In order to clarify the possible mechanisms underlying the inhibition of uterine tumorigenesis in rasH2 mice carrying a human prototype c-Ha-ras gene when treated with ethinylestradiol (EE), mice were given a single intraperitoneal injection of 120 mg/kg N-ethyl-N-nitrosourea (ENU), followed by 2.5 ppm EE for 6 weeks. Many genes involved in estrogen responses and cell proliferation in the uterus of rasH2 mice are activated by treatment with ENU followed by EE. These include genes associated with receptors such as estrogen receptor (ER) alpha, cell cycles such as cyclin E, cyclin dependent kinase (CDK) 4, CDK inhibitors, ubiquitin and smad3, and growth factors such as epidermal growth factor (EGF) receptor, transforming growth factor (TGF) beta, TGF beta receptor and insulin-like growth factor (IGF). In this study, the ENU+EE treated rasH2 mice demonstrated acceleration of estrogen decomposition and down-regulation of the expression of ER alpha. TGF beta, serine/threonine kinase and smad3, which are downstream of the TGF beta signaling pathway, were also down-regulated, indicating signal values such as CDK4 were down-regulated in the ENU+EE treated rasH2 mice. Ubiquitin which indicates CDK inhibitory metabolism was also down-regulated. Finally, several genes involved in growth factors such as EGF receptor, TGF beta receptor and IGF were also down-regulated. On the other hand, the ENU+EE treated ICR mice showed deceleration of estrogen decomposition and up-regulation of the expression of ER alpha, down-regulation of TGF beta, serine/threonine kinase, smad3, CDK4, ubiquitin, EGF receptor, TGF beta receptor and IGF, indicating that these genes appear to act as stimulants of cell proliferation and carcinogenesis. Therefore, we consider that these genes are key genes which are strongly involved in the inhibition of uterine carcinogenesis in ENU-initiated rasH2 mice treated with EE.

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We previously demonstrated that apoptotic cell death occurred in the thymus of rat pups of both sexes after perinatal exposure to methoxychlor (MXC) through dams. Based on that result, we have undertaken a further investigation to determine whether perinatal exposure to MXC persistently influences the rat immune system until a mature age. For the present study, male and female pups were obtained from dams receiving MXC at dietary dose levels of 0, 30, 100, 300 and 1000 ppm. Dietary administration of MXC to the parental rats was started from 3 weeks prior to mating for both sexes and was continued for females (dams) throughout the period of gestation and lactation. The pups of both sexes in all dose groups were maintained on a normal diet from weaning up to 10 weeks of age and then were examined for the effect of MXC on the immune system. Males and females showed a significant increase in thymic CD3⁺/CD4^-CD8^- cells in the 1000 ppm group and a significant decrease in splenic CD3⁺/CD8⁺ cells in the 300 and 1000 ppm groups. In addition, females in the 300 and 1000 ppm groups showed significant decreases and/or decreasing trends in splenic cellularity, splenic CD3⁺ T cells, CD45RA⁺ B cells, and CD3⁺/CD4⁺ T cells. These results suggest that perinatal exposure to MXC may exhibit profound and long-lasting effects on the developing rat immune system, especially in females, which continues into adulthood.

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The authors have previously reported that the combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (the PLP-AMeX method) resulted in better preservation of immunoreactivities and endogenous enzyme activities. In the present study, we investigated whether PLP-AMeX-processed tissue would be useful for in situ hybridization (ISH). The pancreases were removed from rats treated with or without streptozotocin (STZ), and was fixed in PLP fixative. After fixation, the specimens were processed and embedded in paraffin by the AMeX method. Paraffin sections were made on which ISH and immunohistochemical staining were performed. ISH was performed according to the non-radioactive method using digoxigenin-labeled oligodeoxynucleotides probes and anti-digoxigenin antibody. For ISH, a 28S rRNA antisense probe was used for evaluation of the level of hybridizable RNA in the sections. 28S rRNA was clearly detected in the various cells of the pancreas. Using an insulin antisense probe, the presence of insulin mRNA was clearly identified in the pancreatic islet-cells. Furthermore, optimal results for ISH of insulin mRNA and immunohistochemical staining of insulin could be obtained in consecutive sections from a PLP-AMeX-processed block. In the rats treated with STZ, the histological changes of pancreatic islet-cells and the changes in staining intensity of immunohistochemical staining for insulin protein and ISH for mRNA could be observed in the PLP-AMeX-processed sections. These findings suggest that hybridizable RNAs are well preserved by the PLP-AMeX method, and that the method is useful for ISH as well as immunohistochemical and enzyme histochemical analysis.

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The modifying effects of liver injury on male reproductive organ toxicity of di-n-butyl phthalate (DBP) were studied using male F344 rats. Seventy-two male animals, aged 10 weeks at the commencement, were divided into 8 groups of 9 rats each. Groups 1 to 4 were given 200 mg/kg bw of thioacetamide (TAA) intraperitoneally 3 times / week, while Groups 5 to 8 were injected with the PBS vehicle only. From 1 week after the start, groups 1 and 5, 2 and 6, and 3 and 7 were given daily 500, 125, 31.25 mg/kg bw of DBP, respectively (23 times) for 4 weeks, while groups 4 and 8 served as vehicle controls. As the result of our treatment, liver injury was induced in the animals, and the liver injury was confirmed by histopathological findings and serum transaminase levels at sacrifice. Enhancement of rat male reproductive organ toxicity of DBP under liver injury status was also seen. The relative organ weights of prostate, bilateral seminal vesicles, bilateral epididymides and bilateral testes, and the sperm number and motility rate were decreased. Also the sperm abnormality rate was increased under liver injury. Furthermore histopathological abnormality of the testis, such as absence of germ cells and the presence of Sertoli cells only was seen in the rats treated with TAA and high dose of DBP, however neither the serum nor testicular testosterone levels varied in the present study. The sperm number and relative weight of bilateral epididymides were decreased by TAA treatment only, unrelated to DBP treatment. These facts suggest that liver injury can not only enhance the rat male reproductive organ toxicity of DBP, but also induce such toxicity by itself.

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Beagle dogs intravenously treated with alloxan monohydrate at 50 mg/kg and kept for 14 months were subjected to clinicopathological and histopathological examinations in order to characterize the development of diabetic changes. The beagle model of diabetes mellitus with alloxan in the present study was characterized by variegated conditions which markedly involved male animals. All the males in this study either died or were sacrificed in extremis up to Week 20, whereas all the females survived throughout the 14-month observation period. Determination of serum fructosamine level, and the glucose tolerance test proved useful in detecting diabetic signs. Decreased serum creatinine and increased total cholesterol levels, and elevation of alkaline phosphatase activity were important secondary changes; however no consistent signs of ketoacidosis were noticed in the persistently diabetic animals. In the present study, diestrus, as confirmed by estrous bleeding, may have been an important provoking factor in diabetic episodes in female dogs with impaired glucose tolerance. Pathological changes seen in the pancreas, liver, and kidneys were ascribed to direct effects of alloxan and the resulting derangement of carbohydrate metabolism. As early as Week 4 of treatment, ocular changes, especially development of cataract, were recorded as the major secondary complications ascribable to persistent hyperglycemia. There were no signs of structural complications, except for parenchymal atrophy and focal inflammatory changes in various organs. Secondary complications other than the cataract seemed to not easily occur in this animal model.

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We found spontaneous dwarfs in Wistar Hannover GALAS rats, and morphologically and endocrinologically investigated the endocrine systems. Histologically, little colloid formation, decreased follicular size, and huge vacuoles in the follicular epithelium were observed in the thyroid of the affected animals. The vacuoles reacted positively for thyroglobulin. Electron microscopically, dilated rough endoplasmic reticulum, nuclear dislocation and few secretory granules were observed in the thyroid follicular cells. Serum triiodothyronine and thyroxine values were markedly low, whereas the serum thyroid stimulating hormone value was markedly high. These findings indicated that this dwarfism was caused by primary hypothyroidism.

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The present study was conducted with the original purpose of investigating the possibility that α-naphthylisothiocyanate (ANIT) might induce intrahepatic cholangiocellular neoplasms in rats after appropriate carcinogenesis-initiating treatments, based on its known effect of causing intrahepatic bile duct proliferation. Fischer 344 rats (6 weeks old) were given 3 weekly intraperitoneal administrations of vehicle (female and male), N-nitrosobis(2-oxopropyl)amine (BOP) (20 mg/kg body weight, female and male) or N-nitrosodimethylamine (DMN) (10 mg/kg body weight, male only), and fed a basal diet with or without 200 ppm of ANIT from the commencement for up to 24 weeks. Animals were sequentially sacrificed at the ends of weeks 8, 16 and 24 to examine morphological changes in the liver. ANIT caused proliferation of intrahepatic bile duct epithelial cells with no atypia when administered alone or in combination with BOP (female and male) or DMN (male only), while neither BOP nor DMN caused bile duct proliferation per se or altered the magnitude of the effect of ANIT. The degree of bile duct proliferation caused by ANIT was greater in females than in males. No hepatocellular or liver (pre)neoplastic changes were observed. These results indicate that although ANIT does not induce any neoplastic changes in the liver even after initiation with BOP (female and male) or DMN (male), it causes non-neoplastic intrahepatic bile duct proliferation with a clear sex difference.

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A spontaneous ovarian immature teratoma characterized by the presence of tissue derivatives of endoderm, mesoderm and ectoderm was investigated histopathologically in a 6-week-old juvenile Crj:CD(SD)IGS rat. The various kinds of tissue components observed in this teratoma were mainly mature elements derived from three embryonic germ layers, such as nervous tissue, skin, pancreas, salivary gland, gastrointestinal tract, respiratory tracts, striated and smooth muscle, bone with bone marrow, cartilaginous tissue and adipose tissue. Remarkable features were that this teratoma contained immature tissues intermingled with mature tissues, which typically took the form of primitive neuroepithelial rosettes and tubules and islands of immature cartilage, showing evidence of malignancy. A small yolk sac carcinoma-like focus was also observed. Neither invasion nor distant metastases were found in the other organs and tissues. This is the first report of an ovarian immature teratoma in a rat.

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In the present study, a suitable rat model for testing the modifying effect of liver injury on the rat male organ toxicity of endocrine disrupters was researched using thioacetamide (TAA) and carbon tetrachloride (CCl₄) as liver toxicants. Male 8-week-old F344 rats were divided into six groups as follows. Animals in groups 1 and 2 were given 200 and 100 mg/kg body weight of TAA with PBS by intraperitoneal injection (ip.) 3 times a week for 4 weeks, respectively. Group 3 was given PBS only. Animals in groups 4 and 5 were given 2 and 1 mg/kg body weight of CCl₄ with corn oil by subcutaneous injection (sc.) once a week, for 4 weeks, respectively. Group 6 was given corn oil only. Three rats from each group were sacrificed at week 4 and the rest of groups 1 and 4 were sacrificed after a further 4 weeks of withdrawal period. In the animals administered 200 mg/kg of TAA the findings of apparent liver damage on both histopathological and biochemistrical examinations could be seen: fibrosis, enlargement of Glisson's sheath and infiltration of inflammatory cells in the liver, and apparent elevation of serum aspartate aminotransferase and alanine aminotransferase levels. On the other hand, in the animals administered 2 mg/kg of CCl₄ only the histopathological findings of liver damages could be seen: fatty and inflammatory changes in liver. In the animals administered 100 mg/kg of TAA or 1 mg/kg body weight of CCl₄ only the histopathological findings of slight inflammatory changes in their liver samples. However these findings of liver damage were almost completely absent after the 4-week withdrawal period.

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