The carcinogenic potential of 9-(4’-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman: APNH), a newly identified heterocyclic amine, was evaluated in a rapid production model for pancreatic duct adenocarcinoma in hamsters. In experiment 1, the initiation potential of APNH was examined. Hamsters received corn oil, 30 mg/kg body weight N-nitrosobis(2-oxopropyl)amine (BOP) or APNH (totally 50 and 100 mg/kg) as initiators followed by two cycles of augmentation pressure for pancreatic duct carcinogenesis and were sacrificed 75 to 78 days after the beginning of the experiment. Ductal hyperplasia and atypical hyperplasias developed in hamsters receiving APNH but their numbers and incidences did not differ from the corn oil case. In experiment 2, promoting potential of APNH for pancreatic duct carcinogenesis was examined. Hamsters received 30 mg/kg body weight BOP as an initiator followed by augmentation pressures and thereafter diet containing 0, 25 or 50 ppm APNH for 50 days. All hamsters were sacrificed 100 days after the beginning of the experiment. The numbers and incidences of ductal lesions, including adenocarcinomas, did not differ among the experimental groups. The results suggest, APNH may not have initiation or promotion potential for pancreatic duct carcinogenesis in hamsters.

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The effects of D-galactosamine (D-gal) and carbon tetrachloride (CCl₄) administration on induction of liver cell proliferation in a medium term liver bioassay for detection of initiation activity of 1,2-dimethylhydrazine (DMH) were investigated with special emphasis on kinetics of cell proliferation and cytochrome P450 (CYP) expression. In experiment I, fluctuation of cell proliferation and CYP 2E1, which is one of the enzymes involved in bioactivation DMH to ultimate form, levels of liver cells after D-gal administration (4000 mg/kg, i.g.) or CCl₄ administration (1 ml/kg, i.g.) was analyzed by bromodeoxyuridine labeling and western blotting, respectively. In experiment II, induction of glutathione S-transferase placental form (GST-P)-positive foci by DMH in a model for detection of initiation activities using D-gal or CCl₄ administration as a toxicity-regeneration dependent proliferative stimulus was evaluated. Although high BrdU labeling indices continued from 36 h to 72 h after D-gal and CCl₄ treatment, cell proliferation after intragastric D-gal administration was very low compared with the CCl₄ case. Decrease of CYP 2E1 apoprotein content in the microsomes was slight after D-gal administration, whereas decrease up to about 40% of the control level was evident from 12 h until 60 h after CCl₄. When the carcinogen was injected 60 h after D-gal administration, there was appreciable increase in the area and numbers of GST-P positive foci, similar to the case with CCl₄ administration. Sensitivities for detection of initiation activity with intragastric D-gal or CCl₄ administration were equal, despite the kinetics of cell proliferation and CYP 2E1 being very different. With CCl₄ administration, initiation depended on fluctuation of CYP 2E1, reversibly in intragastric D-gal treatment, initiation depended on cell kinetics.

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In order to clarify the cellular and molecular aspects in cirrhotic lesions, we investigated the response of macrophage populations, myofibroblastic cell development, and deposition of extracellular matrices (ECMs), as well as expression patterns of fibrogenic factors, in chronically-developing hepatic cirrhosis induced in rats by thioacetamide (TAA). As an animal model, male F344 rats were rendered cirrhotic by repeated intraperitoneal injection of TAA (100 mg/kg BW; twice a week), and were examined at post-first injection (PFI) weeks 1, 3, 5, 7, 10, 15, and 20. Histologically, hepatocyte degeneration became evident in the perivenular and periportal areas with time, and from PFI weeks 10 micronodular lesions separated by fibrous septa were developed. ECMs (collagen types I, III, and IV, fibronectin and laminin) deposited with advancing lesions. ED1-immunopositive exudate macrophages showed an increased number for 20 weeks. ED2-immunopositive Kupffer cells and antigen-presenting macrophages reacting to OX6 (MHC class II-recognized antibody) revealed a transiently increased number at PFI weeks 1 and 3; interestingly, Kupffer cells became hypertrophic with time. The number of myofibroblastic cells reacting to α-smooth muscle actin was increased from PFI week 1, with a peak at PFI week 10. The semiquantitative analysis by the reverse transcription polymerase chain reaction revealed that expressions of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) mRNAs could be increased for 20 weeks. These results indicated that different macrophage populations participated in chronically-developing rat hepatic cirrhosis with different kinetics patterns, and that these macrophages might be related to myofibroblastic cell development through the production of fibrogenic factors such as TGF-β1 and TNF-α.

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Bone marrow fibrosis and new bone formation were induced by Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injection in the rat. We investigated time course changes of megakaryocyte counts, circulating platelet counts, transforming growth factor-β1 (TGF-β1) levels in the bone marrow and those in platelet-poor plasma (PPP) when rats were injected with PEG-rHuMGDF at a dose of 0.1 mg/kg. Additionally, ultrastructural analysis of the circulating platelet and the bone marrow was performed by electron microscope. PEG-rHuMGDF injection daily for 5 days caused a megakaryocyte hyperplasia on days 5-7[after the commencement of the treatment], myelofibrosis on days 7-10, and new bone formation on days 8-15. TGF-β1 levels in the extracellular fluid of the marrow, megakaryocyte numbers, TGF-β1 levels in the PPP, and circulating platelet counts increased by PEG-rHuMGDF injection, and reached to the maximum level on days 7, 7, 8, and 10, respectively. Ultrastructural analysis showed that circulating platelets had no prominent morphological changes in the PEG-rHuMGDF-treated rats on day 8, compared with vehicle-treated rats. Additionally, there were many platelets or fragments of megakaryocyte around mesenchymal cells, and those fragments deposited in the newly formed bone on day 10. These data suggested that myelofibrosis and new bone formation were induced by the increase of TGF-β1 levels derived from bone marrow megakaryocytes.

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To examine the utility of N-nitrosamines as initiators for a 26-week two-stage rat nasal carcinogenesis model, male F344 rats were treated with 2,6-dimethylaniline (DMA), possessing promoting potential for the rat nasal cavity, after initiation with 3 subcutaneous injections of 1500 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN), 30 mg/kg of N-nitrosobis(2-oxypropyl)amine (BOP), or 30 mg/kg of N-nitrosopiperidine (NPIP). Histopathologically diagnosed nasal epithelial hyperplasias and tumors, including carcinomas, were induced with all three, with significant increase in the DHPN+DMA, BOP+DMA, and NPIP+DMA groups compared with the DHPN alone, BOP alone, and NPIP alone groups, respectively. These results strongly suggested that repeated subcutaneous injections of DHPN, BOP, or NPIP are useful for initiation in the present 26-week nasal carcinogenesis model.

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This study was designed to determine whether intestinal metaplasia can be induced in Mongolian gerbils (MGS) by X-irradiation. Five-week-old animals of both sexes were X-irradiated with two X-ray doses of 10 Gy each at a 3-day interval (total dose: 20 Gy) and killed 24 or 60 weeks thereafter for histopathological examination. Intestinal metaplasia was not detectable in either 11 male or 10 female MGS at 24 weeks and only two lesions were encountered at 60 weeks after X-irradiation. One was type A in a female and the other of type B in a male. These results demonstrated that the glandular stomach of Mongolian gerbils is not susceptible to induction of intestinal metaplasia by X-irradiation.

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The present study was designed to clarify the pathologic features of hyperphosphatasemic conditions in association with drug metabolizing enzyme (DME) induction and cholestatic condition in dogs. Two (2) model compounds were selected; α-naphthylisothiocyanate (ANIT) and phenobarbital (PB). Groups of beagle dogs were orally given ANIT at 30 mg/kg or PB at escalating dose of 20 to 60 mg/kg for 3 weeks and subjected to in-life and postmortem examinations. Determination of hepatic DME and alkaline phosphatase (ALP) activity in liver tissue fractions as well as isoenzyme analysis of serum ALP were performed. Differences between the ANIT and PB groups were: (1) the serum enzyme profile in the ANIT group was characterized by remarkably elevated activity in leakage enzymes (ALT and AST) and ALP accompanied by a remarkable increase in plasma total bilirubin concentration and remarkable inhibition of ICG clearance, (2) the only noticeable finding in the serum enzyme profile in the PB group was remarkable elevation of ALP activity, and (3) microsomal P-450 content decreased in the ANIT group and increased in the PB group. Activities of aminopyrine demethylase and aniline hydroxylase shifted together with the change in microsomal P-450 content in the PB group, and (4) degenerative changes in the biliary system and liver cells were characteristic in the ANIT group and hyperplasia of the smooth surfaced endoplasmic reticulum was prominent in the PB group. The reported hyperphosphatasemia recorded in the PB-treated dogs has to be dealt with separately from the toxicologically significant changes seen in ANIT-treated dogs, since the source of ALP differed between the two conditions.

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Hepatocellular carcinoma which occurred in one male laboratory cynomolgus monkey at 5 years of age was investigated extensively by immunological, histopathological and electron-microscopic examinations. The animal exhibited no abnormalities in clinical laboratory tests including blood-chemistry, except for elevation of protein induced by vitamin K absence or antagonist II (PIVKA-II) in serum, which is one of the hepatic tumor markers. In virus antibody tests, the serum was positive for hepatitis A virus. On necropsy, a mass measuring 60 × 60 × 65 mm was seen in the hepatic left lobe. The tumor mass had a lobulated structure with hemorrhagic and necrotic areas, and was demarcated by thin fibrous capsules. Histopathologically, the tumor was composed of hepatocyte-like cells, having irregular trabecular structure with various thickness, lined by vascular endothelial cells. Cellular atypia such as polynucleated cells, mitotic figures, and invasion into the vascular cavity were also observed. Immunohistochemically, the tumor cells showed positive reaction for anti-PIVKA-II, anti-epithelial membrane antigen (EMA), anti-carcinoembryonic antigen (CEA), and anti-cytokeratin 18, as well as for proliferating cell nuclear antigen (PCNA). On electron-microscopic examination, the tumor cells had a number of tight junctions and formations of bile canaliculi between adjacent cells, basically resembling hepatocytes. This is the first case of hepatocellular carcinoma with PIVKA-II production in monkeys. Serological and immunohistochemical analyses for PIVKA-II are, therefore, practicable for diagnose as hepatocellular carcinoma in nonhuman primates.

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Cardiac rhabdomyoma was found as an incidental finding in a 6-month-old male beagle dog on microscopic examination. The lesions took a spongy appearance and were located in the subepicardial area of the juncion of the left ventricular wall and the interventricular septum. The cells comprising the lesions had a large vacuole surrounded by a narrow rim of cytoplasm. The cytoplasm of these cells was positive for periodic acid-Schiff’s reaction, but negative after digestion with diastase. Cross-striation was seen in the cytoplasm by phosphotungstic acid hematoxylin staining. No abnormalities were detected by clinical examination, including blood chemistry and electrocardiography. This case might be the first reported cardiac rhabdomyoma in laboratory beagle dogs.

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A mammary fibroadenoma with prominent sebaceous gland-like structures in a female Donryu rat aged 67 weeks was immunohistochemically investigated. The tumor demonstrated a mixture of both epithelial and connective tissue components, the former having both single- and multi-layered patterns. Characteristic sebaceous gland-like structures were apparent in connection with multi-layered epithelium. Area of single-layered epithelium comprised well-differentiated myoepithelial cells positive for alpha-smooth muscle actin (alpha-SMA), similar to mammary ducts. In contrast, many epithelial cells on the basal sides of multi-layers were negative for alpha-SMA, but positive for cytokeratin 14 (CK14), suggesting pluripotency. From these results, we diagnosed this case as a mammary fibroadenoma with sebaceous gland metaplasia.

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