Male F344 rats were administered piperonyl butoxide (α[2-(2-butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltoluene), mixed in their diet for up to 4 weeks, and phenobarbital was administered to rats for the same period in drinking water as a positive control. We examined the changes in relative liver weight, cytochrome P450 contents, and various drugmetabolizing enzyme activities i.e., 7-alkoxycoumarin O-dealkylase activities, glutathione S-transferase activities to 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene as a substrate, and UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate in the livers of those animals. All parameters examined were significantly elevated by treatment of 2% or both 0.2 and 2% piperonyl butoxide. As for P450 isozymes, induction of CYP1A1, 2B1/2, 3A, and 4A was revealed by both immunoblot analysis and immunohistochemistry. Immunohistochemistry also demonstrated that these isozymes were induced in the central zone of the hepatic lobule. These results, except for CYP1A1 induction, observed in the piperonyl butoxide-treated rats were exceedingly similar to those in the phenobarbital-treated rats used as a positive control in the present study. Phenobarbital is well-known as a promoter of liver tumor development, and consequently it has been postulated that there is a strong relationship between the promoter activity and the induction of CYP2B1/2. The present results suggest that piperonyl butoxide may act as a promoter in liver tumor development, as has recently been reported in a rat study.

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Rats treated with L-NNA develop stroke to serve as an animal model of multiple infarct dementia. A study of behavioral pharmacology and histopathology was conducted to clarify this characteristics in detail. In this study, male, 4-week old Wistar rats were fed with SP food containing L-NNA at 0.023%, and thus development of stroke was induced. This was followed by a wash-out period to observe behavioral changes such as learning impairment, and remaining cerebrovascular disease in the rats which had recovered most of neurologic symptoms which usually accompany stroke. Body weight, food consumption, and blood pressure were determined, while neurologic symptoms were observed during the test period. A cerebrovascular damage was detected non-invasively by Magnetic Resonance Imaging, and examined histologically. As a result, it was found that L-NNA induced in rats significant body weight decrease and severe hypertension, aggravated offensiveness and sensitivity, also led to the occurrence of stroke. The wash-out period set after onset of stroke helped to mitigate neurologic symptom and body weight decrease; however, learning impairment remained. The area of high-level MRI signal in the cerebral cortex corresponded to a broad range of cerebral infarct covering histologic edema and vascular damage. Thus, the study results imply that L-NNA-induced stroke rats can be used as an animal model of cerebrovascular dementia, and a wide range of histologic edema can be detected non-invasively by MRI.

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Neoplastic and proliferative hepatic lesions observed in 500 male and 499 female DBF1 mice used as untreated controls in the past ten 2-year carcinogenicity studies (each 50 males and 50 females for one study) for industrial chemicals at the Japan Bioassay Research Center. The incidences of altered cell foci (ACF) were 11.6% in males and 2.8% in females and those of each type of ACF were as follows; basophilic, eosinophilic, clear, vacuolated, and mixed cell foci were 4.0%, 1.2%, 4.6%, 0.2%, and 1.6% in male mice, respectively, and 1.2%, 0%, 0.8%, 0.4%, and 0.4% in females, hepatocellular adenoma (HCA) was 15% in males (minimum 4%, maximum 30%), and 4.4% in females (minimum 2%, maximum 8%), hepatocellular carcinoma (HCC) was 22.8% (minimum 2%, maximum 36%) in males and 2.0% (minimum 0%, maximum 4%) in females, vascular tumors were 6.6% (minimum 0%, maximum 6%) in male mice and 15.0% (minimum 6%, maximum 20%) in females. Compared with the data in rats, incidences of HCA and HCC in BDF1 mice were remarkably higher, however, the incidence of ACF in BDF1 mice was lower than those of rats. The different incidence of ACF noted in rats and in mice may suggest the different implications of ACF as the preneoplastic role in hepatocarcinogenesis.

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Six transplantable rat prostate carcinoma lines were established from primary tumors developing in the dorsolateral prostate and a metastatic lesion in the liver of F344 rats receiving a combined treatment with 3,2'-dimethyl-4-aminobiphenyl (DMAB) and testosterone propionate (TP). Growth in the subcutis of nude mice (ICR-nu/nu) is rapid with a doubling time of approximately 10 days and transplantability is 100%. The histological appearance of the transplanted tumors (well- to moderately differentiated and poorly differentiated adenocarcinomas) is very similar to that of the primaries and their structures have not been altered by after 8 passages. Lung metastasis has been observed at frequencies of 30 to 100%. Androgen receptor immunohistochemistry revealed all to be negative. These data indicate that the lines should prove useful for studies of invasion, metastasis, and hormone dependency of prostate carcinomas.

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Phenobarbital sodium (PB) was administered intramuscularly once a day to beagle dogs aged 10 to 13 months old, and measurement of hepatic drug-metabolizing enzyme (aminopyrine-N-demethylase and aniline hydroxylase) activites, histopathology, and electron microscopy were carried out on liver samples collected after 4 and 14 days treatment. After 4 days treatment, marked increases in hepatic drug-metabolizing enzyme activities were noted. Morphologically, hypertrophy of hepatocytes, accompanied by a ground glass-appearance, was observed on light microscopy, and proliferation of smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER), with wave- or horseshoe-shaped arrangements of the proliferated rER, were observed by electron microscopy. After 14 days, hepatic drug-metabolizing enzyme activities were increased compared with those after 4 days treatment, and eosinophilic intracytoplasmic inclusion bodies were observed in the hepatocytes. The inclusion bodies showed a positive reaction for sudan black B staining, indicating the presence of phospholipids. On electron microscopy, these inclusion bodies corresponded to myelin figures (MFs), and continuity of the membranes between MF and proliferated sER or rER was noted. When the animals were treated with cobalt chloride which is an inhibitor of P-450 synthesis, increases in hepatic drug-metabolizing enzyme activities induced by PB were suppressed; however, proliferation of sER and rER was not inhibited and horseshoe-arranged rER, a precursor of the inclusion bodies, was still observed. From these results, it was considered that the inclusion bodies in the dog hepatocytes induced by PB were related to sER proliferation.

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The carcinogenicity of sodium metaphosphate (SMP) was examined in F344 rats of both sexes. SMP was mixed in basal diet at levels of 0, 1.5 or 3.0%, and groups of 50 male and 50 female rats were administered each of these diets ad libitum. At final sacrifice no statistically significant differences between treated and control groups regarding mean body weight, or total intake were observed in either male or female. The results of urine and serum analysis, or hematological determinations studies show that there was no significant difference between test group and control group. Many tumors developed in all groups including the controls and the organ distribution of neoplasms and their histological characteristics did not differ significantly from those reported to occur spontaneously in this strain of rats. From these findings, it was concluded that sodium metaphosphate does not induce tumours, when given orally at doses of up to 3.0% mixed in basal diet for 108 weeks. With regard to non-neoplastic lesions, there were non-neoplastic lesions in kidneys, mineralization, cast formation and basophilic tubular cell proliferation present in greater proportion in female rats treated with sodium metaphosphate than in non-treated female rats. Mineralization was seen as marked calcium deposition in the pars intermedia of the kidney in female rats treated with 3.0% sodium metaphosphate.

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The distribution of sialic acid residues in renal tubule of streptozotocin-induced diabetes rats was investigated using lectin histochemical method to compare with specimens from control animals. Sialic acid specific lectins, Limax flavus (LFA), Sambucus nigra (SNA), and Maackia amurensis (MAA) were applied. This sialic acid residues of the renal tubule in diabetes rats were markedly different from those in the control. The epithelial cells of the entire proximal tubule in normal rats were positive for LFA and SNA, and negative for MAA. Meantime, the proximal tubule in diabetes rats was negative for these lectins. The medullary thick ascending limb in normal rats showed negative for LFA, SNA and MAA. In contrast, that limb in diabetes rats showed positive for these lectins. Moreover, the degenerated epithelial cells in the cortical thick ascending limb were intensely positive for LFA and MAA, and negative for SNA. The present study revealed that the different distribution of epithelial sialic acid residues in the renal tubule might be related to the functional diversity exhibited by these tubular regions between diabetic and normal rats.

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Etiologic research has shown that sodium chloride contributes to gastric carcinogenecity. We investigated the initial changes of gastric mucosa in the cytoplasmic mucin of superficial epithelium and the surface mucous layer of Beagle dogs after an oral dose of sodium chloride. Animals were sacrificed at 0.5, 1.5, 3.5 and 5.5 hours after dosing with 5 g of sodium chloride filled gelatin capsules. Sodium chloride caused lesions such as degeneration and detachment of epithelium, and erosion of mucosa as early as 0.5 hours after administration. ConA type III mucin, the evidence for the origin from gland mucous cells, increased after treatment and was found to be thick at the area of the lesion with progression of decreased mucin in the surface mucous layer, or where epithelial detachment occurred. Gland mucous cell mucin might be responsible for the protection and healing of gastric epithelium. Sulfomucin, demonstrated by high iron diamine staining, originally in the lower foveolar cells of the gastric pits was widely observed in the superficial epithelium of the luminar surface during the time course of the study. PCNA positive generative cells originally in the ithmus and parietal cells originally below the gastric pits were also found among superficial epithelial cells of the luminar surface. It was suggested that these cells might cover the lesion as well as superficial epithelium next to the detaching part.

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Unilateral hypoplasia of the retina and optic nerve was found in a male Sprague-Dawley rats. The lesion was first suspected on the electroretinographic examination. There was very little response from the left eye to the light stimulus, whereas the right eye was normal. On the ophthalmic examination, the fundus of the left eye had faint retinal vessels. At necropsy, the left optic nerve and right optic tract were thin. Microscopically, the retina of the left eye was not fully developed showing undifferentiated photoreceptor, outer and inner granular, inner plexiform and ganglion cell layers, and a complete lack of the outer plexiform layer.

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We examined cell kinetics of bone cells and chondrocytes in the femur of young growing rats using flash and cumulative labeling with bromodeoxyuridine (BrdU). As flash labeling, rats received an intravenous injection of BrdU. As cumulative labeling, rats were implanted with osmotic pumps filled with BrdU into the peritoneal cavity. The BrdU-labeled nuclei were immunohistochemically identified in decalcified paraffin-embedded specimens using anti-BrdU antibody. In the articular cartilage, BrdU-positive chondrocytes were observed in the middle of the cartilage by flash labeling, and the positive cells were constantly increased with the cumulative labeling days. In the growth plate, BrdU-positive chondrocytes were observed in the proliferating zone by flash labeling, and almost all chondrocytes were positive for BrdU by cumulative labeling. In the bone tissues, by flash labeling, the undifferentiated mesenchymal cells which seem to be a source of osteoblast were positively stained for BrdU. By cumulative labeling, BrdU-positive osteoblasts, osteocytes, and osteoclasts were observed and these cells were mainly localized in the metaphysis. The results suggest that the technique using the combination of flash and cumulative labeling the BrdU is useful for analysis of cell kinetics of bone cells and chondrocytes, and will be applicable to the analysis of bone and cartilage changes induced by various compounds and observed in animal models of skeletal diseases.

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Since granulomatous gastritis and infection with spirilla were observed in most of the Beagle dogs sacrificed for a certain toxicity study, we investigated the stomach histopathologically. The lesions were characterized by degeneration and erosion in the mucosal and crypt epithelium, diffuse and focal infiltration of inflammatory cells containing foreign body giant cells in the lamina propria and hyperplasia of lymphoid follicles in the submucosa. Spirilla were adhered to the epithelium and seen in the crypt lacunae without invading into the tissues. However, they invaded into the cytoplasm of the parietal cells and induced mild degeneration and necrosis. The granulomatous lesions and spirilla were more markedly observed in the pylorus than in the fundus. There was no clear relationship between granuloma and spirilla, but granulomas were considered to be formed by foreign materials invading from the gastric lumen through the destroyed epithelium.

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