The objective of this investigation was to elucidate the role of sex hormones in the progression and subsequent hormone dependency of mammary carcinomas induced by single or multiple administration of 7, 12-dimethylbenz[a]anthracene (DMBA). Ten milligrams of DMBA were administrated orally to female Sprague-Dawley (SD) rats once at the age of 42 days (Group A) and three times at the ages of 28, 42 and 56 days (Group B). All 70-day-old rats in Groups A and B were divided into 4 groups (I, II, III and IV), respectively. Rats in Groups I of A and B were intact controls. Rats in Groups II of A and B were ovariectomized (OVEX) at the age of 70 days. Rats in Group II of Group A and in Groups III of Groups A and B were divided into 4 subgroups (Group II-1, II-2, II-3 and II-4) and 3 subgroups (Group III-1, III-2 and III-3), respectively. Rats in Groups II-2 of A and III-1 of A and B, rats in Groups II-3 of A and III-2 of A and B and rats in Groups II-4 of A and III-3 of A and B were given injections of 0.01, 0.1 and 1 mg 17β-estradiol (E₂), respectively. Rats in Groups IV of A and B were given injections of 4 mg progesterone (P). Injections of E₂ or P were given 3 times a week between the ages of 70 and 217 days in Group A and the ages of 70 and 105 days in Group B. In Group A, there was no difference of incidence of mammary carcinomas among Groups I, II-2, II-4, III-1 and IV but the incidence of mammary carcinomas in Groups II-1, II-3, III-2 and III-3 was extremely low, compared with that in Group I. In Group B, a large number of mammary carcinomas developed and although there was no difference in the incidence of mammary carcinomas among Groups I, II, III and IV, the number of mammary carcinomas per rat decreased in Groups II, III-2 and III-3, compared with Group I. These findings indicate that the progression of mammary carcinomas induced by a single or multiple administration of DMBA was suppressed in ovariectomized rats and in rats with injections of high doses of E₂. DMBA-induced mammary carcinomas in Group B were transplanted into OVEX rats (R-1), OVEX rats with injections of 0.01 mg E₂ (R-2) and OVEX rats with injections of 1 mg E₂ (R-3). According to tumorigenesis in those rats with various hormonal treatments (R-1, 2, 3), mammary carcinomas were classified into one of the four categories (Type A, B, C and D). Type A tumor: tumor developed greatly in R-2 but slightly or not in R-1 and R-3. Type B tumor: tumors developed greatly in R-1 but slightly or not in R-2 and R-3. Type C tumor: tumors developed greatly in R-3 but slightly or not in R-1 and R-2. Type D tumor: tumors developed greatly in R-1, R-2 and R-3. In Group B, rates of type A, B, C, D tumor were 43.2, 13.5, 24.3 and 18.9% in Group I, 26.3, 10.5, 21.1 and 42.1% in Group II, 40.0, 5.0, 45.0 and 10.0% in Group III-1, 38.5, 15.4, 30.8 and 15.4% in Group III-2, 28.6, 0, 71.4 and 0% in Group III-3 and 44.4, 33.3, 11.1 and 11.1 in Group IV, respectively. These results suggested that the hormonal status of the development process may be one of factors which affects the characteristics of hormone dependency and the multiple administration of DMBA may induce large numbers of carcinoma cells with varying dependency on hormones and carcinoma cells may be selected in hormonal conditions during progression.

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Inbred, female Sprague-Dawley rats were neonatally androgenized at the age of 2 days by subcutaneous injections of 1.25 mg testosterone propionate and divided into 4 groups. At the age of 50 days, rats of all groups were given 20 mg 7, 12-dimethylbenz[a]anthracene (DMBA). Four weeks after the administration of DMBA, the rats in Group I remained intact, those in Group II were subjected to ovariectomy, while the animals in Groups III and IV were given 1 mg 17β-estradiol (E₂) at 2-day intervals for 3 weeks. Additionally, the rats in Group IV were given 1 mg E₂ at 2-day intervals for 3 weeks by intramuscular injection, starting from the 105th day after the administration of DMBA.The ovaries of the neonatally androgenized rats contained numerous large vesicular follicles and no corpora lutea, and the mammary glands of the animals in Groups I, III and IV consisted mainly of dilated acini containing milky secretions. In rats in Group I, mammary carcinomas and mammary dysplasias were induced. Rat mammary dysplasias were grossly characterized by gross cysts and solid masses. Solid masses were heteromorphic lesions and commonly consisted of several nodules with various microscopic features. These masses were subclassified into the 2 types of histological features that were predominant among the nodules: acinar adenosis and fibrotic adenosis. The induction of mammary carcinomas and mammary dysplasias was strongly suppressed by ovariectomy (Group II) as the development of mammary carcinomas and mammary dysplasias induced in neonatally androgenized rats are dependent on estrogens. The induction of mammary carcinomas was suppressed by injections of high doses of E₂ for 21 days (Once in Group III and twice in Group IV). Conversely, the induction of mammary dysplasias was promoted by one time (Group III) but was suppressed by two times (Group IV) of E₂. The results indicate that carcinoma cells and dysplasia cells induced by DMBA in neonatally androgenized rats differ in response to high doses of E₂.

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Humans encounter exposure to arsenic (As) in numerous ways. Dimethylarsinic acid (DMA), one of the major metabolites of inorganic arsenics in most mammals promotes kidney, urinary bladder, liver, and thyroid carcinogeneses in F344 rats. Dimethylarsenic peroxyl radical ((CH₃)₂ AsOO.) has been postulated to be responsible for the DNA damage induced by DMA. In a preliminary experiment conducted in our laboratory, subacute doses of DMA induced formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and increased proliferation indices in the renal cells of NCI-Black Reiter (NBR) rats which lack α_2u-globulin, which has been attributed to influence renal carcinogenesis. This prompted us to study the promoting effects of DMA on renal carcinogenesis in this strain of rats by employing the initiation-promotion model. Animals were divided into two groups (11 rats in each group). Group 1 was treated with N-ethyl-N-hydroxyethylnitrosamine (EHEN), at a concentration of 0.1% in the drinking water for 2 weeks followed by basal diet for 30 weeks. Group 2 was treated with EHEN with the same dosage as Group 1 for 2 weeks followed by 200 ppm DMA in the drinking water for 29 weeks. Neither atypical tubules nor renal cell tumors were induced in both groups, suggesting that absence of α_2u-globulin may contribute to the resistance to the above stimulus by this strain of rats. Since EHEN also initiated liver carcinogenesis, we analyzed the liver for glutathione S-transferase placental form (GST-P)-positive foci, which is a marker in rat liver carcinogenesis. There was no significant change between the two groups. Thus, DMA lacked the potential to promote kidney and liver carcinogeneses in male NBR rats.

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Our previous data showed that the F344/DuCrj rat strain is sensitive to the promoting activity of sodium Lascorbate in two-stage urinary bladder carcinogenesis, whereas the WS/Shi rat strain is resistant. In the present study, we found that the latter also lack susceptibility to three other urinary bladder promoters of differing type: NaHCO₃, butylated hydroxyanisole, and melamine. Melamine formed urinary calculi, whereas NaHCO₃ and butylated hydroxyanisole did not. NaHCO₃ alone increased urinary pH and sodium ion concentration. Male rats, 6-week-old, were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks, and then fed basal diet supplemented with 3% NaHCO₃, 2% butylated hydroxyanisole, or 3% melamine for 32 weeks. Melamine, which gives rise to urinary calculi, promoted urinary bladder carcinogenesis, whereas NaHCO₃ did not, despite significant elevation of urinary pH and sodium ion concentration. Butylated hydroxyanisole was also without effect. The results indicate that WS/Shi rats are sensitive to the promotion of two-stage urinary bladder carcinogenesis by melamine, but not NaHCO₃ or butylated hydroxyanisole.

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Controversial results have been reported about the change in anionic charge density on the glomerular basement membrane (GBM) and podocytes in relation to glomerular protein leakage induced by puromycin aminonucleoside (PAN). So we examined the anionic charge on the glomerular wall of rats with PAN-induced proteinuria. PAN, 15 mg/kg, was intraperitoneally injected daily into female Wistar rats for 4 to 14 days. Examinations performed were as follows: Urinary levels of protein and sialic acid, protein fraction analysis of urine by SDS-PAGE, serum chemistry, cytochemistry for assessment of anionic charge by a cationic poly-L-lysine gold (PLG) method (pH 3.3), digestion tests with neuraminidase and heparitinase, immunocytochemistry for the localization of serum proteins and actin, and conventional light and electron microscopy. In proteinuric rats, the anionic sites on podocytes consisting mainly of sialic acids were decreased from the early stage of protein leakage. The urinary sialic acid level was elevated in advanced proteinuria. The density of anionic sites on the GBM remained mostly unchanged even in severe proteinuria. Serum proteins were identified in dense bodies and vacuoles in the podocytes in proteinuric rats. Disarrangement of actin was seen in flattened foot processes. No detachment of foot processes was observed even in severe proteinuric rats. In conclusion, the decrease in anionic charge density on podocytes is attributable to protein leakage through the glomerulus in PAN-induced proteinuric rats. The anionic charge on GBM and the detachment of foot processes seem to be irrelevant to the primary change leading to protein leakage.

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To establish a method of lung processing suitable for morphological, immunohistochemical and enzyme histochemical analyses, we examined the combination of inflated-fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method. The lungs were removed from rats with or without bleomycin (BLM) treatment and the airways were instilled with PLP fixative. After inflated-fixation, the specimens were processed and embedded in paraffin by the AMeX method. Paraffin sections were stained immunohistochemically and enzyme histochemically. Hematoxylin and eosin (HE)-stained sections were also subjected to evaluation of tissue architecture. In the HE-stained sections, the bronchioles, alveolar ducts, and alveolar sacs were uniformly inflated, and the architecture was well preserved. In addition, detailed histopathological findings of lung lesions induced by BLM could be observed. In immunohistochemical staining, all of the antibodies used in this study (anti-CD3, -CD45RA, -ED-1, and -alpha-smooth muscle actin monoclonal antibodies) stained the cells positively according to their specificities. It is noteworthy that CD3 and CD45RA, which are not detected in routinely formalin-fixed paraffin sections, were clearly detectable. In enzyme histochemical staining, alkaline phosphatase activity was very well preserved. These findings suggest that the present method is useful for immunohistochemical and enzyme histochemical analyses of the lung.

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Brown Norway (BN) rats are known to be hypersensitive to inhaled allergen or ozone. We compared the histological responses of the respiratory system following 2-week-inhalation of cigarette smoke between BN and Fischer 344 (F344) rats which have been widely used in long-term toxicity studies. F344 rats showed salivation and/or palpitation after every cigarette smoke-exposure while BN rats showed no clinical signs. Histologicaly, in the nasal mucosa near the open air, mild stratification of epithelial cells was seen in both strains. In the epiglottis, apparent stratification of hypertrophic epithelial cells was found in BN rats while squamous metaplasia with a distinct keratinized layer of mucous epithelium was observed in F344 rats. The number of alveolar macrophage in F344 rats was larger than that in BN rats. Serum VA level which is known to control epithelial cell differentiation did not show significant change by cigarette smoke challenge. These results suggest that the responses of the respiratory system were severer in F344 rats than in BN rats.

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A 15-week-old, male Fischer344 rat clinically had bilateral buphthalmos. Grossly, both globes were enlarged showing protrusion of the cornea with an increase in anterior chamber depth. Histologically, similar findings were observed in both globes. The cornea and sclera were slightly thinned. The iris was displaced forward, resulting in sealing of filtration angle. The root of the iris was partly connected with the corneal endothelium, in which the Descemet’s membrane was lost. The trabecular meshwork was hypoplastic and covered completely with the deformed iris tissue. Neither inflammatory reaction nor fibrosis was detected in the affected angle and anterior uvea. The ciliary body was hypoplastic and dysplastic. The retina, particularly the inner layers, was atrophic and degenerative. The optic disc was deeply cupped and the optic nerve fibers were degenerated. These findings led us to diagnose the present case as primary congenital glaucoma in the rat.

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A hematopoietic neoplasm was found in a 14-week-old male Sprague-Dawley (SD) rat. Blood smear examination revealed the existence of numerous blastic cells with large, round or oval nuclei, and scant pale cytoplasm containing no granules. At autopsy, the liver and spleen were markedly enlarged and the right femur showed swelling and hemorrahge. Histopathologically, neoplastic cells infiltrated diffusely in the bone marrow, liver, and spleen, and destroyed their normal architectures. In the bone marrow neoplastic cells invaded into the surrounding tissues, while in the spleen they proliferated from the red pulp. Immunohistochemically, neoplastic cells were positive for lysozyme, while they were negative for hemoglobin, ED-1, OX-7, OX-8, and OX-12. Based on these findings, the present neoplasm was diagnosed as myeloblastic leukemia.

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A 9-month-old male beagle suddenly died without any clinical signs. Histopathologically, he showed acute circulatory insufficiency due to diffuse degenerative changes of blood vessels and exudation from arterioles and myocardial contraction band necrosis of the heart. The cardiac lesions were marked in the left ventricle and the vascular lesion consisted of medial degeneration of the arterial system extending from the main branches of the extramural coronary arteries to the arteriolar segments. These lesions showed no inflammatory changes.

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Multiple chondromas were observed in one young adult (18 weeks old) male B6C3F₁ strain mouse used in toxicity tests for safety assessment of chemicals. Solitary chondroma composed of mature cartilage cells was found in different sites of the ribs. In addition, chondrosarcoma was observed in the lumbar vertebra and abdominal cavity of one old (109 weeks old) male mouse of the same strain. The sarcoma was composed of mixed atypical chondrocytic and spindle-shaped cells, extending into the surrounding tissue. This is a report on spontaneous multiple chondromas occurring in the B6C3F₁ mouse.

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