Hepatocellular Cytoplasmic Inclusions in a Cynomolgus Monkey

J Toxicol Pathol 2006; 19: 191–194

Case Report Hepatocellular Cytoplasmic Inclusions in a Cynomolgus Monkey

Hisashi Ikegami1, Yoshimasa Okazaki1, Masahiro Matsumoto1, Shunji Nakatsuji1, Shiro Fujihira1, Katsuhiko Yoshizawa1, Kenjiro Tsubota1, Yuichi Murakami1, Akiko Anagawa1, and Yuji Oishi1

1Toxicologic Pathology, Drug Safety Research Labs., Astellas Pharma Inc., 2–1–6, Kashima Yodogawa-ku, Osaka 532–8514, Japan

Abstract: Spontaneous hepatocellular intracytoplasmic inclusions were observed in a 4-year old male cynomolgus monkey used in a toxicity study. Histopathological findings of the inclusions revealed an eosinophilic ground glass appearance. The inclusions were positive for periodic acid Schiff and colloidal iron under diastase or hyaluronidase treatment, and they included microvacuoles which stained positive in oil red O. Ultrastructurally, the inclusions consisted of many lipid droplets with granular substances and disintegration of organelles, but there were had no limiting membrane around the inclusions. Any abnormal findings such as necrosis, inflammation or virus particles were not detected. Our case was considered to be a kind of a disease categorized under metabolic diseases with unknown mechanisms, and it might provide information as a historical control in the assessment of monkey toxicity studies. (J Toxicol Pathol 2006; 19: 191–194) Key words: cytoplasmic inclusion, hepatocyte, cynomolgus monkey, historical control data

Hepatocellular cytoplasmic inclusions are often virus, simian T-cell leukemia virus-I, simian retrovirus-I, -II, observed in animals and humans, and they have been and -V, ebola, hepatitis A and B], bacterial examinations suggested to be caused by a wide variety of etiologies using medium culture (Mycobacterium tuberculosis, including viral infection1 and metabolic disorders2,3. In the shigella, salmonella), and microscopic and feces-smear safety assessment studies of new chemicals and drugs, it is examination for parasites (lice, flea, tick, giardia, important to recognize spontaneous lesions in laboratory strongyloides, Entamoeba) were conducted before the monkeys, especially in young adult monkeys that are commencement of study, and the absence of infection was routineIy used. In this case report, we describe the confirmed. The animal was handled during the study in occurrence of spontaneous cytoplasmic inclusions of accordance with Guideline for Animal Experimentation hepatocytes without viral association in a young adult issued by Astellas Pharma Inc., which is based on the laboratory monkey and discuss the pathogenesis with regard Guideline for Animal Experimentation published by the to the results of immunohistochemistry and electron Japanese Association for Laboratory Animal Science4. microscopy. In clinical observation, vomiting associated with drug The male cynomolgus monkey (Macaca fascicularis), administration was observed sporadically in the first week of which was born and raised in primate colonies in China, was dosing. There were no significant changes in body weight, purchased from Guangxi Grandforest Scientific Primate food consumption and hematological parameters. In blood Company, Ltd (Guangxi, China) at 4 years of the age. The chemistry, the monkey showed elevated activities of monkey was allocated to the mid-dose group of a 4-week aspartate aminotransferase (AST), alanine aminotransferase repeated oral toxicity study for a new drug and was (ALT) and alkaline phosphatase (ALP) before the start of sacrificed after the completion of the treatment. Serological dosing (48, 64 and 2840 mU/mL, respectively) compared to examination for viruses [B-virus, simian immunodeficiency those of animals in the control group given vehicle. The values of AST and ALT increased slightly during the dosing Received: 4 September 2006, Accepted: 7 November 2006 (77 and 194, respectively). Changes in these parameters Mailing address: Yuji Oishi, Toxicologic Pathology, Drug Safety were not detected in other animals in this study. Research Labs., Astellas Pharma Inc., 2–1–6, Kashima Yodogawa-ku, The monkey was euthanized under ketamin anesthesia Osaka 532–8514, Japan and complete necropsy was performed. For TEL: 81-6-6390-1167 FAX: 81-6-6304-5716 histopathological examination, systemic organs and tissues E-mail: [email protected] 192 Hepatocellular Cytoplasmic Inclusions in Monkey were fixed with 10% neutral buffered formalin, embedded in membrane at the margins of the inclusions (Fig. 2b). Neither paraffin, sectioned and stained with hematoxylin and eosin degeneration/necrosis of the hepatocytes nor inflammatory (H&E). The sections from the liver were also stained with reaction in the liver were observed. Similar cytoplasmic periodic acid Schiff (PAS) with and without diastase inclusions were not detected in any other organs or tissues. treatment, colloidal iron with and without hyaluronidase Special stains suggested the presence of polysaccharides treatment, toluidine blue (pH 4.1 and 7.0), alcian blue (pH except normal glycogen and hyaluronic acid in the area of 1.0 and 2.5), periodic acid-methenamine-silver (PAM), the granular substance. Immunohistochemical and electron phosphotungstic acid hematoxylin (PTAH) and Masson’s microscopical findings indicated that the granular substance trichrome. Further, frozen sections of the liver were did not include intermediate filaments, e.g. vimentin and prepared and stained with oil red O, Nile blue and Sudan cytokeratins. The results also indicate that the granular black. For immunohistochemistry, paraffin sections of the substance was not formed by accumulation of fibrinogen or liver were reacted with antibodies as follows: rabbit anti- α-1-antitrypsin, which are often produced by hepatocytes11. human lysozyme (dilution 1/200, DAKO A/S, Glostrup, The lipid droplets were considered to correspond to the Denmark), mouse anti-vimentin (dilution 1/50, DAKO A/S), microvacuoles seen in H&E sections. mouse anti-human pancytokeratin (dilution 1/100, In the present case report, cytoplasmic inclusions were Novocastra laboratories Ltd., Newcastle, U.K.), rabbit anti- observed in a young adult cynomolgus monkey. It was not human fibrinogen (dilution 1/1000, DAKO A/S), or rabbit noticed in other animals used in this toxicity study, even in anti-alpha-1-antitrypsin (dilution 1/200, Zymed laboratories the highest dose group which showed higher plasma Inc., CA, U.S.A.). Subsequently, they were incubated with concentrations of the test compound than this monkey. biotinylated antibodies followed by labeling with Therefore, it must be judged that the finding was not related streptavidin-biotin-peroxidase complex using kits (LSAB®2 to the treatment with the test compound and that it was a System-HRP, DAKO A/S), and visualized by 3-3’- spontaneously occurring change. Spontaneously occurring diaminobenzidine. For electron microscopy, small pieces of eosinophilic inclusion bodies in hepatocytes have been the formalin-fixed liver were refixed with 2.5% reported in cynomolgus monkeys (incidence: 5.9% in males, glutaraldehyde in 0.1 M phosphate buffer for 2 hr and post- 4.5% in females) used in toxicity studies5. Detailed fixed with 2% osmium tetroxide in 0.1 M phosphate buffer pathological examination of the inclusion bodies was not for 2 hr. After dehydration through a graded ethanol series, conducted in that report, therefore, it is unclear whether our the sample were embedded in Epon 812 resin (Nacalai present case is of the same type of inclusion as previously Tesque Inc., Kyoto, Japan). Ultrathin sections were reported or of a different type. prepared and stained with uranyl acetate and lead citrate, and Cytoplasmic inclusions of ground-glass appearance examined with a transmission electron microscope (H-7600, were observed in hepatocytes of patients infected with Hitachi High-technologies Corp., Tokyo, Japan). hepatitis B virus1. In this monkey, viral particles were not At necropsy, no noteworthy findings were observed in detected in hepatocytes in the electron microscopical the liver and other systemic organs, and no significant examination and neither degeneration/necrosis of the change was detected in the organ weight of the liver. hepatocytes nor inflammatory reaction were noticed in the Histopathologically, cytoplasmic inclusions were observed liver. Additionally, it was reported that viral replication did in the perilobular hepatocytes of the liver (Fig. 1a). The not occur and cytoplasmic inclusion was not induced by inclusions were eosinophilic, showed a ground-glass hepatitis B virus infection in the cynomolgus monkey6, appearance, and often included microvacuoles in H&E stain whereas the anti-viral antibody titer in blood was elevated. (Fig. 1b). The inclusions were surrounded by a clear halo, Taken together, it suggests that the hepatocellular inclusions which might have been a shrinkage artifact. In PAS and of our case were not caused by hepatitis B virus infection. colloidal iron stains with and without diastase or Cytoplasmic inclusions in hepatocytes are often hyaluronidase treatments, positive reactions were detected in observed in some metabolic diseases2,3 including the inclusions (Fig. 1c and 1d). The inclusions showed glycogenosis2,7, Lafora’s disease2,3,8–10 and endoplasmic positive reactions in toluidine blue and negative reactions in storage disease (ERSD)11–14 in monkeys and humans. alcian blue, PAM, PTAH and Masson’s trichrome stains. Glycogenosis is a congenital disease in which glycogen or The microvacuoles in the inclusions were stained red, pale abnormal synthetic materials of glycogen accumulate due to red to reddish violet and bluish black in oil red O, Nile blue defects in the enzymes related to glycogen synthesis or and Sudan black stains, respectively (Fig. 1e to 1g), degradation. Type IV glycogenosis is reported to induce suggesting that small neutral fat droplets were included in cytoplasmic inclusions in perilobular hepatocytes similar to the vacuoles. In the immunohistochemical examination, the the present case. Accumulation of glycogen granules in inclusions showed no reaction with any antibodies for inclusions and severe fibrosis around hepatocytes were lysozyme, vimentin, pancytokeratin, fibrinogen, or alpha-1- observed in type IV glycogenosis2,7, but our case showed no antitrypsin. Ultrastructurally, many small lipid droplets, similar features. Lafora’s disease is a non-lysosomal storage granular substances, and disintegrated materials, mainly disease characterized by accumulation of polyglucosan, a derived from mitochondria and endoplasmic reticulum, were type of glycoprotein, and formation of polyglucosan bodies observed in the inclusion (Fig. 2a). There was no limiting observed mainly in the central nervous system3,10. In Ikegami, Okazaki, Matsumoto et al. 193 hepatocytes of patients with Lafora’s disease, cytoplasmic examination. In our present case, fibrillar or microfibrillar inclusions similar to the present case are observed, and materials in inclusions and cytoplasmic inclusions in the fibrillar or microfibrillar materials in the inclusion are central nervous system, characteristic findings of Lafora’s characteristically detected in electron microscopical disease, were not observed. ERSD, in which accumulation

Fig. 1. Liver of the monkey. H&E (a, b), PAS without diastase (c), colloidal iron without hyaluronidase (d), oil red O (e), Nile blue (f), Sudan black (g). a) Cytoplasmic inclusions in perilobular hepatocytes (arrows). b) Eosinophilic, ground glass appearance with microvacuoles in inclusion. c, d) Positive reaction in inclusion. e–g) Positive reaction in microvacuoles. 194 Hepatocellular Cytoplasmic Inclusions in Monkey

mechanism, since polysaccharides were accumulated in the cytoplasm without other characteristic intracellular changes. Inclusions are usually present in multi-organs in metabolic diseases, but they were present only in hepatocytes in this case. This uneven distribution might be due to the type of metabolic pathway and metabolic activity related with this case, since hepatocytes are one of the most metabolically- active cells and they have many unique metabolic pathways compared with other cells.

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