Toru Hoshiya

Inhibition of mammary gland carcinogenesis by green tea catechins and other naturally occurring antioxidants in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[a]anthracene

Effects of the naturally occurring antioxidants on mammary gland carcinogenesis were examined in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[alpha]anthracene (DMBA). Groups of 15-16 7-week-old rats received a 50 mg/kg body weight intra-gastric dose of DMBA, and starting one week thereafter placed on diet containing 0.4% catechol, 1.0% gamma-oryzanol, 2.0% phytic acid, 1.0% green tea catechins (GTC), 1.0% tannic acid or basal diet alone for 35 weeks. Although the final incidences and multiplicities of mammary tumors were not significantly different between DMBA-treated groups, the numbers of survivors in the antioxidant-treated groups at the end of the experiment at week 36 were significantly higher than in the basal diet group. In particular, the survival rate of the GTC group at 93.8% strongly contrasted with that of only 33.3% for rats on the basal diet. At the end of week 18, when all the animals were still alive, the average size of palpable mammary tumors was significantly smaller in the catechol, phytic acid and catechins groups. These results indicate that antioxidants, and GTC in particular, inhibit rat mammary gland carcinogenesis after DMBA initiation.

Induction of Adenocarcinomas in the Glandular Stomach of BALB/c Mice Treated with N‐Methyl‐N‐nitrosourea

Male 6‐week‐old BALB/c strain animals (groups 1 and 2) received 10 weekly intragastric intubations of 0.5 mg/mouse of N‐methyl‐N‐nitrosourea. At week 11 the forestomachs were resected in group 1 but not group 2. Although many animals in group 2 died due to development of squamous cell carcinomas in the forestomach, development of cancers in the glandular stomach was quite similar in both groups. Well‐differentiated adenocarcinomas in groups 1 and 2 were found at low incidence at week 20, rising to 100% at week 40, with two lesions metastasizing to the lymph nodes. Four poorly differentiated adenocarcinomas and 5 signet ring cell carcinomas were also found in 27 glandular stomach tumor‐bearing animals.

Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci development initiated with diethylnitrosamine in the rat

The potential hepatocarcinogenicity of seven pesticides was examined using a rapid bioassay based on the induction of glutathione S-transferase placental form positive foci in the rat liver. Rats were initially injected with diethylnitrosamine and two weeks later were fed on diet supplemented with one of the pesticides for 6 weeks and then killed; all rats were subjected to a partial hepatectomy at week 3. Positive results were seen with chlorobenzilate (2000 ppm), vinclozolin (2000 ppm), malathion (4000 ppm), tecnazene (2000 ppm) and isoproturon (2000 ppm). S,S,S-tributylphosphorotrithioate (DEF, 200 ppm) and dicloran (2000 ppm) were negative in both number and area analyses. Although chlorobenzilate is carcinogenic in mice, malathion and vinclozolin have been reported as non-carcinogens in both rats and mice. Since the present system is based on the two-stage carcinogenesis hypothesis, it is possible that the chemicals showing positive results in this system possess at least tumor-promoting activity in the rat liver. This is very significant, as most carcinogens show tumor-promoting activity in their target organs.

Medium-term bioassay for the hepatocarcinogenicity of hexachlorobenzene.

Hexachlorobenzene (HCB) is an important environmental contaminant derived mainly from industrial and agricultural sources. It is carcinogenic in mice, rats and hamsters. It has now been studied in a medium-term bioassay for carcinogenicity based on the induction of preneoplastic lesions in the liver. We report here that the bioassay can rapidly detect carcinogenic doses of HCB and that there is a clear dose-response relationship. At the lowest dose of HCB administered, the incidence of preneoplastic lesions in the liver was no different from that in controls.

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

Recently, RccHanTM: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 RccHanTM: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 RccHanTM: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 RccHanTM: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.

A Rapid in vivo Bioassay for the Carcinogenicity of Pesticides

Eight pesticides were tested in a bioassay based on the induction of preneoplastic lesions in the liver. Rats were given diethylnitrosamine intraperitoneally at 200 mg/kg bw and two weeks later were treated with pesticides for six weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione s-transferase (placental form) -positive foci In the liver with those of controls given diethylnitrosamine alone. Positive results were seen with Chinomethionat, Phosmet and Propiconazole; the results obtained with Captan and Prochloraz were borderline; Benomyl, Daminozide and Folpet gave negative results. Our findings provide enough experimental evidence to indicate that great care should be exercised in the use of these compounds.

Sequential Morphological and Biological Changes in the Glandular Stomach Induced by Oral Administration of Catechol to Male F344 Rats

Histogenesis and mechanisms of catechol-induced rat glandular stomach carcinogenesis were investigated in male F344 rats. Groups of 5 or 6 rats were treated with dietary catechol at doses of 1, 0.5, 0.1, and 0.01% for 12 hr or for 1, 2, 3, or 7 days or at a dose of 0.8% for 1, 2, 4, 12, and 24 wk; rats were then euthanatized. The initial morphological changes were edema of the gastric wall, inflammatory-cell infiltration, erosion in the pyloric region close to the duodenum, and considerable increase in apoptosis at 12 hr; later, changes included augmented DNA synthesis and cell proliferation, as evaluated by bromodeoxyuridine labeling index and thickness of mucosa, respectively, on day 1. Downward hyperplasia due to excess regeneration appeared at edges of ulceration at week 2. This lesion disappeared, and then submucosal hyperplasia appeared in the course of adenoma development. Only slight expression of c-myc or c-fos was apparent after 30-min oral administration or 1-, 3-, and 6-hr oral administration of catechol. No increase in lipid peroxide levels was evident in gastric epithelium fed catechol for 1 wk. The amount of catechol distributed in the glandular stomach and forestomach epithelium, which is not a target for carcinogenesis, did not differ 1, 3, 6, and 24 hr after a single intragastric dose of 75 mg/kg body weight. Amounts of catechol bound to tissue protein were also not specifically high in the glandular stomach. These results indicate that regenerative cell proliferation due to toxicity plays an important role in catechol-induced glandular stomach carcinogenesis. Protein binding and free radicals may not be largely responsible for the toxicity.

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.

Reversibility of Carcinogen‐induced Rat Forestomach Basal Cell Hyperplasia Is Due to Squamous Cell Differentiation

The mechanisms of reversibility of basal cell hyperplasia in the rat forestomach were investigated. Male F344 rats were given an initial single gastric intubation of N‐methyl‐N′‐nitro‐N‐nitoroso‐guanidine and then received 2% butylated hydroxyanisole in the diet from the third week to the 26th week. Rats were killed at weeks 26 and 46 after return to basal diet and their forestomachs were removed. Bromouracil deoxyriboside (BUdR) was administered as a single i.p. injection 1 h before death or by osmotic minipump (120 μg/h) continuously for 7 days before death. Additional animals were maintained for 2 or 4 weeks after removal of osmotic minipumps to allow assessment of the fate of proliferating populations. In each case BUdR‐labeled cells were demonstrated by immunohisto‐chemistry. At week 26, hyperplastic changes were more pronounced than at week 46. Squamous cells above basal cell hyperplasias were strongly labeled even 4 weeks after cessation of continuous BUdR administration, in clear contrast to those in normal‐appearing epithelium. Three‐dimensional reconstruction of persisting basal cell hyperplasias showed almost all basal cells limited to a thin sheet in direct contact with the squamous cell layer, occasional separate islands demonstrating differentiation to squamous cells and formation of epidermal cysts. The results thus showed that the mechanism of reversibility of basal cell hyperplasia involves differentiation of basal cells to squamous cells.

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.