We investigated the naturally occurring calcification of the aortic orifice (ostium aortae), which is the opening from the left ventricle into the ascending aorta, in dogs. Twenty-one beagle dogs were examined at the age of 6 weeks (N=6), 8 months (N=12), and 7-8 years (N=3). Typical calcification exhibiting a characteristic purplish color with hematoxylin and eosin staining was observed in 3 out of 12 dogs at the age of 8 months. The changes were found in the aortic wall above the seputal semilunar cusp. In addition to the typical calcification, small eosinophilic particles with a positive reaction for von Kossa technique was frequently observed in all dogs except for 2 animals in the 6-week-old group. The deposition of the eosinophilic particles increased with aging, and some particles had basophilic cores in 8-month-old and older dogs. These particles were preferentially localized at the aortic medial elastic fibers above the seputal semilunar cusp. Energy dispersive X-ray analysis revealed that the particles were composed of calcium phosphate. These results suggest that the small eosinophilic particles demonstrate the minimal calcification. The lesion deteriorates with aging and seems to be a based lesion for typical calcification.

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2, 2, 4-Trimethylpentane (TMP) was administered orally to male Sprague-Dawley rats for 26 weeks to induce α2u-globulin nephropathy and histopathological examinations of the kidney as well as urinary sediment morphology and 5-bromo-2'-deoxyuridine (BrdU) incorporation were performed. The number of exfoliated renal epithelial cells in the urine increased with the TMP treatment until week 4, when a peak of approximately 90-fold as compared with the matched control values was observed. Significantly elevated levels then persisted throughout the study. Deposits of α2u-globulin and BrdU labeling in the kidney also reached maxima at week 4. A correlation among the amount of α2u-globulin deposits, the BrdU labeling indices, and the numbers of exfoliated cells in the urine was noted at each time point. The results thus suggest that damaged cells become detached following excessive accumulation of α2u-globulin and this stimulates regenerative cell proliferation in the kidney. Quantitative examination of exfoliated cells in the urine may thus provide a reliable estimate of cell proliferation induced by α2u-globulin nephropathy during rat toxicology studies.

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The B6C3F₁ (C57BL/6 × C3H/He) mouse has commonly been used for various chronic toxicity and carcinogenicity studies. This mouse develops various types of spontaneous tumors during its lifetime. The incidence of ras gene mutation has been reported in tumors of various epithelial tissues including the liver, lung, and Harderian gland of the B6C3F₁ mice. However, there are few reports on the frequency of ras activation in non-epithelial tumors in spite of their high incidence similar to that of epithelial tumors. We examined ras gene mutations in 114 non-epithelial tumors (hemangioendothelial cell tumors, malignant lymphomas, and histiocytic sarcomas), generated spontaneously in B6C3F₁ mice by non-RI single strand conformation polymorphism analysis and direct sequencing of DNA, which was isolated from paraffin-embedded sections and amplified by polymerase chain reaction. Only seven non-epithelial tumors in the B6C3F₁ mice contained mutation of the H-ras codon 61, all of which were C to A transversion (CAA → AAA). The present studies suggest that activation of the ras gene is involved only a little in occurrence of the primary spontaneous non-epithelial tumors and that the pathways other than ras gene activation lead to the development of most non-epithelial tumors in the B6C3F₁ mouse.

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The usefulness of precision-cut liver slices for toxicological evaluation in vitro was confirmed with a representative hepatotoxic agent acetaminophen. Liver slices obtained from normal and in vivo phenobarbital-treated rats were incubated with 0, 1.25, 2.5, 5, and 10 mM of acetaminophen for 24 hours. Intracellular potassium concentration dose-dependently decreased and lactate dehydrogenase (LDH) leakage increased in the slices from phenobarbital-treated rats, whereas no changes were detected in the slices from normal rats. The cytotoxicity of acetaminophen was also shown in the slices from normal rats when the slices were preincubated with phenobarbital in vitro for one hour. The acetaminophen cytotoxicity in the liver slices was enhanced by the preincubation with diethyl maleate, a depressant of hepatic glutathione, tributyltin acetate, an inhibitor of glutathione S-transferase, and 2, 6-dichloro-4-nitrophenol, an inhibitor of sulfotransferase. These data suggest that the formation and accumulation of the toxic metabolite are essential to induce the acetaminophen hepatotoxicity. Histopathological observation revealed centrilobular hepatocyte necrosis on the slices from phenobarbital-treated rats 24 hours after the exposure to acetaminophen. These results coincide with those reported in in vivo acetaminophen toxicity. The application of precision-cut liver slices is considered to be useful in toxicological evaluation of chemicals.

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T-2 toxin is a kind of trichothecene mycotoxin produced by species of the genus Fusarium, and it attacks the tissues containing a large number of actively dividing cells such as lymphoid and hematopoietic tissues. The development of apoptosis and the changes in mRNA expressions of apoptosis-related cell-surface antigen (Fas) and genes (p53, bcl-2, c-myc, and c-fos) were investigated in the thymus up to 9 hours after oral inoculation (HAI) with 10 mg/kg body weight of T-2 toxin in female ICR: CD-1 mice. The number of TUNEL-positive lymphocytes clearly increased in the thymus of T-2 toxin-inoculated mice at 6 and 9 HAI. By agarose gel electrophoresis, DNA ladder was clearly detected at 9 HAI. The c-fos mRNA expression increased prior to the progression of apoptotic cell death in the thymus of T-2 toxin-inoculated mice. As to Fas, p53, bcl-2, and c-myc, their mRNA expressions showed no significant changes through the observation period. The results using p53 knockout mice and Fas disfunctional mutant mice also demonstrated that p53 and Fas had no relation to T-2 toxin-induced apoptosis. These findings suggest that c-fos mRNA induction may be associated with T-2 toxin-induced lymphocyte apoptosis.

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Dermatotoxicity of topical application of chemicals for hair-dyeing (CHD) was clinically and histopathologically investigated in hairless dogs. CHD examined were as follows: 1. Resorcinol (RCL), 2. pyrogallol (PGL), 3. tolylene-3, 4-diamine (T34D), 4. tolylene-2, 4-diamine (T24D), 5. ammonium persulfate (AMPS), 6. potassium persulfate (PTPS), 7. hydrogen peroxide (HDPO), and 8. vehicle (control). Severe contact dermatitis characterized by swelling, intense edema, and blister formation was seen at 4 or 5 days application of PGL. Test sites applying T34D and T24D showed striking contact dermatitis from 7 to 10 days application. Especially, the T34D-applying sites showed prominently edematous vesicular reactions followed by moderate pigmentation. At the test site applying AMPS provoked delayed contact dermatitis. At the test sites applying HDPO, severe dermatitis was immediately exhibited. Histopathologically, test sites applying RCL showed remarkable hyperkeratosis. At the test site 5 days application of PGL, intraepidermal blisters contained abundant inflammatory cells. At the test site 10 days application of T34D and T24D, prominent hypertrophy of epidermal cells and infiltration of inflammatory cells in the dermis were observed. These histopathological findings were severer in the test sites applying T34D than T24D. At the site 40 days after the end of topical application of T34D, a few epidermal cells with pyknotic nuclei and eosinophilic cytoplasma were seen in some repairing epithelium. By 40 days after the end of application of T34D, pigmentation was observed in the stratum basale and spinosum. From 20 to 30 days application of AMPS, focal necrosis in the dermal-epidermis junction was noted. In the dermis, prominent inflammatory changes were observed. At the test site 5 days application of HDPO, the epidermis was thickened by intracellular edema and hyperplasia of component cells. Additionally, the epidermis showed prominent parakeratosis. These results revealed that the skin of hairless dogs gave a definitive response to topical application of CHD. Hairless dogs were suggested for laboratory animals to evaluate immediate-and/or delayed-type dermatotoxicity of CHD.

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