Time-course changes in 5-fluorouracil-induced neural progenitor cell damages in the developing rat brain.

5-Fluorouracil (5-Fu) is a DNA-damaging agent and teratogenic in rodents. This study aimed to investigate its influence on neural progenitor cells (NPCs) in the developing fetal rat brain. Dams were intraperitoneally injected with 5-Fu (50 mg/kg b.w.) on gestation day 13 and its effects on fetal NPCs were observed from 3 to 72 hours after treatment (HAT), via periodic examination at six intervals. In NPCs of the fetal brain, the p53-labeling index (LI%) was markedly elevated at 3 HAT. Pyknosis and cleaved caspase-3-LI% also increased at 3 HAT, reaching peak values at 9 and 12 HAT. These parallel changes suggested the induction of apoptosis through a p53-mediated pathway. Pyknotic NPCs were distributed across the ventricular zone (VZ) of the telencephalic wall until 12 HAT, and became localized in the medial and dorsal layers at 12 and 48 HAT. Significant decreases in the numbers of mitotic NPCs and BrdU-LI% were noted from 3 HAT and 24 HAT, respectively. BrdU-positive NPCs were located in the ventral and middle layer at 24 and 48 HAT. p21-positive cells were detected at 12 and 24 HAT. The present results demonstrated that p53-mediated apoptosis was induced in all phases of the cell cycle of the NPCs in the early stage after 5-FU treatment. Furthermore, apoptosis of NPCs and suppression of cell proliferative activity are the events that take place in parallel leading to prominent reduction in the width of the telencephalic wall.

Spontaneous extraskeletal osteosarcoma in the duodenum of a Crlj:CD1 (ICR) mouse.

Extraskeletal osteosarcoma is a very rare tumor in humans and animals. This paper describes a case of extraskeletal osteosarcoma observed in the duodenum of a male ICR mouse. Grossly, a solid mass pushing up the tunica serosa was observed in the duodenal wall. Histologically, the tumor was located in the lamina propria mucosae and tela mucosa. Neoplastic cells densely proliferated in these areas, and replaced of the normal tissue components. A small amount of osteoid and a small clump of bone tissue were observed in the area of neoplastic cell proliferation, especially in the lamina propria mucosae. Neoplastic cells consisted of atypical polygonal cells and pleomorphic spindle-shaped cells, and the former were predominant. Mitotic figures were occasionally observed. Neither invasion of vessels in the duodenum nor metastasis to distant organs was observed. There were no skeletal tumors in the body. Immunohistochemically, the neoplastic cells were positive for anti-osteocalcin, osteonectin, vimentin, and S-100 protein. Judging from these results, the present tumor was diagnosed as extraskeletal osteosarcoma. This is the first report of spontaneous extraskeletal osteosarcoma arising from the duodenum of a mouse.

Ocular lesions induced in infant rats by busulfan.

Although busulfan, a bifunctional alkylating agent, is known to induce cataracts in infant rats, the full nature of busulfan-induced ocular lesions has not yet been shown. In order to clarify this point, 6-day-old rats were treated with a single dose of 20 mg/kg busulfan and the ocular tissue was histopathologically and immunohistochemically examined at 1, 2, 4, 7 and 12 days after treatment (DAT). As a result, in the nuclear layer (NL) of the peripheral retina, apoptotic cells significantly increased at 1 DAT and peaked at 2 DAT when cell proliferating activity was depressed. At 4 DAT, the NL showed wavy deformation with formation of rosette-like structures, and these changes progressed prominently at 12 DAT. In addition, a significant reduction in the retinal thickness due to decreased thickness of NL or inner NL was detected at 2 and 4 DAT. On the other hand, in the germinative zone of the lens equator, apoptotic lens epithelial cells significantly increased from 2 to 7 DAT, resulting in partial loss of lens epithelial cells at 7 and 12 DAT. At 12 DAT, prominent swelling and vacuolation of lens fibers were observed in the area from the equatorial zone to the posterior pole, indicating the development of cataract. The present results strongly suggest that prominent apoptosis in component cells was the initial and essential event underlying the development of busulfan-induced ocular lesions in infant rats.

Spontaneously Occurring Formation of Intranuclear and Cytoplasmic Inclusions in Renal Proximal Epithelium Due to Accumulation of D-Amino Acid Oxidase in Wistar Hannover Rats.

Intranuclear and cytoplasmic inclusions in the renal proximal tubular epithelium were observed in nontreated male and female Wistar Hannover rats in a 26-week study (32 weeks of age) and a 104-week study (110 weeks of age). The incidence rates were less than 5% in these two studies. In affected animals, the inclusions were observed in more than 60% of proximal tubular epithelium as various sized (approximately 1-8 µm in diameter) round and eosinophilic materials, but not in distal tubules, Henle's loop, or collecting ducts. Ultrastructurally, inclusions appeared finely granular, homogenous with middle-electron density, and without a limiting membrane. These inclusions were determined to be protein histochemically stained by Azan-Mallory and immunoreactive with an antibody against D-amino acid oxidase (DAO). There was no abnormality in in-life observations or in clinical test values suggestive of renal dysfunction. There were no associated degenerative or inflammatory changes in the kidneys, and no similar inclusions were observed in the other organs. These inclusions are very similar to propiverine hydrochloride (propiverine) and norepinephreine/serotonin reuptake inhibitor-induced inclusions. This is the first report of accumulation of DAO and formation of inclusions occurring spontaneously in rat kidneys. The data are important for toxicological studies using Wistar Hannover rats.

Systemic histopathology of infant rats exposed to busulfan.

Busulfan is an antineoplastic bifunctional alkylating agent. We previously reported the busulfan-induced systemic histopathological changes in fetal rats and the sequence of brain lesions in fetal and infant rats. In the present study, in order to clarify the nature and sequence of busulfan-induced systemic histopathological changes in infant rats, 6-day-old male infant rats were subcutaneously administered 20 mg/kg of busulfan and histopathologically examined at 1, 2, 4, 7 and 14 days after treatment (DAT). As a result, histopathological changes characterized by pyknosis of component cells were observed in the heart, lungs, stomach, intestines, liver, kidneys, testes, epididymides, hematopoietic and lymphoid tissues, dorsal skin and femur as well as in the brain and eyes (data not shown in this paper). Such pyknosis transiently appeared until 7 DAT with prominence at 2 and/or 4 DAT in each tissue, except for the thymus, in which pyknosis peaked at 1 DAT. Most of the pyknotic nuclei were immunohistochemically positive for cleaved caspase-3, indicating that pyknotic cells were apoptotic. Different from the reports of fetal and adult rats, apoptosis was also found in cardiomyocytes and osteoblasts in infant rats.

Occurrence of Spontaneous Tumors in the Central Nervous System (CNS) of F344 and SD Rats.

In order to accurately assess the carcinogenicity of chemicals with regard to rare tumors such as rat CNS tumors, sufficient information about spontaneous tumors are very important. This paper presents the data on the type, incidence and detected age of CNS tumors in F344/DuCrlCrlj (a total of 1363 males and 1363 females) and Crl:CD(SD) rats (a total of 1650 males and 1705 females) collected from in-house background data-collection studies and control groups of carcinogenicity studies at our laboratory, together with those previously reported in F344 and SD rats. The present data on F344/DuCrlCrlj rats (F344 rats) and Crl:CD(SD) rats (SD rats) clarified the following. (1) The incidences of all CNS tumors observed in F344 rats were less than 1%. (2) The incidences of malignant astrocytoma and granular cell tumor were higher in male SD rats than in female SD rats. (3) The incidences of astrocytoma and granular cell tumor were higher in SD rats than in F344 rats. (4) Among astrocytoma, oligodendroglioma and granular cell tumor, oligodendroglioma was detected at the youngest age, followed by astrocytoma, and ultimately, granular cell tumor developed in both strains. The incidences observed in our study were almost consistent with those previously reported in F344 and SD rats.

GFAP-positive neoplastic astrocytes in spontaneous oligodendrogliomas and mixed gliomas of rats.

It is generally said that neoplastic cells are immunohistochemically negative for glial fibrillary acidic protein (GFAP) in rat spontaneous astrocytomas, and there are no reports describing the existence of GFAP-positive neoplastic astrocytes in rat spontaneous oligodendrogliomas and mixed gliomas which contain neoplastic astrocytes. In the present study, to clarify whether GFAP-positive neoplastic astrocytes exist in rat spontaneous oligodendrogliomas and mixed gliomas or not, immunohistochemical examination was performed on spontaneous oligodendrogliomas (26 cases) and mixed gliomas (5 cases) collected from the carcinogenicity studies and short-term toxicity studies. The neoplastic cells that constitute oligodendrogliomas and mixed gliomas were morphologically classified into five types: round A, round B, round C, spindle, and bizarre. The cells of round A, B, and C types were thought to be neoplastic oligodendrocytes because of their positive immunostainability for Olig2.  The origin of bizarre cells was obscure because they were negative for Olig2, GFAP, and nestin. The spindle cells were considered to be neoplastic astrocytes, because some of them were positive for GFAP or nestin, and GFAP-positive spindle cells could be morphologically distinguished from reactive astrocytes.  In conclusion, the present study clarified for the first time that GFAP-positive neoplastic astrocytes exist in rat spontaneous gliomas.

Spontaneous renal tumors suspected of being familial in sprague-dawley rats.

Spontaneous renal tubule tumors (RTTs), with a distinctive morphological phenotype, were present in three Sprague-Dawley rats, 1 male and 2 females, out a total of 120 animals of each sex from untreated and placebo control groups in a 2-year carcinogenicity study. One female had one carcinoma, adenoma and hyperplasia, and the other female had five adenomas and many hyperplastic lesions; the male case had one carcinoma. From these cases, a biological continuum of hyperplasia, adenoma and carcinoma could be recognized. The tumors were present in the renal cortex and appeared as solid lobulated growths with occasional central necrosis. The lobules were divided by a small amount of fibrovascular tissue. Occasionally the larger tumors contained a cystic area. Tumor cells appeared distinctive and exhibited variable amounts of eosinophilic/amphophilic and vacuolated cytoplasm. Nuclei were round to oval with a prominent nucleolus. Mitotic figures were uncommon, and no distant metastasis was noted. The tumors were seen as multiple and bilateral lesions in two animals and had no apparent relationship to chronic progressive nephropathy (CPN). Foci of tubule hyperplasia were also noted to contain the same type of cellular morphology. The morphological and biological features of these 3 cases resembled the amphophilic-vacuolar (AV) variant of RTT that has been posited to be of familial origin. This is a report of spontaneous familial renal tumors in Sprague-Dawley rats from Japan.

Background Data for General Toxicology Parameters in RccHan:WIST Rats at 8, 10, 19 and 32 Weeks of Age.

Recently, RccHan(TM):WIST (Wistar Hannover) rats were introduced to toxicity studies in Japan. The present study was performed to obtain control data for general toxicological parameters as an aid for interpretation of results in toxicity studies using this strain of rats. Four test groups comprising of 25 male and 25 female RccHan(TM):WIST rats were housed for 2, 4, 13 or 26 weeks from 6 weeks of age and observed and examined for clinical observation, body weight, food consumption, urinalysis, hematology, blood chemistry, organ weight, necropsy and/or histopathology. Ophthalmological examination was not conducted in this study, and the data in this report were obtained from an ongoing 104-week background study in RccHan(TM):WIST rats. These data were compared with the historical control data of CD(SD) (Sprague-Dawley) and/or F344 (Fischer) rats. The body weights of RccHan(TM):WIST rats were lower than those of CD(SD) rats and higher than those of F344 rats. The ophthalmological examination revealed a greater incidence of focal corneal opacity. Histopathology revealed focal mineralization of the cornea and Berlin blue-positive pigmentation in the epididymal interstitium as well as hepatocytes. Other than the above, some minor differences were found in urinalysis, hematology, blood chemistry and organ weights as compared with CD(SD) rats.

Spontaneous extraskeletal osteosarcoma in the stomach of an aged f344 rat.

Extraskeletal osteosarcoma is a very rare tumor in humans and animals including rats. This paper describes a case of extraskeletal osteosarcoma observed in the glandular stomach of an aged female Fischer 344 rat. Grossly, a whitish solid mass was observed at the greater curvature of the glandular stomach. Histologically, the tumor consisted of both atypical polygonal and pleomorphic spindle-shaped cells, with pleomorphic nuclei, and it contained variable amounts of osteoids and small clumps of mature bone tissue. In addition, mitotic figures were frequently observed. Neither invasion of the muscle layer or vessels in the stomach nor metastasis to distant organs was detected. There were no skeletal tumors in the body. Immunohistochemically, the tumor cells were positive for osteocalcin, osteonectin, vimentin and S-100 protein. Judging from these results, the present tumor was diagnosed as extraskeletal osteosarcoma. This is the first report of spontaneous extraskeletal osteosarcoma arising from the stomach in a rat.

Age-dependent changes in the activity of serum alkaline phosphatase in laboratory beagle dogs.

In order to prepare background data for toxicity studies, serum alkaline phosphatase activity in a total of 5,242 male and female beagle dogs was surveyed for the sequence of changes in activity through aging. About 95% of the beagle dogs surveyed were 5 to 12 months of age, corresponding with the age usually employed in toxicity studies. Serum alkaline phosphatase (ALP) activity, about 460 IU/l at 5 months of age, steadily decreased and reached a level about one third of that (about 160 IU/l) at 12 months of age, and remained unchanged thereafter. The above findings were essentially the same irrespective of sex and breeding colony. The present results are useful information in the evaluation of blood chemistry data in toxicity studies.

An occurrence of multiple carcinomas in both kidneys of a 5-year-old female cynomolgus monkey (Macaca fascicularis).

An occurrence of multiple carcinoma in both kidneys of a 5-year-old female cynomolgus monkey is described in the present paper. Macroscopically, the kidneys were enlarged with multiple solid and grayish-white masses and cysts mainly in the cortex and outer medulla. Light microscopically, carcinomas showed either tubulopapillary or tubulosolid growth patterns. Carcinoma cells were pleomorphic cuboidal to columnar cells. Cytoplasm was abundant and either granular and eosinophilic or clear, and nuclei were large and vesicular with prominent nucleoli. Electron microscopy revealed the existence of a brush border-like structure on the cell surface of carcinoma cells, suggesting that carcinoma cells arose from proximal tubular epithelial cells. Small foci of tubular epithelial cell hyperplasia and adenomas were also observed independently of carcinomas. There was no evidence of transition from adenoma to carcinoma, and no metastases were detected.