Ultrastructural Changes Produced by Triparanol in Morris 5123 and Novikoff Hepatomas1

[CANCER RESEARCH 27, 165-171,January 1967]

WILLIAM C. RUBRICHI, OWEN KELLER,AND ZDENEK HRUBAN Zoller Dental Clinic, University of Chicago, Chicago, Illinois 60037

Summary 215-240 gm and allowed to grow for approximately 1 month. At that time the tumor size ranged from 1.6 to 2.3 cm in diameter. Force feeding of large amounts of triparanol to rats bearing Holtzman rats weighing 315-340 gm were similarly inoculated transplanted hepatomas results in appearance of myeloid bodies with the Novikoff hepatoma cells. Experimental feeding was in liver cells and in hepatoma cells. The myeloid bodies are most started on the 7th day when the tumor was about 1.8-2.3 cm in numerous in liver cells, less abundant in the cells of Morris 5123 diameter. The individual groups are listed in Table 1. hepatoma, and rare in the cells of Novikoff hepatoma. The Following treatment, the rats were fasted overnight, and quantity of myeloid bodies can be correlated with development samples of liver and hepatoma were taken from each rat under of endoplasmic reticulum and with duration of treatment. light ether anesthesia. The tissues were fixed in 1% osmium tetroxide in s-collidine buffer (4), pH 7.4, at 5Â°Cfor 1 hr. The Introduction tissues were dehydrated in graded alcohols and embedded in Triparanol is a known inhibitor of the final stages of cholesterol methacrylate with 2% benzoyl peroxide as catalyst. Thin sec tions were cut on a Porter-Blum microtome and stained with synthesis (2, 25). Administration of triparanol is followed by accumulation of desmosterol in various tissues (9) and by cyto lead by Karnovsky Method A (16). The specimens were photo logie alterations in the adrenal cortex (28). Large doses of tri graphed in the RCA EMU 2 and RCA EMU 3 electron micro paranol produce characteristic changes in the ultrastructure of scopes. liver cells and of pancreatic acinar cells (15). Clinical studies have Additional samples of livers and hepatomas were used for light shown that triparanol may influence the growth of some carci microscopic studies. The formalin-fixed tissues were used for hematoxylin and eosin stains. Livers fixed in formaldehyde- nomas (6, 18). calcium solution were used for Baker's acid hematein test to Triparanol could be of value as a chemotherapeutic agent in demonstrate phospholipids (3). Other pieces of livers fixed in spite of its toxic side effects (1, 5, 19, 29) if a significant dif weak Bouin's solution (3), treated with pyridine, and subse ference was shown between its cytotoxic effects on normal and on neoplastic cells. An attempt to demonstrate differences in quently stained with acid hematein were used as controls. ultrastructural changes produced by large doses of triparanol in 2 transplantable hepatomas and livers was the purpose of the Results present study. Alterations in the cytoplasm were most pro The ultrastructural changes produced by large doses of tri nounced in the liver cells, less marked in the cells of Morris 5123 paranol in hepatoma cells and in liver cells were similar struc hepatoma, and slight in the cells of Novikoff hepatoma. turally, but differed quantitatively. The cells of animals treated with triparanol contained characteristic myeloid bodies, which Materials and Methods were more abundant in liver cells than in the cells of hepatomas. Adult male rats of comparable age and weight were used in each experiment. The experimental and control diets were force- Light Microscopy Studies fed by a stomach tube (30) in 3 equal daily doses at 6-hr intervals Clear cytoplasmic vacuoles were found in hematoxylin and for 3 or 5 days, respectively. The experimental diets contained eosin stained sections of liver cells (Fig. 1). The number of these 250 mg of triparanol/kg body weight/day in a liquid diet pre vacuoles was increased with duration of treatment. Cytoplasmic viously described (30). The proteins in the diet were supplied in inclusions were seen in thick sections of methacrylate-cmbedded the form of casein hydrolysate. Triparanol was supplied in livers when viewed in the phase microscope. These inclusions powder form by the Wm. S. Merrell Company. Triparanol was were also demonstrated by the Baker's acid hematein method lacking in the control diet. The rats were fasted overnight before (Fig. 2), and they contained phospholipids extractable by the onset of experimental feeding and before killing. pyridine. Adult male Buffalo rats were used for studies on the Morris 5123 hepatoma. The tumor was implanted s.c. into rats weighing Electron Microscopic Studies

1Supported by a fellowship of the Zoller Memorial Dental The characteristic alteration in the cytoplasm of liver cells Clinic. and hepatoma cells of rats fed triparanol was the appearance of Received April 6, 1966; accepted August 19, 1966. myeloid bodies. These bodies were most abundant in liver cells,

JANUARY 1967 165

TABLE 1 body. Granular material was found in the center of some myeloid SCHEDULEOP EXPERIMENTS bodies (Fig. 7). Continuation of treatment with triparanol resulted in a moderate increase in number of hepatoma cells con of ofkilling4646taining myeloid bodies, but even at 5 days some cells were free TumorMorris rate42324232Triparanol(mg/kg/ offeeding(days)3535Day day)250None250None250None250NoneDurationof these bodies. The myeloid bodies become more complex (Fig. 6), but in contrast to liver cells lipoid material was not found in hepatomaMorris5123 them. While most myeloid bodies were intracytoplasmic, in a few instances whorls of myeloid membranes or myeloid bodies were seen in bile canaliculi. These bodies may have formed hepatomaNovikoff5123 within the canaliculi. hepatomaNovikoff Novikoff Hepatoma The fine structure of the previously described Novikoff hepa toma (12,14) was least affected by triparanol treatment. Myeloid hepatomaNo. bodies were seen rarely in nonnecrotic cells even after 5 days of treatment. These bodies often contained granular material (Fig. 8), and some were very large. The myeloid bodies were distinctly moderately numerous in the cells of Morris 5123 hepatoma, and different from the vacuoles containing granules and vesicles (Fig. infrequent in the cells of Novikoff hepatoma. Within the same 9, inset), which are present also in untreated Novikoff hepatoma tissue, the number of myeloid bodies increased with duration of cells (14). These vacuoles were seen more frequently, and were treatment. Differences in the structure of myeloid bodies were often larger after triparanol treatment (Fig. 9), than seen in also observed. untreated Novikoff hepatoma cells.

Discussion Liver Cells The myeloid bodies in the liver cells of rats treated with tri- Ultrastructural studies show a marked difference between the response of liver cells and hepatoma cells to administration of paranol for 3 days were about the size of mitochondria or larger large doses of triparanol. Although the myeloid bodies appear (Fig. 3). They contained concentric layers of smooth membranes characteristically as a response to triparanol in both normal and and were surrounded by a separate membrane. Connections neoplastic cells, they are rare in the cells of Novikoff hepatoma between the myeloid membranes and endoplasmic reticulum were and less numerous in Morris 5123 hepatoma cells than in the not found. The myeloid bodies were seen in many, but not in all, liver cells. Formation of myeloid bodies is not restricted to liver liver cells, and only a few bodies were found in a given cell. Most cells and their neoplastic derivatives, as they were found also in hepatocytes of rats fed triparanol for 5 days contained myeloid other tissues (13, 15). bodies, and the number of myeloid bodies in a single cell was The formation of myeloid bodies after treatment with tri markedly increased. In comparison with the simple myeloid paranol may be related to (a) detoxification of triparanol, (6) bodies seen on the 3rd day, the myeloid bodies observed on the direct injury of endoplasmic reticulum by triparanol, and (c) 5th day were often more complex. Two or more groups of con accumulation of desmosterol in place of the lacking cholesterol centric myeloid membranes appeared to form a large complex in the membranes of endoplasmic reticulum. myeloid body (Fig. 4). Many myeloid bodies also contained It is known that various drug-metabolizing enzymes are homogeneous, moderately electron dense material which was localized in the endoplasmic reticulum (8). The usual response occasionally connected with the myeloid membranes (Fig. 5). Other changes observed in the hepatocytes of triparanol- of the liver cell to administration of drugs is hyperplasia of treated rats were a diffuse distribution of the usually peri- smooth endoplasmic reticulum (23) and formation of smooth fingerprints, as exemplified by phÃ©nobarbital (11, 23). While canalicular dense bodies, an increase of smooth endoplasmic reticulum, and a variable degree of disorganization of rough endo- triparanol is localized in endoplasmic reticulum (10), the altera tions produced by triparanol are quite different from those pro plasmie reticulum. duced by phÃ©nobarbital (11). The well-formed myeloid bodies are usually sequestered by a limiting membrane and do not show Morris 5123 Hepatoma transitions to the neighboring endoplasmic reticulum. The structure of this hepatoma from untreated rats was the Inhibition of drug-metabolizing enzyme systems by triparanol same as that previously described (7, 14). Myeloid bodies ap (10, 17) may be explained either by a direct injury of endoplasmic peared in nonnecrotic Morris 5123 hepatoma cells after treat reticulum by triparanol, which has been shown to be present in ment with triparanol. These bodies were present in moderate the liver (20), or by the effect of triparanol on cholesterol synthe numbers after 3 days of treatment and were more abundant after sis. Diethylaminoethyldiphenyl-n-propylacetate (SKF 525-A) 5 days of treatment, but their quantity was in general smaller and triparanol are both derivatives of /3-diethylaminoethanol. when compared with liver cells similarly treated. Both compounds inhibit drug-metabolizing enzymes (17) and Myeloid bodies from Morris 5123 hepatoma cells also showed interfere with formation of hepatic cholesterol (26). Formation minor structural differences. The membranes in the center of the of myeloid bodies was, however, not observed after administra body were often more densely packed than in the periphery of the tion of SKF 525-A (24).

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The present observations and the previously described stages 10. Gillette, J. R., and Davenport, L. Inhibition by Triparanol in the formation of myeloid bodies (27) suggest that the cyto- (MER-29) of Microsomal Enzyme Systems that Metabolize plasmic changes produced by triparanol represent an overproduc Drugs. Federation Proc., 20: 173, 1961. tion of lipoproteins by the endoplasmic reticulum and their 11. Herdson, P. B., Garvin, P. J., and Jennings, R. B. Fine Structural Changes in Rat Liver Induced by PhÃ©nobarbital. subsequent sequestration. Triparanol interferes with formation Lab. Invest., IS: 1032-37, 1964. of cholesterol from desmosterol (2, 25) in endoplasmic reticulum 12. Howatson, A. F., and Ham, A. W. Electron Microscope Study (10) and results in accumulation of desmosterol (9). The altered of Sections of Two Rat Liver Tumors. Cancer Res., 15: 62-69, composition of lipoprotein membranes appears to be followed by 1955. an overproduction of membranes. The newly formed membranes 13. Hruban, Z., Spargo, B., Swift, H., Wissler, R. W., and Klein apparently have abnormal composition, because they are se feld, R. G. Focal Cytoplasmic Degradation. Am. J. Pathol., questered. The persistence of myeloid membranes in myeloid 42: 657-83, 1963. bodies suggests inadequate degradation of sequestered material. 14. Hruban, Z., Swift, H., and Rechcigl, M. Fine Structure of The hepatoma cells contain in general fewer myeloid bodies Transplantable Hepatomas of the Rat. J. Nati. Cancer Inst., SB: 459-95, 1965. than the liver cells. The neoplastic cells are either more resistant to injury by triparanol than is the normal liver cell, or the neo 15. Hruban, Z., Swift, H., and Slesers, A. Effect of Triparanol and Diethanolamine on the Fine Structure of Hepatocytes plastic cells do not react to the injury as markedly as does the and Pancreatic Acinar Cells. Lab. Invest., 14: 1652-72, 1965. liver cell. The myeloid body is a cytoplasmic lesion which can be 16. Karnofsky, M. J. Simple Methods for "Staining with Lead" interpreted as an attempt of the cell to compensate for the injury at High pH in Electron Microscopy. J. Biophys. Biochem. to membranes which is unsuccessful and is followed by the process Cytol., //: 729-32, 1961. of focal degradation (13). 17. Kato, R., Chiesara, E., and Vassanelli, P. Further Studies on The differences in the quantity of myeloid bodies in liver cells the Inhibition and Stimulation of Microsomal Drug Metab and hepatoma cells can be well correlated with the amount of olizing Enzymes of Rat Liver by Various Compounds. Bio rough endoplasmic reticulum in respective cells. Different metab chem. Pharmacol., IS: 69-83, 1964. olism of cholesterol in tumors (22) and absence of drug-metaboliz 18. Kraft, R. O. Triparanol in the Treatment of Disseminated Mammary Carcinoma. Cancer Chemotherapy Rept., 25: 113- ing enzymes in tumors (21) may be contributory factors. Whether 15, 1962. the diminished response of the hepatoma cell to triparanol also 19. Laughlin, R. G., and Carey, T. F. Cataracts in Patients represents a greater resistance to injury must be decided in Treated with Triparanol. J. Am. Med. Assoc., 181: 339-40, chronic experiments. 1962. 20. MacKenzie, R. D., and Blohm. T. R. Distribution and Excre References tion of Radioactive Triparanol (MER/29) in the Albino Rat. Federation Proc., 19: 14, 1960. 1. Anchor, R. W. P., Winkelman, R. K., and Perry, H. O. Cuta 21. Morris, H. P. Some Growth, Morphological and Biochemical neous Side Effects from Use of Triparanol (MER-29) : Pre Characteristics of Hepatoma 5123 and Other New Trans liminary Data on Ichthyosis and Loss of Hair. Proc. Staff plantable Hepatomas. Progr. Exptl. Tumor Res., 3: 370-411, Meetings Mayo Clinic, 36: 217-28, 1961. 1963. 2. Avignan, J., Steinberg, D., Thompson, M. J., and Mosettig, E. 22. Novikoff, A. B. Enzyme Localization in Tumor Cells. In: Inhibition of Conversion of 24-Dehydrocholesterol to Cho R. W. Cumley, M. Abbott, and J. McCay (eds.), Cell Physiol lesterol by MER-29. Federation Proc., 19: 239,1960. ogy of Neoplasia. Austin: University of Texas Press, 1960. 3. Baker, J. R. The Histochemical Recognition of Lipine. Quart. 23. Remmer, H., and Merker, H. J. Drug-induced Changes in the J. Microscop. Sci., 87: 441-70, 1946. Liver Endoplasmic Reticulum: Association with Drug- 4. Bennett, H. S., and Luft, J. H. S-Collidine as a Basis for metabolizing Enzymes. Science, 143: 1657-58, 1963. Buffering Fixatives. J. Biophys. Biochem. Cytol., 6: 113-14, 24. Rogers, L. A., and Fouts, J. R. The Effects of SKF 525-A on 1959. Hepatic Ultraatructure. Federation Proc., 2S: 537, 1964. 5. Burchenal, J. H. Improvement of Cancer Chemotherapy in 25. Schroepfer, G. J. Studies on the Conversion of Zymosterol to Man. Multiple Drug Therapy. Acta UniÃ³ Intern. Contra Cholesterol. Ibid. 19: 240, 1960. Cancrum, eO: 48-51, 1964. 26. Smith, R. L. The Metabolism and Disposition of Hypo- 6. Davis, P. L. Decrease in X-ray Evidence of Bone MÃ©tastases cholesteremic Drugs. In: R. Paoletti (ed.), Lipid Pharmacol in Prostatic Cancer during Use of Triparanol. Clin. Med., 8: ogy >PP- 461-88. New York: Academic Press, 1964. 2360-62, 1961. 27. Swift, H., and Hruban, Z. Focal Degradation as a Biological 7. Essner, E., and Novikoff, A. B. Cytological Studies on Two Process. Federation Proc., #3: 1026-37, 1964. Functional Hepatomas. Interrelation of Endoplasmic Reticu lum, Golgi Apparatus and Lysosomes. J. Cell Biol., 15: 289- 28. Volk, T. L., and Scarpelli, D. G. Alterations of Fine Structure of the Rat Adrenal Cortex after the Administration of Tri 312, 1962. paranol. Lab. Invest., IS: 1205-14, 1964. 8. Fouts, J. R., and Rogers, L. A. Morphological Changes in the Liver Accompanying Stimulation of Microsomal Drug Metab 29. Wetzel, R. Beitrag zur Therapie der Hand-SchÃ¼ller-Christian- olizing Enzyme Activity by PhÃ©nobarbital, Chlordane, Benz- schen Krankheit. Med. Welt, 46: 2368-69, 1963. pyrene or Methylcholanthrene in Rats. J. Pharmacol. Exptl. 30. Wissler, R. W., Frazier, L. F., SoÃ»les,K. H., Barker, P., and Therap., 147: 112-19, 1965. Bristow, E. C. The Acute Effects of /3-3-Thienylalanine in the 9. Frantz, I. D., Mobberley, M. L., and Schroepfer, G. J. Effects Adult Male Albino Rat; Observations on Nitrogen Balance, of MER-29 on the Intermediary Metabolism of Cholesterol. Antibody Formation, and Tumor Growth. Arch. Pathol., 62: Progr. Cardiovascular Diseases, 2: 511-18, 1960. 62-73, 1956.

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Fio. 1. Portion of liver from a rat treated with tripanuiol for 3 days. Numerous vacuoles are seen in the cytoplasm of hepatic cells. H & E, X 380. FIG. 2. Portion of liver from a rat treated with triparanol for 3 days. The cytoplasm of liver cells shows numerous dark bodies of various sizes. Nuclei (x) are pale. Acid hematein stain, X 380. FIG. 3. Myeloid bodies from a hepatocyte of a rat treated with triparanol for 3 days. The bodies consist of concentric layers of smooth membranes. X 37,000. FIG. 4. Portion of 2 hepatocytes from a rat treated with triparanol for 5 days. Several myeloid bodies form complex bodies. Limiting membrane is seen at arrows. Other structures seen are microbodies (m), Golgi complex (G), mitochondria, and smooth and rough endoplasmic reticuluni. X 37,000. FIG. 5. Myeloid bodies containing amorphous material in a hepatocyte of a rat treated with triparanol for 5 days. Limiting mem brane is seen at arrows. Other structures seen are multivesiculate bodies (v), microbodies (m), and mitochondria. X 26,000. FIG. 6. Complex myeloid body from a cell of Morris 5123 hepatoma from a rat treated with triparanol for 5 days. A simple myeloid body is seen at the top. Other structures seen are nucleus (JV), microbodies (m), mitochondria, and endoplasmic reticuluni. X 37,000. FIG. 7. Portion of a cell from Morris 5123 hepatoma from a rat treated with triparanol for 3 days. Myeloid bodies (B) are small and often fused with other bodies into larger complexes. Granular material (x) in the center of myeloid bodies. Single membrane (arrows) surrounds 1 group of myeloid bodies. Microvilli cover canalicular surfaces (L). X 32,000. FIG. 8. Portion of a cell from the Novikoff hepatoma from a rat treated with triparanol for 3 days. Myeloid bodies (B) are small and contain granular material. Other structures seen are nucleus (jV), annulate lamellae (A), Golgi complex (G), endoplasmic reticulum, and free ribosomes. X 25,000. FIG. 9. Portion of a cell from the Novikoff hepatoma from a rat treated with triparanol for 5 days. Large vacuoles contain vesicles, granules, and membranous material. Other structures seen are mitochondria, annulate lamellae (A), fibrillar material (F), endoplasmic reticulum, and free ribosomes. X 37,000. Inset: Small vacuoles containing vesicles, granules, and membranous material from another cell. Rat treated as in Fig. 9. X 26,000.

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JANUARY 1967 169

William C. Rubright, Owen Keller and Zdenek Hruban

Cancer Res 1967;27:165-171.

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