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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.

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.

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.

An immunohistochemical staining method for prohibitin, a 30-kDa protein located in the inner membrane of mitochondria, is used for detection of mitochondria on formalin-fixed paraffin-embedded sections. Methapyrilene hydrochloride (MP), which is known to increase mitochondria in periportal hepatocytes, was administered once by oral gavage to male rats at a dose of 100 mg/kg. Hypertrophy with eosinophilic granules was noted in the periportal hepatocytes treated with MP. Immunohistochemical staining with prohibitin antibody demonstrated a positive reaction to eosinophilic granules. Electron microscopy showed an increased number of mitochondria in the periportal hepatocytes treated with MP compared to the control. Although electron microscopy is a useful tool to detect ultrastructural changes, an immunohistochemical examination with anti-prohibitin antibody is a simple and easy method for detecting cytoplasmic mitochondria quantitatively.

To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment.

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.

The mechanism by which the specific β3-adrenoceptor agonist AJ-9677 relieves insulin resistance in vivo was investigated by studying its effects in the white and brown adipose tissues of the KK-Ay/Ta diabetic obese mouse model. AJ-9677 reduced the total weight of white adipose tissues by reducing the size of the adipocytes, an effect associated with the normalization of tumor necrosis factor-α (TNF-α) and leptin expression levels. The levels of uncoupling protein (UCP)-1 mRNA in brown adipose tissue were increased threefold. AJ-9677 caused a marked increase (20- to 80-fold) in the expression of UCP-1 in white adipose tissues. The levels of UCP-2 mRNA were increased in both the white and brown adipose tissues of diabetic obese mice, and AJ-9677 further upregulated UCP-2 mRNA levels in brown adipose tissue, but reduced its levels in white adipose tissue. UCP-3 mRNA levels were not essentially changed by AJ-9677. However, AJ-9677 significantly (two- to four-fold) upregulated the GLUT4 mRNA and protein levels in white and brown adipose tissues and the gastrocnemius. The generation of small adipocytes, presumably mediated by increased expression of UCP-1 in addition to increased lipolysis in response to AJ-9677, was associated with decreased TNF-α and free fatty acid production and may be the mechanism of amelioration of insulin resistance in KK-Ay/Ta diabetic obese mice.

An 18-month-old male Brown Norway (BN) rat showed a grayish-white subcutaneous mass in the right cheek. Histologically, the mass was composed of highly pleomorphic cells producing collagen. Immunohistochemical analysis showed that the tumor cells were strongly positive for vimentin and partially positive for Ki-67; however, they were negative for ED-1, ED-2, S-100, cytokeratin, desmin and myoglobin. Ultrastructurally, the cytoplasms of the tumor cells contained well-developed rough endoplasmic reticulum. Thus, the tumor had no characteristic feature other than collagen production and was diagnosed as a fibrosarcoma.

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.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the rising "Nihon" rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.

Glomerular expression of cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha), together with leukocytic infiltration, are prominent features in crescentic glomerulonephritis. Because these cytokines are targets for nuclear transcription factor-kappaB (NF-kappaB), the use of NF-kappaB decoy oligodeoxynucleotide (ODN) treatment was evaluated in an experimental disease model. Crescentic glomerulonephritis was induced in primed Wistar rats by injection of sheep antiglomerular basement membrane serum. Thirty minutes after injection, rats were anesthetized and the left kidney was perfused with NF-kappaB decoy ODN or scrambled ODN control mixed with a virus-liposome complex, and then killed 7 d later. Animals given the scrambled control ODN developed severe glomerulonephritis by day 7 with heavy proteinuria, glomerular crescents and interstitial lesions, marked leukocytic infiltration, and upregulated renal expression of cytokines (IL-1 and TNF-alpha) and adhesion molecules (intercellular adhesion molecule-1). In contrast, NF-kappaB decoy ODN treatment substantially inhibited the disease with a 50% reduction in proteinuria, a threefold reduction in histologic damage, a 50% reduction in leukocytic infiltration, and a 50 to 80% reduction in the renal expression of cytokines and leukocyte adhesion molecules. In conclusion, this study has demonstrated that NF-kappaB plays a key role in cytokine-mediated renal injury and that NF-kappaB decoy ODN treatment has clear therapeutic potential in rapidly progressive glomerulonephritis.

Male F344 rats received diet containing 3,000 ppm 2,6-dimethylaniline (DMA) after initiation with a single subcutaneous injection of 2,400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN), and histological and electron microscopic examinations of the nasal cavity were performed at 4, 13, 26 and 52 weeks to examine sequential changes induced by DMA. Severe atrophy of Bowman's glands and epithelial disarrangement were apparent from week 4, followed by dilatation and/or proliferation of Bowman's glands, degeneration of epithelial cells, and proliferation of undifferentiated epithelial cells from week 13. Focal glandular hyperplasias, dysplastic foci, and adenomas were observed from week 26, and carcinomas at 52 week. These nasal lesions were mostly evident in the olfactory mucosa in the nasal cavity, and their severity and/or incidences, other than atrophy of Bowman's glands, increased with the treatment period. Electron microscopically, carcinoma cells demonstrated desmosomes, dense secretory granules identical to those in normal Bowman's glands, a basement membrane, and microvilli. These results suggest that Bowman's glands are the target of DMA, giving rise to nasal carcinomas after DHPN-initiation.

A mucinous adenocarcinoma was found in the jejunum of an untreated male F344 rat of 126 weeks old. The neoplasm was primarily composed of cuboidal to columnar goblet cells that were arranged in a glandular form. Alcian blue-PAS staining revealed mucin in neoplastic cells and in the lumen. Paneth cells were found scattered among the goblet cells together with metaplastic cartilage and bone formation in the interstitium.

This paper describes spontaneous ovarian choriocarcinoma in two B6C3F1 mice used in toxicity studies. One animal (case 1) was sacrificed at eight weeks of age, and the other one (case 2) was found dead at sixty-seven weeks of age. Macroscopically, a dark red dot measuring approximately I mm in diameter (case 1) and hematoma-like enlargement (case 2) were observed in the ovary. Microscopically, in case I, the neoplasm was fully packed with tumor cells and erythrocytes. The tumor cells were round to oval giant cells with abundant, faintly basophilic cytoplasm containing PAS positive granules. In case 2, some small and two large hematocysts were present. The hematocysts contained a large number of erythrocytes and a small number of tumor cells. The tumor cells were oval to elongated giant cells with abundant, basophilic cytoplasm. Immunohistochemically, tumor cells in both cases were positive or weakly positive for human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and human specific beta-1 glycoprotein (SP-1). Both cases were considered to be originated from germ cells since those female animals were virgin.

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 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.

Granular cell tumors were found in 18 female reproductive tract out of 300 Wistar rats. The tumor cells were histologically polygonal and arranged in sheets or cords separated from each other by collagenous septa. The cytoplasm was filled with periodic acid-Schiff reaction positive and diastase-resistant coarse granules, which showed electronmicroscopically characteristics of lysosomes. Metastatic lesions were not seen in any of the cases. The nest of tumor cells were occasionally embedded within the uterine stromal cells, and no discernible capsule was present. Immunohistochemically, they were positive for neuron specific enolase in all cases and vimentin in some cases, and negative for S-100, desmin or myoglobin in any of the cases. Biological relationship with meningeal granular cell tumors was not evident. These findings supported the notion of neuroectoderm origin of granular cell tumors in the female reproductive tract, and reminded us of a large variation of the tumor incidence among different colonies.

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.

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.

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.

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 Bowman's 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 Bowman's 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 Bowman's glands to glandular hyperplasias and adenomas. These results suggest that dysplastic foci arise from Bowman's glands and progress to carcinomas, while proliferation of Bowman's glands result in glandular hyperplasias and adenomas.