We have developed a medium-term liver bioassay system for rapid detection of carcinogenic agents using male F344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system is fundamen-tally based on the two-stage hypothesis with regard to tumor production, employing initiation by diethylnitrosamine (200mg/kg, ip) in the first stage and test chemical administration during the second, in combination with 2/3 partial hepatectomy. It requires only 8 weeks for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically-demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 277 chemicals/substances have already been analyzed in our laboratory and the efficacy of the system for hepatocarcinogens has thereby been well established. This bioassay is increasingly becoming regarded as an appropriate alternative test system for carcinogenicity risk assessment. It is particularly useful for studies which require many experimental groups and for evaluation of chemopreventive agents.

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The codon 61 of the activated Ha-ras gene is detected most frequently among mutations of proto-oncogenes in spontaneously-occurring hepatocellular tumors in B6C3F₁ mice. In the present study, the ras mutations and their patterns in normal liver tissue, foci of cellular alteration (FCA), and hepatocellular tumor from untreated mice were analyzed using the non-RI PCR-SSCP method. The Ha-ras gene with mutated codon 61 was detected in 1.6% (2/122) from normal liver tissues, 16.7% (6/36) from spontaneous FCA tissues, 30.4% (48/158) from spontaneous hepatocellular tumor tissues, and 7.7% (3/39) from normal portion in spontaneous hepatocellular tumor tissues. Furthermore, mutation in the Ha-ras gene was compared between spontaneous and induced hepatocellular proliferative lesions including hepatocellular tumor from archival materials at our Center. Although no clear difference was found between genotoxic carcinogen and non-genotoxic carcinogens in the incidence of Ha-ras gene mutation in the induced tumors, the incidence of Ha-ras gene mutation in FCA, which was induced by genotoxic carcinogen, was higher than that in FCA induced by non-genotoxic carcinogens. These data suggested that to analyze the incidence of Ha-ras gene mutation in proliferative lesions in the livers of B6C3F₁ mice, especially in the FCA is useful in evaluation of hepatocarcinogenicity of the chemicals.

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The numbers of argyrophilic nucleolar organizer regions (AgNOR) stained by the one-step silver colloid method were counted in background hepatocytes and within eosinophilic foci, amphophilic foci, and neoplastic nodules developing in rats given diet containing one of the peroxisome proliferators, clofibrate, di-(2-ethylhexyl)phthalate (DEHP), dehydroepian-drosterone (DHEA), and 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14, 643), for 32 weeks after a single i.p. injection of diethylnitrosamine. The frequency of AgNORs was highest in amphophilic foci, followed by neoplastic nodules, non-lesional areas, and eosinophilic foci in the rats receiving basal diet alone or treated with clofibrate, DEHP or DHEA. In the Wy-14, 643 case the amphophilic foci and neoplastic nodules were again highest but the value for eosinophilic foci was greater over that for the background parenchyma. Mean numbers of AgNORs in non-lesional hepatocytes were elevated in rats treated with DEHP and Wy-14, 643 to 1.81 and 1.82, respectively, significantly different (p<0.05) from the control value of 1.69. The present results suggest that AgNORs may be a useful marker for predicting the hepatocarcinogenic potential of peroxisome proliferators with amphophilic foci demonstrating a particularly high proliferative activity judging from the observed AgNOR scores.

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The incidences and lesions of foreign body rhinitis due to hairs and/or diet powders were investigated in Fischer 344 DuCrj rats subjected to 13 week-toxicity and 2 year-carcinogenicity studies. During the 2 year-carcinogenicity studies, rhinitis was disclosed in 207 of 600 (34.5%) male and 71 of 600 (11.8%) female rats of the untreated control groups. In all cases the foreign bodies concerned were animal hairs. The lesions were detected only in aged rats but not in those younger than 4 months of age. The incidence was higher in males than females, and most lesions were located at the level of the incisive papilla.

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Neovascularization was studied during the development of epithelial proliferative lesions of rat urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) using an immunohistochemical and electron microscopical approach. Male 6-week old F344 rats were treated with 0.05% BBN in the drinking water for 12 weeks and then maintained without BBN for 8 weeks (total of 20 weeks observation). At intervals of 2 weeks during carcinogen administration period and at 16 and 20 weeks, groups of rats were killed and urinary bladder lesions were examined. Neovascularization appeared early in simple hyperplasia and capillary intrusion into the mucosal layer, accompanied by stromal cells and extracellular matrix formation, occurred in papillary or nodular (PN) hyperplasia, and with pronounced growth of tumor stroma within papillomas and carcinomas. However, bFGF, a potent angiogenic growth factor could not be demonstrated in the epithelial lesions at any time point's during the course of carcinogenesis. An important feature was that the basement membranes between epithelial and endothelial cells were maintained at the base of the mucosal layer but became obscure with more intrusive neovasculariza-tion. The findings indicate that stromal cells and the extracellular matrix are important factors for neovascularization during the course of rat bladder carcinogenesis.

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The incidence of spontaeous mammary tumors in female rodents was increased in 2-year oncogenic studies of (-)-(R)-5-[2-[[2-(o-ethoxyphenoxy)ethyl] amino] propyl-2-methoxybenzenesulfon-amide hydrochloride (tamsulosin hydroch-loride) administered with diet. This study was carried out to clarify mammary carcinogenic potential of tamsulosin hydrochlor-ide, in particular whether these mammary tumors are prolactin dependent or not, by a two-stage carcinogenesis study. Sprague-Dawley rats were initially given a single oral dose (20mg/rat) of 7, 12-dimethylbenzanthracene (DMBA). Two weeks later, they were divided into 4 groups as follows: 1) DMBA control, 2) 0.1% tamsulosin hydrochloride in diet, 3) 0.1% tamsulosin hydrochloride in diet and daily s.c. injections of 1mg/rat 2-bromo-alpha-ergocryptine mesylate (CB-154), a potent suppressor of pituitary prolactin secretion, and 4) daily s. c. injections of 1mg/rat CB-154. The non-treated control group was also prepared. Administration periods of the compounds were 15 weeks in females and 39 weeks in males. At the end of the treatment period, all surviving rats were necropsied, and then all mammary nodular lesions were fixed in phosphate buffered 10% formalin, and were weighed after fixation. Histopathological examination revealed that tamsulosin hydrochloride at 0.1% dietary dose caused a slight promoting effect on mammary tumorigenesis, whereas the combination of CB-154 with tamsulosin hydrochloride inhibited this effect. The results strongly suggest that mammary tumors induced by tamsulosin hydrochloride administration were not due to direct effect of this compound but were possibly secondary effect through the pharmacological effect increasing the serum prolactin level of this drug.

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Possible modifying effects of verapamil on 2-amino-l-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) car-cinogenicity were investigated. Male and female F344 rats were administered both PhIP, 0.02% in the diet, and verapamil, 0.05% in the diet (group 1), or either PhIP (group 2) or verapamil (group 3) alone for 52 weeks. Development of mammary tumors induced by PhIP in females was delayed by verapamil coadministration, although the final incidence of adenocar-cinomas was not statistically significant between groups l and 2. These findings were not related to the changes in body weight gain. In males, PhIP induction of intestinal tumors was not affected by verapamil coadministration. In other organs, verapamil did not change the appearance of toxic and neoplastic lesions induced by PhIP. Thus, verapamil exerted only slight inhibitory effect on mammary tumor development.

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To evaluate the early toxic lesions induced by DEHP and 2, 5-hexanedione, ten-week-old male Sprague-Dawley rats were given a single oral dose of DEHP or daily doses of 2, 5-hexandione. The single DEHP dose induced disruption of the ectoplasmic specialization of Sertoli cells at 3 hours after dosing. The lesion was characterized by marked dilatation of the endoplasmic reticulum facing the tight junction and by disappearance of the actin filament bundles associated with ectoplasmic specialization. The lanthanum trace study suggested a functional disturbance of the blood-testis barrier. Daily administration of 2, 5-hexanedione induced failure of spermatocyte and spermatid migration to the apical portion of the Sertoli cells. Degeneration or death of germ cells and multinucleated giant cell formation were also recognized after the 2, 5-hexanedione administration. Some of the degenerated germ cells and the apical processes of the Sertoli cells sloughed into the lumina of the seminiferous tubules. Therefore, it is suggested that DEHP affect the ectoplasmic specialization in Sertoli cells and 2, 5-hexanedione impair the Sertoli to germ cell contact and continual movement of germ cells from the base to the lumen of the tubules.

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Lymphocytic apoptosis in Peyer's patches in the small intestine of mice orally inoculated with T-2 toxin (10mg/kg b.w.) were examined for up to 48 hours after inoculation (HAI). The number of apoptotic lymphocytes observed by in situ detection method of fragmented DNA increased significantly from 6HAI and reached a plateau at 24HAI. Ultrastructurally, lymphocytes characterized by shrinkage of the cell body and condensation of nuclear chromatin and/or margination of condensed chromatin along the nuclear membrane were observed from 6HAI. Some cell nuclei were fragmented into small pieces, and these small pieces were sometimes ingested by macrophages.

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Granular cell tumors were found in 18 female reproductive tract out of 300 Wistar rats. The tumor cells were histologically polygonal and arranged in sheets or cords separated from each other by collagenous septa. The cytoplasm was filled with periodic acid-Schiff reaction positive and diastase-resistant coarse granules, which showed electronmicroscopically characteristics of lysosomes. Metastatic lesions were not seen in any of the cases. The nest of tumor cells were occasionally embedded within the uterine stromal cells, and no discernible capsule was present. Immunohistochemically, they were positive for neuron specific enolase in all cases and vimentin in some cases, and negative for S-100, desmin or myoglobin in any of the cases. Biological relationship with meningeal granular cell tumors was not evident. These findings supported the notion of neuroectoderm origin of granular cell tumors in the female reproductive tract, and reminded us of a large variation of the tumor incidence among different colonies.

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