Correlation among species, sex, and tumor-site of neoplastic responses in 25 chemicals tested for carcinogenicity at the An-Pyo Center in 1981-1993 were evaluated. Of the 25 chemicals tested in rats and mice, 44 percent (11 of 25) were positive in at least one species and 56 percent (14 of 25) were negativein both rats and mice. Rats and mice exhibited a similar sensitivity to carcinogens as evidenced by 8 positive studies in rats and 9 positive studies in mice for a total of 17 positive studies. Three of the 25 chemicals evaluated were genotoxic and all tested positive in the carcinogenic bioassays in all groups and in both species and sexes. In addition, target tissues were similar between males and females. However, species specific or sex-related responses were evident at different dose levels with the non-genotoxic compounds tested.
Compilation ofhistorical tumor development data has revealed biological mechanisms whereby three classes of compounds produce characteristic positive results in rodent bioassays. Criteria for defining genotoxic, non-genotoxic and promotor/pseudo-carcinogens are presented and discussed.
Results of current bioassays together with mechanistic and refined, short-term studies may be evaluated collectively to aidin interpretation of results and in the mechanistic identification of these three classes of compounds.

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An integrated toxicologic pathology system named “MacMic” has been developed and expanded for use in toxicological studies. By utilizing a personal computer Local Area Network (LAN), high user interface and easy data sharing were achieved at low cost. The studies covered by this system are all of the general toxicological studies including carcinogenicity studies. It was designed for the purpose of easy handling from the standpoint of pathologists, and it offers high usability. At histopathological examinations, screeners can refer to the data entered from other subsystems: body/organ weight measurement, urinalysis, hematology, and blood chemistry, and also those data summaries, which clarify the differences at a given rate from controls by different color. Entry of gross and histological findings is done by referring to the specialized dictionaries, which are maintained routinely, and the selection of the findings and copy from those entered before are easy. Movement to the next step is quick with function keys, and the proceeding status of histological examinations is obvious with a key for fixing and color distinction. There is a memo function for communication between screeners and exclusive control function to allow multiple access to the same study but not to the same organ at a time for protection of data. “MacMic” was originally the system using personal computer LAN for a certain laboratory, but it was found to be applicable to other laboratory/institute.

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Suitable conditions of bleomycin (BLM)-treatment to effectively induce pulmonary fibrosis without high mortality were investigated in Syrian golden hamsters. Male 6-week-old hamsters were divided into 5 groups, each consisting of 5 animals. Groups 1-4 were intratracheally instilled with 2.5U/kg of BLM on day O and then repeatedly given BLM as follows: group 1, 2.5U/kg intratracheally on day 14; group 2, 1.0U/kg intratracheally on day 14; group 3, 1.0U/kg intratracheally each on days 7 and 14; group 4, 20U/kg intraperitoneally each on days 10and 14. Group 5 served as a vehicle control. The survival rate was 4/5 (80%) in groups l and 3, and 5/5 (100%) in other groups. One animal each in groups I and 3 was found dead on day 22, of which lungs histopathologically showed evident fibrosis accompanied with congestion, edema, inflammatory cell infiltration, and hemorrhage. The lung weight at the termination of the experiment on day 28 was the highest in group 1, and significantly higher in groups 1 (p<0.01) and 2 (p<0.05) than in group 5, well correlating with the histopathological severity of lungfibrosis. The lung weight of group 3 was also higher as compared to the group 5 value although this was not statistically significant and the lung weight of group 4 was almost comparable to that of group 5, again being in line with the histopathology of the lung. The results in the present study thus suggest that the repeated intratracheal instillation with 2.5U/kg BLM at intervals of 2 weeks may offer a favorable condition to induce diffuse lung fibrosis in hamsters.

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Macrophages and α-smooth muscle actin immunopositive myofibroblasts are thought to be crucial cells in granuloma formation. In this study, the reactive cell kinetics was investigated in Sephadex bead-induced rat pulmonary granulomas (1mg/ml saline/head; intravenously) from l hour to 90 days after the injection. Intravenously administered Sephadex beads occluded pulmonary arteries and caused granulomatous pulmonary arteritis. The inflammatory cells appeared first around the trapped beads and then granuloma lesions developed within 24 hours. Three days later, the granulomas mainly consisted of neutrophils, macrophages, eosinophils, and giant cells. Immunohistochemically, the number of ED1-positive macrophages quickly increased 24 hours after the injection, and a large number of macrophages were still found 3 days after the injection. Alpha-smooth muscle actin immunopositive myofibroblasts increased in numbers from 3 to 7 days after theinjection. These results indicate that the appearance of myofibroblasts in pulmonary granulomas might be mediated by macro-phages, which were the most predominant inflammatory cells in the present pulmonary granuloma.

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We investigated the presence of α_2u-globulin, which has generally been thought to be a protein specific to male rats, in the kidneys of female rats. Immunohistochemical, immunoelectron microscopic and immunochemical studies were performed on the kidneys of male and female F344 rats using rabbit anti-serum prepared with α_2u-globulin purified from male rat urine. In male rats, hyaline droplets containing granules positive for immunohistochemical staining of α_2u-globulin were observed in the proximal convoluted tubular epithelium. In females, fine granules positive for staining of α_2u-globulin were observed in the same region, although so-called hyaline droplets were not observed in HE-stained specimens. Upon immunoelectron microscopic examination, gold particles indicating the presence of α_2u-globulin were found localized in the lysosomes of proximal convoluted tubular epithelial cells in both sexes. Furthermore, immunohistochemistry revealed that α_2u-globulin was localized in the submaxillary glands in both sexes and in the liver in males. In the immunochemical study, a double immunodiffusion test, precipitin lines were formed between the anti-serum and the kidney preparation and between the anti-serum and the submaxillary gland preparation from female rats. Spur formation was observed between the precipitin lines for the anti-serum and the submaxillary gland preparation and the precipitin lines for the anti-serum and urine in female rats; therefore, a partial difference in the antigenicity of α_2u-globulin between males and females was suggested. From these results, we concluded that α_2u-globulin exists in the kidneys in the female rats, and it is considered to originate mainly from the submaxillary glands, not the liver.

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Experiment I was conducted to examine the optimum duration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for studying promoters and inhibitors of mouse bladder carcinogenesis in mice. When the mice were treated with 0.05% BBN for 12 weeks and subsequently observed without any treatment until the 28th week, the incidence of CIS and invasive carcinoma were 20-80% and 20-60%, respectively. These results suggest that an experiment involving 12-week treatment with 0.05%BBN is useful in exploring inhibitors. Using this model, the effects of three regimens of combination chemotherapy (CAP, M-VAC, and MEC regimens) in supressing the development of bladder carcinoma were examined in Experiments 2 and 3. The animals treated with CAP or MEC had significantly lower incidences of invasive carcinoma than BBN alone groups. As for M-VAC treated group, the incidence of superficial bladder carcinoma was significantly lower than BBN alone group. Thus each of the three combination chemotherapy regimens suppressed the development of mouse bladder carcinomas. These results suggest that CAP, M-VAC, and MEC regimens are useful in adjuvant chemotherapy for invasive bladder carcinoma.

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Localization of erythropoietin messenger RNA was studied by an in situ hybridization procedure using anti-sense oligonucleotide probes of murine erythropoietin in the kidneys of mice with acute anemia. Acute anemia was induced by depletion of blood. Five hundred microliters of blood were withdrawn form orbital plexus 6, 24, and 30 hours before necropsy. Animals were sacrificed 6 hours after the last bleeding. Erythropoietin levels in the plasma, renal cortex, and medulla were 797.0mU/ml, 216.7, and 11.3mU/g protein, respectively, and these values were 50, 16, and 4.5times higher than those in the untreated control mice. Silver grains indicating erythropoietin messenger RNA expression were observed in the interstitial cells of the renal cortex and outer stripe of the outer medulla in the mice with acute anemia, but not in the renal tubules or glomeruli. Silver grains were not detected in the kidneys of the untreated control mice. From these results, it was concluded that the main area of erythropoietin synthesis in the kidneys was the interstitial cells in the renal cortex and outer stripe ofthe outer medulla in the mice with acute anemia.

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Immunolocalization of glutathione-peroxidase (GSH-PO), apoptosis, and bcl-2 protein in the rat ventral prostate was investigated under the presence or absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group I consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were administered subcutaneously 1mg/animal of testosterone-propionate daily for three or seven days after two days of castration. The intensity of GSH-PO staining in the glandular epithelial cells of the ventral prostate was remarkably decreased after castration (Group 2), and it was clearly recovered by testosterone-administration (Groups 3 and 4) in the castrated rats. Furthermore, castration (Group 2) induced apoptosis in the prostatic glandular epithelial cells and apoptosis was reduced by testosterone-administration (Groups 3 and 4) in the castrated rats. In groups 3 and 4, expression of bcl-2 protein was clearly detected in the glandular epithelial cells of the ventral prostate. These findings strongly suggest thatexpression of GSH-PO and bcl-2 protein in the glandular epithelial cells of the rat ventral prostate is considered to be testosterone-dependent.

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Changes resulting from toxicity to the rat testis and epididymis induced by ethylene-1, 2-dimeth-anesulfonate (EDS) were histomorphometrically studied. In experiment I, ten-week old male rats received a single dose of 100mg/kg body weight EDS, and at 1, 3, 7, 14, and 28 days thereafter were sacrificed for histopathological evaluation of the testis, epididymis, prostate, and seminal vesicle. Lesions involving Leydig cells and seminiferous tubules in the testis wereanalyzed using a quantitative method. In the treated animals, Leydig cells disappeared within 3 days of the EDS treatment, and decreases of pachytene spermatocytes and round spermatids were subsequently observed after 7 days. In contrast, necrosis of epithelia was already observed in the epididymis at 1 day after the EDS treatment, and the severity of the lesion increased up to day 3. At day 7, although loss of Leydig cells was still evident, recovery of epithelia in the epididymis was observed. In the prostate and seminal vesicles, atrophic changes wereseen without necrotic lesions. In experiment II, castrated animals were injected with EDS and sacrificed after 3 days for histophathological observation. Changes similar to those in uncastrated animals treated with EDS were recognized in the epididymis, and with more pronounced severity than those observed in castratedanimals without EDS treatment. These results suggest that toxic lesions of the epididymis induced by EDS are not secondary to those in the testis, but rather are due to direct effects of the compound.

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To identify initial changes and progression of late onset cataract of UPL (Upjohn Pharmaceuticals Limited) rat, cataractous lenses were evaluated ophthalmoscopically, histologically, and by Raman spectroscopy. At 2 to 4 weeks of age, intracellular vacuoles in the ends of the lens fibers and stratified epithelial cells were observed in the anterior suture of the lens. At 4 to 7 weeks of age, stratified epithelial cells and large vacuoles in lens fibers were present in the equator region. In the posterior cortex, small vacuoles were observed. Raman spectroscopic measurement indicated an increase in water content and the lens nucleus shift to the anterior. At approximately 7 weeks of age, mature cataracts, including rupture and liquefaction of lens fibers in the entire lens cortex, were present. In conclusion, the initial changes were observed in lens fibers at the anterior suture. Hydration of the posterior cortex might be correlated with lens opacification.

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For determination of the effect of light stimulation on the distribution of the lesions in N-methyl-N-nitrosourea (MNU)-induced retinopathy, MNU-treated (70mg/kg) rats with sutured closed right eyelid were housed under constant light (150 lux for 24 hours). In spite of the unilateral eyelid closure, retinopathy was observed in bilateral retinas by 24 hours after MNU treatment and the retinal lesion was severe in the area centralis. In addition, the retinopathy wasmore pronounced in the non-closed left retina than that in the closed right retina. In the morphometrical analysis of the retina at 21 days after MNU treatment, the ratio of the outer nuclear layer thickness to the whole retinal thickness was markedly reduced in the area within 2 nun distance from the optic nerve head in both retinas. These results indicate that the light stimulation does not critically affect the distribution of the MNU-induced lesions but does enhance the severity of the lesion.

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The present study was undertaken to clarify the strain differences between ICR and BALB/c mice and sex differences in ICR mice in the sequential changes by the photic damage to the Harderian gland and the correlation between Harderian gland damage and porphyrin content. The animals were exposed to fluorescent light at 3, 000 lux for 12 hr a day for 2, 4 or 8 days. On day 2, the Harderian glands showed marked necrosis and luminal dilation with secretion and inflammatory edema. Photic damage was observed in a restricted area just behind the eye in both strains. ICR mice showed extensive and diffuse necrosis of the glandularcells, but BALB/c mice showed sparsely scattered necrosis of glandular cells, that is, the extent of damage was more marked in ICR mice than in BALB/c mice. With prolongation of exposure period the glandular necrosis was improved and cell regeneration was accompanied by squamous metaplasia. The red fluorescence of the glands under ultraviolet light was intense in ICR mice and faint in BALB/c mice correlating with the porphyrin contents in Harderian glands. The fluorescence faded completely in the damaged acini and the intraluminal porphyrin accretions were also significantly decreased after light exposure. The photohemolytic activity of the Harderian gland homogenate was much stronger in ICR mice than in BALB/cmice. There was no obvious sex difference in any of these variables in ICR mice.In conclusion, there was a marked strain difference between ICR and BALB/c mice in Harderian gland vulnerability to photic damage, and this difference highly correlated with the porphyrin contents in the Harderian glands. The photic damage to the Harderian glands may be due to photodynamic action of the porphyrins present in the glands.

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A 10-month-old male beagle dog had a congenital tumor in the right kidney. Histopathologically, the tumor was characterized by fingerlike extensionsprojecting into the adjacent normal tissue, and often entrapping the normal glomerulo-tubular structure. The tumor was composed of spindle cells arranged in an interlacing fascicular pattern with some collagen fibers. Ultrastructurally, thetumor cells had long projections on the surface, rarely in contact with the junctional structures among the neighboring cells, and formed thin discontinuous basement membranes at the cell ends. Immunohistochemically, the tumor cells showed positive reactions with vimentin and S-100 protein antisera, and some weak reactions with actin, desmin, or myoglobin, but no reactions with cytokeratin.
This present tumor should be diagnosed as a congenital mesoblastic nephroma (CMN) on the basis of characteristic features in HE staining, other immunohistochemical reactions, and electromicroscopic findings, as well as the young age of the dog.

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Light microscopic, histochemical and ultrastructural observations were conducted to examine a spontaneous malignant schwannoma arising from the intracranial trigeminal nerve in a 19-week-old female virgin Sprague-Dawley rat. The tumor, which extended from the right trigeminal ganglia and nerve to the bottom of the cerebellum macroscopically, was composed of sheets of small fusiform cells with rod-shaped nuclei and rather abundant eosinophilic cytoplasms. Some tumorcells were arranged in roughly parallel arrays with nuclear palisades or in a whirling pattern. The tumor had invaded the subarachnoid and Virchow-Robin's spaceof the cerebrum and thoracic spinal cord, and the pars distalis of the pituitary gland. In immunohisto-chemistry, the tumor cells showed positive reaction to anti-S100 protein and anti-vimentin, but were negative for anti-GFAP. Ultrastructurally, interdigitating cytoplasmic processes, a few junctional complexes, and fragmented basal lamina-like structures were observed. These findings closely resembled the malignant schwannoma described in the soft tissues of rats and human. From our review of the literature, we believe our case to be the first report of a spontaneous malignant schwannoma arising in the intracranial trigeminal nerve of a young rat.

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A desmoplastic malignant melanoma appearing on the forelimb in a dogwas histopathologically examined. The tumor cells, having an oval nucleus and a spindle-shaped abundant cytoplasm, showed perivascular whorled and storiformed proliferative pattern and rarely formed syncytium. They also loosely proliferatedin area of myxomatous matrix occupying a part of the tumor. No melanin granules were visible in the neoplastic tissue of HE stain, but a small quantity of melanin could be seen by Fontana-Masson stain in storiformed areas and locally by DOPA reaction. These granules were never recognized in the whorled proliferative area. The tumor cells were immunohistochemically positive for S-100 protein, and ultrastructurally contained cell organellas such as rER, dense body, and microfilament. Immature melanin, however, could not be detected in the tumor cells. Numerous collagen fibers were present around the neoplastic cells. These findings described above appeared to be essentially similar to those of desmoplastic melanoma of human.

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