Kaoru Toyosawa

Interlaboratory Comparison of Short-Term Carcinogenicity Studies Using CB6F1-rasH2 Transgenic Mice

In order to evaluate a short-term carcinogenicity testing system using CB6F1-Tg rasH2 ( rasH2-Tg) mice carrying a human prototype c-Ha-ras gene, 26-week studies were conducted in 12 different facilities as a part of an International Life Science Institute Health and Environmental Science Institute (ILSI HESI) international collaborative project. In each study N-methyl-N-nitrosourea (MNU) was administered to a separate group of rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. We herein have summarized the mortality, body weight change, and neoplastic and nonneoplastic lesions detected in these positive control groups as representative historical positive control data. Also, we performed an interlaboratory comparison of the response of rasH2-Tg mice to MNU based on the data of 11 positive control groups from these studies. Although the body weight of rasH2-Tg mice showed lower values than that of non-Tg mice during the experimental period, body weight gain in the rasH2-Tg mice was similar to that in non-Tg mice. The mortality of rasH2-Tg mice during the study period was very low, the same as for the non-Tg mice. Incidences of spontaneous alveolar/bronchiolar adenomas and splenic hemangiomas/hemangiosarcomas were also low in the rasH2-Tg mice. Nonneoplastic lesions detected in the rasH2-Tg mice were similar to those in non-Tg mice, excluding the incidence of myopathy. There were interlaboratory differences in mortality and incidence of some lesions in the MNU-treated groups. However, the causes of death were common among the 11 laboratories and almost all the MNU-treated rasH2-Tg mice developed forestomach squamous cell papillomas/carcinomas or malignant lymphomas. This suggests that there is no appreciable difference in the response of the rasH2-Tg mouse to MNU used as a positive control. Therefore, it is concluded that MNU would be an adequate positive control compound in this testing system.

Di(2-ethylhexyl)phthalate Induces Hepatocellular Adenoma in Transgenic Mice Carrying a Human Prototype c-Ha-ras Gene in a 26-Week Carcinogenicity Study

To evaluate the transgenic mouse carrying a human prototype c-Ha-ras gene (rasH2 mouse) as a model for 26-week carcinogenicity tests, Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, was administered to 15 rasH2 mice/sex/group at concentrations of 1,500, 3,000 or 6,000 ppm, and to 15 wild-type (non-Tg) mice/sex/group at a concentration of 6,000 ppm in their diets for 26 weeks. Survival rates and food consumption in the groups treated with DEHP and in the control group were similar. Body weight gain in rasH2 and non-Tg mice at 6,000 ppm in the terminal week decreased about 10% as compared to the control group. Common findings related to treatment with DEHP in rasH2 and non-Tg mice included hypertrophy with coarse granules and deposit of pigment in the liver, hydronephrosi s and tubular regeneration in the kidney, focal atrophy in the testis, and increased eosinophilic body in the nasal cavity. Hepatocellular adenoma was induced by treatment with DEHP, and was confined to male rasH2; mice the incidence being 7%(1/15), 13%(2/15), and 27%(4/15) in the 1,500-, 3,000-, and 6,000-ppm group, respectively. Point mutation was not detected in codon 12 and 61 of human c-Ha-ras transgene upon DNA analyses on frozen samples taken from these hepatocellular adenomas. From the results obtained in this 26-week carcinogenicity study, it is concluded that DEHP is a hepato-carcinogen for transgenic mouse carrying a human prototype c-Ha-ras gene.

The First Case of Feline Infectious Peritonitis-like Pyogranuloma in a Ferret Infected by Coronavirus in Japan.

A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for anti-mouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan.

Mutation and Overexpression of the Transgene in Ethylnitrosourea-Induced Tumors in Mice Carrying a Human Prototype c-Ha-ras Gene

To investigate mechanisms underlying accelerated carcinogenesis in mice carrying a human prototype c-Ha- ras gene (rasH2 mouse), mutations and the expression profile of the transgene were evaluated in 14 tumors induced by a single injection of ethylnitrosourea (ENU), with or without additional beta-estradiol 3-benzoate (EB) treatment. Although no codon 12 mutations were detected, changes in codon 61 were evident in all lung adenocarcinomas, skin squamous cell carcinomas and forestomach squamous cell carcinomas examined. The mRNA levels of the transgene in these lesions were also elevated 1.71- to 4.77-fold, 3.04- to 5.18-fold, and 3.00- to 5.67-fold, respectively, in comparison with those in the normal livers of rasH2 mice. The results obtained in this study suggest that mutations in codon 61 and amplification of the transgene play key roles in the carcinogenesis induced by ENU in rasH2 mice.

Immunohistochemical and Ultrastructural Studies of 2,6-Dimethylaniline-induced Nasal Proliferative Lesions in a Rat Two-stage Nasal Carcinogenesis Model Initiated with N-bis(2-hydroxypropyl)nitrosamine

Proliferative lesions induced by 2,6-dimethylaniline (DMA) in a two-stage rat nasal carcinogenesis model were immunohistochemically and ultrastructurally investigated. Male F344 rats received diet containing 3,000 ppm DMA for 52 weeks after initiation with a single subcutaneous injection of 2400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN). Histopathologically, proliferation of Bowmans glands, glandular hyperplasias, dysplastic foci, adenomas, and carcinomas were observed in treated rats. These nasal lesions mostly arose in the olfactory mucosa of the nasal cavity. Immunohistochemically, they were positive for cytokeratin and/or collagen type IV antibodies. Ultrastructurally, intracytoplasmic dense secretory granules (200—850 nm in diameter), identical to those in normal Bowmans glands, were observed in all the lesions, providing further support from an origin from these glands. Based on their cellular characterization, growth pattern and/or proliferative activity, two morphological continua were evident, one from dysplastic foci to carcinomas and the other from proliferation of Bowmans glands to glandular hyperplasias and adenomas. These results suggest that dysplastic foci arise from Bowmans glands and progress to carcinomas, while proliferation of Bowmans glands result in glandular hyperplasias and adenomas.

Overexpression of the peroxisome proliferator activated receptor α or the human c-Ha-ras transgene is not involved in tumorigenesis induced by di(2-ethylhexyl)phthalate in rasH2 mice

The mRNA profiles for peroxisome proliferator activated receptor (PPAR) alpha and human c-Ha-ras genes were determined by real-time semi-quantitative polymerase chain reaction analysis of hepatocellular adenomas induced by di(2-ethylhexyl)phthalate (DEHP) in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice). The mRNA levels were essentially equal in hepatocellular adenomas and adjacent non-neoplastic hepatocytes, in spite of a remarkable elevation in the cell proliferation index in tumors determined by anti-Ki-67 immunohistochemistry. From the results, it is concluded that overexpression of PPARalpha or the transgene is not associated with the liver tumorigenesis induced by DEHP in rasH2 mice.

Spontaneous Rhabdomyosarcoma in a Common Marmoset (Callithrix jacchus)

The common marmoset (Callithrix jacchus) is now widely used in various research fields, including toxicology. However, information about the background pathology of this species is scarce. Here, we report a case of rhabdomyosarcoma that spontaneously occurred in a common marmoset. A 44-month-old male common marmoset was euthanized due to bilateral hind limb paralysis. At necropsy, a 2×2×5-cm intramuscular mass was observed in the lower right back. Histologically, the mass was mainly composed of interlacing bundles of spindle-shaped tumor cells. Immunohistochemically, the tumor cells were positive for myogenin, desmin, vimentin and alpha-smooth muscle actin. Ultrastructurally, the tumor cells contained bundles of myofilaments with Z-band-like structures. Thus, the tumor was diagnosed as a rhabdomyosarcoma. To our knowledge, this is the first report of spontaneous rhabdomyosarcoma that was definitely diagnosed in the common marmoset.

Hyaline Glomerulopathy with Tubulo-Fibrillary Deposits in Young ddY Mice

Hyaline glomerulopathy with tubulo-fibrillary deposits was observed in two young female ddY mice. One of the mice showed gross systemic edema and bilateral enlargement and pale color of the kidneys, whereas no significant gross findings were noted in the other mouse. Microscopically, a large number of the glomeruli in both mice were enlarged because of diffuse and global deposition of amorphous eosinophilic materials. The deposits were negatively stained with Congo red and positively stained with IgG, IgM, IgA, C3, and periodic acid–Schiff. Electron microscopic examination revealed microtubular and fibrillary deposits with diameters of 80–100 and 9–16 nm, respectively, in the subendothelial space of the glomeruli. These features are histopathologically similar to immunotactoid glomerulopathy or fibrillary glomerulonephritis according to the classification of human glomerular lesions. Understanding of these characteristics of hyaline glomerulopathy in ddY mice is essential when evaluating pharmacological, pharmacokinetic, and toxicological studies using this mouse strain.

Cystic Cholangioma in the Thoracic Cavity of a Rat

A female congenic rat produced by repeated backcrossing of Nihon rats, a model for hereditary renal cell carcinoma, to Brown Norway rats was necropsied at 24 months of age. At necropsy, a white mass about 1 centimeter in size was observed in the thoracic cavity, and the mass partly adhered to the esophagus and the diaphragm. Histologically, the mass was clearly circumscribed by connective tissue, and consisted of neoplastic cuboidal epithelial cells that showed cystic tubular proliferation. Some islands of well-differentiated hepatocytes and some vessels were observed in the mass. Immunohistochemically, the tumor cells were strongly positive for cytokeratin and partly positive for vimentin but were negative for mesothelin and Von Willebrand Factor. The positive rate for Ki-67 was 2.4%. Based on these histological and immunohistochemical evidences, we diagnosed this tumor as a cystic cholangioma that might have arisen from the ectopic hepatic tissue in the thoracic cavity.

Immunohistochemical and Ultrastructural Analyses of Cytoplasmic Blood Plasma Inclusions of Rat Hepatocytes

In the present study, we investigated the histological, immunohistochemical and ultrastructural characteristics of cytoplasmic blood plasma inclusions that spontaneously occurred in a rat liver. Histologically, a number of cytoplasmic inclusions were observed in the liver of an 8-week-old female SD rat. These inclusions were strongly positive for PAS staining and resistant to diastase digestion. Immunohistochemical analyses revealed that these inclusions were positive for albumin and IgG; however, most of them were negative for LAMP-1 and LAMP-2. Ultrastructurally, the inclusions were surrounded by limiting membranes and composed of moderately electron dense, homogenous materials. These characteristics described here represent valuable information for pathological examination in toxicity studies.