implications without necessarily becoming apparent on routine examination. In order to evaluate minimal mitochondrial changes in hepatic parenchymal cells, experiments were designed using a drug with known hepatotoxic properties in rats and dogs. A potential nonsteroid, anti-inflammatory agent, 1%benzylideneyohimbol, has been found in preliminary studies in our laboratories to induce well defined liver lesions including cell necrosis. This compound, when administered to rats and dogs under controlled dietary conditions and at 10 % of the damaging dose level, does not induce easily detectable alterations in the liver; it provides, therefore, the possibility of studying the minimal reaction of the liver to the drug effects (de la Iglesia et al., 1968b, 1969). The purpose of the present investigation is to describe and discuss the morphological ultrastructural changes of the liver cells of rats and dogs receiving the drug, with emphasis on the changes taking place in mitochondria. METHODS Male Wistar albino rats, 95-150 g, were allotted to two groups, experimental and control. Control animals were pair-fed individually. Purebred beagle dogs of either sex, 6-8 kg, were assigned also to an experimental and control group. The animals were housed in individual stainless steel cages in air-conditioned, well ventilated rooms. A semisynthetic, well balanced diet was designed in accordance with the nutritional requirements of the animals, containing (in grammes per 100 g of diet): 26.50 g of casein, 2.00 g of corn oil (Mazola), 43.60 g of sucrose, 21.80 g of corn starch,40.54 g of cod liver oil, 1 g of vitamin fortification mixture. 4 Five grams of salt mixture4 (Hubbell Mendel and Wakeman), was added to every 100 g of final mixture of diet. The diet was mixed once a week in a Hobart food mixer and the complete diet was stored at 4” in closed containers. The compound benzylideneyohimbols (1 S-benzylidine-I 7-hydroxyyohimbane, W7166), was mixed with the diet at a level calculated to give a dose of 30 mg/kg of body weight daily and given to the experimental group of rats. A dose of 60 mg/kg in gelatin capsules was given orally each day to the experimental groups of dogs. These dose levels were selected to be one-tenth those that produce overt liver damage. Food intake was recorded every day, and the concentration of drug in the feed was adjusted to achieve the required dosage on a body weight basis for each animal. Body weights were recorded weekly. At the end of 4,8, and 16 weeks of drug administration, 5 rats from each group were sacrificed. Dogs were sacrificed after 12 weeks. The animals were anesthetized with ether (rats) or pentobarbital (dogs), and their livers were removed and quickly blotted and weighed. Then, the animals were killed by exsanguination. Immediately after the livers were weighed, small blocks of tissue from the median lobe were immersed in osmic fixative (Dalton, 1955), and subsequently dehydrated in alcohols and embedded in Epon (Luft, 1961). Ultrathin sections cut with Reichert and MT2 Porter-Blum ultramicrotomes were stained with lead (Reynolds, 1963a) and photographed in a Philips 300 electron microscope. 4 Obtained from General Biochemicals, Chagrin Falls, Ohio. 5 Supplied by Warner-Lambert Research Institute, Morris Plains, New Jersey.

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Liver portions from the same lobe were fixed in neutral formalin. Frozen sections were stained with oil red 0, and paraffin sections were stained in hematoxylin-eosin, 1~x01 fast blue (Laqueur, 1950) chromotrope 2R (Roque, 1953) and periodic acidSchiff. Selection of lobular areas to be examined under the electron microscope was done in 1 p sections of plastic embedded tissue and stained according to Trump et al. (1961). RESULTS Light Microscopic

Findings

The architecture of tho liver lobule was well preserved in the rats killed at 4,8, and 16 weeks and in dogs killed after 12 weeks. No evidence of pathologic alterations could be seen in the paraffin-embedded tissues. Glycogen was moderately diminished in experimental rats and dogs, and no fatty changes were observed at the intervals studied. Histologic evaluation of thin sections of plastic-embedded material permitted detailed observations of the hepatocytic cytoplasm. In the rats killed at 8 and 16 weeks, as well as the dogs killed at 12 weeks, a moderate degree of mitochondrial enlargement was observable in those groups of animals receiving the drug. This enlargement, between 2 and 2.5 p was not constantly present in every cell. In rats, it was located in the center of the liver lobule, and in dogs it was exclusively in the periportal and midzonal areas. Male dogs receiving the drug evidenced a moderate degree of cytoplasmic enlargement. Electron Microscopic

Findings

For comparative purposes, the ultrastructural hepatocytic changes found in the different areas of the liver lobule have been grouped according to the different cytoplasmic organelles. Mitochondria. The ultrastructural changes in mitochondria in the liver of rats receiving the drug were located in the center of the lobule, and these were consistently found at the experimental periods studied. Figure 1 represents an example of the normal mitochondrial configuration observed in the control animals from the central portion of the hepatic lobule. Hepatocytes from the centrilobular zones in experimental rats showed misshapen mitochondria usually diffused throughout the cell. Almost every mitochondrion was affected in some degree (Fig. 2), and about five to six circular rows of cells surrounding the central vein showed these altered organelles. Among the variety of distortions, there were annular, club-shaped, and elongated forms (Figs. 3-6). Some mitochondria had coalescent membranes and the ring-shaped varieties were usually found encircling small droplets of fat, portions of endoplasmic reticulum, glycogen rosettes, microbodies or sometimes other mitochondria (Figs. 5-8). Cristae mitochondriales were found shortened, curled, and decreased in number. Matrical rarefaction and spurious condensations appeared as an early change in these organelles (4 weeks) (Fig. 9) and persisted at later stages of the observations (8 and 16 weeks). Mitochondria from the periphery of the liver lobule in these experimental animals were not affected. In the liver of dogs, the bizarre-shaped mitochondria of the type described for rats were seldom present (Fig. 10). A distinct feature from the canine liver was that mitochondria from the central areas of the liver lobule were unaffected. Peripheral lobular 9

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FIG. 1. Control rat liver killed at week 16. Liver cell from the centrilobular area shows normal configuration of mitochondria, microbodies, and rough endoplasmic reticulum. Lead stain. x 9000.

zones were the only areas in which mitochondrial involvement was seen.There were no

differences referable to sex. The mitochondrial changes observed in dogs receiving the drug consisted of 3 main types: deviations in the arrangement of cristae mitochondriales, enlargement of the

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FIG. 2. Liver of rat treated with benzylideneyohimbol for 4 weeks. Bizarre-shaped mitochondria, diminished rough endoplasmic reticulum lamellae and incipient development of smooth endoplasmic reticulum are observed in cells from the center of the liver lobule. Lead stain. z 7500.

organelleswith concomitant changesin cristae and matrix, and inclusions of unknown nature (paracrystalline or crystalline-like or filamentous inclusions). Parallel stacks of cristae were common in some mitochondria and coexisted with

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FIGS. 3-8. Mitochondrial changes present in centrilobular hepatocytes from rats receiving benzylideneyohimbol and killed at 4.8, and 16 weeks. Figures 3 and 4 show mitochondrial elongation together with apposition of rough endoplasmic reticulum membranes (arrows) and mitochondrial membranes. found after 4 weeks. Ring-shaped mitochondria encompassing a microbody (arrow, Mi, Fig. 5), small droplets of fat (F, arrows in Fig. 6), portions of cytoplasm including endoplasmic reticulum and glycogen (ER and Gfy, indicated by the arrows in Fig. 7), and other mitochondria (A4, arrows in Fig. 8), observed in central hepatocytes of rats killed at 8 weeks. Lead stain. Figs. 3 and 4, ~26,000; Figs. 5-8, ~21,500.

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F‘IG. 9. Portion of hepatocyticcytoplasm of a rat receiving benzylideneyohimbol and killed at 4 wf:eks. The : arrows indicate areas of matrical rarefaction in mitochondria. Lead stain. ~20,6CO. FIG. 10. Canine liver. Altered (ring-shaped) mitochondrion embracing another mitochond rion (arr ow) found in the cytoplasm of a periportal hepatocyte. Lead stain. ~20,600. F‘IG. 11. Parallel arrangement of cristae in mitochondria from treated dog liver. Lead stain. ‘20. ,600.

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other organelles of normal configuration (Fig. 11). Enlargement of several mitochondria within a cell was observed frequently (Fig. 12). The matrices were rarefied, and sometimes dark, homogeneous inclusions were seen (Fig. 13). “Compartmentalization” of mitochondrial matrices was observed occasionally. Cristae adopted conspicuous and peculiar configurations, some resembling a braidlike disposition and others a coiled appearance (Figs. 14 and 15). Crystalline or filamentous inclusions were a prominent finding in the canine specimens. Usually 3-5 mitochondria containing these inclusions were seen in a given cell (Fig. 16). These structures had a periodicity of approximately 80 A intervals with dark filaments of similar thickness (Figs. 16 and 17). These parallel arrays of proteinaceous material were often arranged along the main mitochondrial axis. Mitochondria containing these crystalline substances were not always increased in size, but some reached IO-15 p in axial diameter. Shortening of the cristae was usually associated with the presence of this substance (Figs. 15 and 16). In livers of control animals, only two mitochondria containing these filamentous inclusions were found after searching all the blocks of tissues processed. Endoplasmic reticulum and microbodies. Well developed endoplasmic reticulum cisternae were observable in the livers of control rats and dogs. In canine hepatocytes, these cisternae were occasionally visualized close to the bile canaliculus (Fig. 18). In central hepatocytes from rats treated for 4 weeks, an incipient development of focal areas in the cytoplasm was observed, and it usually appeared as aggregates of smoothsurfaced vesicles and contorted tubules (Figs. 19 and 20). These small foci of developing smooth-surfaced membranes were closely associated with rough-surfaced membranes found in their vicinity. This close relationship suggesting continuity was clearly evident in some of the photographic material (Fig. 20). At later stages of the experiment (8 and 16 weeks) this development of areas of smooth-surfaced endoplasmic reticulum was significantly increased and sparse areas of rough endoplasmic reticulum were observed. Development of large cytoplasmic areas occupied by smooth-endoplasmic reticulum membranes was extreme in the liver cells of dogs receiving the drug for 12 weeks. These areas were usually devoid of other cytoplasmic organelles, and glycogen rosettes were encompassed among the membranes of hypertrophied endoplasmic reticulum (Figs. 21 and 22). Several areas of rarefaction of membranes resembling foci of hyaline substance were found, and more detailed description of this subject will be found elsewhere (de la Iglesia et al., 1968a). The presence of microbodies with or without nucleoid wascommon to the liver cells of rats and dogs. However, these particles were gradually increasing in number parallel to the development of smooth endoplasmic reticulum proliferation in drug-treated animals (rats and dogs). This absolute increase in the population of particles was significantly higher in the canine liver (Fig. 23). There were no alterations related to changes in size, shape, or content of these particles. The significance of the increase in the population of these organelles has been recently discussed (de la Iglesia, 1969). Golgiapparatus andlysosomes. Liver cells from treated rats at 4,8, and 16 weeks and dogs at 12 weeks evidenced a well developed Golgi apparatus usually in areas close to the bile canaliculus. The saccules were found sometimes dilated containing a material of light density. Golgi-related vesicles were numerous surrounding the saccules (Figs.

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F ‘IG. 12. Mitochondrial enlargement in periportal canine liver cells after 12 weeks of treatmen it. Lea .d stain. x I 1,700. F'IGS. I3- 15. Combined alterationsof size, cristaemitochondriales, and matrices inmitochondria fro]m doa liver. Zonal loss of matrical density is marked by (*) in Fig. 13. Coiled and braidlike arrangement (3f cris tae shown in Fig. 14 (arrows); in Fig. 15 they are shown in a perpendicular section (arrows). LeEtd stai n. Fig. 13, ~43,000; Figs 14and 15, ~28,800.

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3~s. 16 and 17. Filamentous inclusions in mitochondria from dog liver. Several mitocho In dria 7,2,3,4,5) displaying this material, are shown in Fig. 16. Figure 17 is included to demonstra te ) the ,iodicity displayed by the filaments (Fi). Lead stain. Fig. 16, ~27,000; Fig. 17, x35,ooO. 3~. 18. Cytoplasmic portion of a hepatocyte from a control dog. Areas of glycogen clusters (GUY> 1 parallel stacks of endoplasmic reticulum cisternae (arrow) are shown. Lead stain. ~21,000.

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FIG. 19. Rat liver, treated for 4 weeks. Well-defined areas of smooth endoplasmic reticulum (SER) are seen. Lead stain. x6200. FIG. 20. High power view of an area of contorted tubules of smooth endoplasmic reticulum (SER). The arrows indicate the transition point of rough-surfaced cisternae into the smooth-surfaced variant. Microbodies (Mi) are frequently observed in close association with these areas. Lead stain. ,~22,500.

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FI IGS. 21 and 22. Large areas of smooth endoplasmic reticulum development (SER) in canine hepartocytic: cytoplasm from the periportal lobular area. Figure 22 shows a higher power view demonstrat ing the 6mcompassment of glycogen rosettes by smooth membranes. Lead stain. Fig. 21, x9000; Fig. 22. x21, ,300.

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FE. 23. Canine liver. The illustrated area shows the parallel increase of microbodies (m), some of which do not display the characteristic nudeoid. Lead stain. Y 10,800. FIG. 24. Ultrastructural appearance of Golgi apparatus (Go) in rat liver after 16 weeks of treatment. Microbodies (Mi) appear closely associated. Lead stain. ~20,500. FIG. 25. Golgi apparatus (Go) in the pericanalicular area of a canine liver cell cytoplasm. A moderate increase in the number of multivesicular bodies (mvb) is observable. Lead stain. ~15,000.

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24 and 25). The paucity in the number of lysosomes and autoplagic vacuoles precluded further interpretations of significance for this study. DISCUSSION These results indicate that, in the liver of rats and dogs, mitochondria are the organelles primarily affected by the administration of benzylideneyohimbol. It has also been shown that hypertrophic development of smooth endoplasmic reticulum occurs, which represents the morphological basis for induction of drug-metabolizing enzymes. Previous studies from our laboratories have shown that after prolonged administration of the yohimbane derivative, 18-benzylideneyohimbol, there are no significant changes in the basic composition of liver fractions, but some decrease occurs in succinic dehydrogenase (de la Iglesia et al., 1969). Although mitochondria are affected in both canine and murine livers, these ultrastructural modifications are different. It is possible that, even when the nature of the changes are different in these animals, the mechanisms by which they are induced is the same. Mitochondria in the murine species have been shown to be altered in numerous experimental conditions, some of which were nutritionally induced (Hartroft, 1958) or metabolically related (Porta et al., 1967). Other types of experimental hepatic injury also mediate conspicuous mitochondrial changes (Roullier and Bernhard, 1956; Steiner and Baglio, 1963; Minick et al., 1965). A search of the literature has revealed only one report in which bizarre-shaped mitochondria were described in a single normal rat. (Stephens and Bils, 1965). In this case, the authors were unable to establish the origin of these changes. In the canine liver the mitochondrial changes are morphologically different from those in the rat, and they include paracrystalline intramitochondrial inclusions of unknown origin. It has been suggested that crystalline inclusions are degenerative phenomena (Laguens and Bianchi, 1963), and some investigators have assumed that they are derived from cristae (Svoboda and Manning, 1964) or result from aberrant molecular interactions of lipids with proteins (Svoboda and Higginson, 1964) which modify the lipoprotein complexes (Watrach, 1964). Mitochondria displaying paracrystalline inclusions in their matrices have been found in the liver of humans affected by a heterogeneous variety of pathologic conditions, and these have been discussed recently by Haust (1968). In these pathologic states, the inclusions were numerous and the affected mitochondria were usually enlarged and irregular. Wills (1965) found this type of mitochondrial inclusion in three humans without signs of hepatic disease and considered them as variants of the normal human pattern. He pointed out, however, that, in contradistinction to those found in pathologic conditions, they occurred in otherwise normal mitochondria and were considerably less frequent. Similar types of structures have not been reproduced in the liver of rats, although they were induced by lead poisoning in pig liver (Watrach, 1964) and by other means in canine hepatocytes (de la Iglesia et al., 1968b, 1969; Ericsson et al., 1966; Richter et al., 1966; Stein et al., 1966). Changes in the morphology of the particles are associated with uncoupling of oxidative phosphorylation (Wilson and Leduc, 1963; Smith and DeLuca, 1964), but impairment of the respiratory activity is not commonly present (Reynolds, 1963b). The change in shape has been assumed to provide an increased area to facilitate meta-

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bolic exchange with the surrounding cytoplasm (Stephens and Bils, 1965). This would result in a functional change by increasing the surface area without increasing the level of energy required within the framework of the thermodynamic principles. Ethionine induces protein synthesis inhibition due to a relative ATP deficiency (Villa-Trevino and Farber, 1962), and associated changes occur in mitochondria (Wood, 1965) which are preventable by ATP administration (Meldolesi et al., 1966). In addition, studies have shown the incorporation of labeled amino acids into protein by mitochondria (McLean et al., 1958; Truman and Korner, 1962). These systems contain a certain amount of DNA (Granick and Gibor, 1967), and it seems that they synthesize only the insoluble structural protein (Roodyn et al., 1962; Truman, 1964). The soluble enzymatic fraction is provided by microsomal synthesis with subsequent transfer to mitochondria, a process that also requires ATP (Kadenbach, 1966). Thus, alterations in those equilibria that include mitochondrial participation may produce toxic steady states that are evidenced very early by morphological and biochemical changes within mitochondria. It seems likely, therefore, that mitochondrial function and conformation depend on the availability of ATP, both for reactions which provide the rest of the cell with “energy currency” as well as reactions considered to be localized within themitochondria themselves. Such an order of priorities or “triage in mitochondria” would represent, therefore, a compensatory activity. In conclusion, readily identifiable mitochondrial lesions are induced by benzylidineyohimbol in the liver of dogs and rats at a dose level where no other significant alterations can be detected. The mechanism of production of these changes is currently under investigation. It seems certain that identifiable morphological changes occur in mitochondria early in the sequence of changes which lead to intracellular biochemical dysfunction. This study has shown the importance of careful evaluation of different zones of the liver lobule. This experimental model, which produces consistent mitochondrial changes in the species examined, possessespotential for establishing correlation with biochemical tissue changes. It may also serve as a possible predictive tool in toxicologic interpretation. ACKNOWLEDGMENTS The authors are grateful to Mrs. C. Burgess,Mr. C. Wall, and Mr. K. Stever for active collaboration in theseexperiments. REFERENCES DALTON,A. J. (1955).A chrome-osmiumfixative for electron microscopy.Amt. Record121, 281-282. DE LA IGLESIA,F. A. (1969). Comparative analysisof hepatic microbodies.A review. Acta Hepato-splenol16,141-160. DELA IGLESIA,F. A., SOSA-LUCERO, J. C., and LUMB,G. (1968a).On the experimentalreproduction of intracytoplasmichyaline in liver cells.FederationProc. 27,605. DE LA IGLESIA, F. A., SOSA-LUCERO, J. C., and LUMB,G. (1968b).Subcellularhepaticchanges I’ollowingdrug administration.Am. J. Puthol. 52,4a. DE LA IGLESIA, F. A., SOSA-LUCERO, J. C., and LUMB,G. (1969). The significanceof ultrastructural mitochondrial changesin toxicology. Proc. EuropeanSot. Study Drug Toxicity, Excerpta Med. Found. Intern. Cong. Ser. No. 181,Vol. 10.101-121.

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