To clarify the effects of a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on bone, rhG-CSF (100, 1,000, and 5,000 μg/kg) was subcutaneously given to 6-week-old rats, and the femur and tibia were evaluated histopathologically at 2, 4 and 7 days after the injection. A significant increase in WBC counts related to the major pharmacological activity of G-CSF was observed in the rhG-CSF-treated groups at 1 to 2 days after the injection. Bone changes were found only in rats treated with 5,000 μg/kg of rhG-CSF. The development of bone changes lagged behind the increase in WBC counts, and the bone changes occurred at 4 and 7 days after the injection. At 4 days after the injection, accelerated osteoclastic bone resorption was observed in the metaphyseal spongy trabeculae of femur and tibia. At 7 days after the injection, a small amount of newly formed bone due to intramembranous ossification was seen in the metaphysis of femur. These results suggest that the higher dose of rhG-CSF may intrinsically induce bone changes with a particular histopathological nature in rats.

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In the present study, establishment of a rat model for progression in the large bowel carcinogenesis by combined exposure of methylazoxymethanol (MAM) acetate and 1,2-dimethylhydrazine (DMH) was tried, and the modifying effect of α-difluoromethylornithine (DFMO), a chemopreventive agent, was examined in this model. A total of 200 F344 male rats were divided into 8 groups. At 6 weeks of age, the rats of Groups 1 (30 rats), 2 (30 rats), 3 (30 rats), and 4 (30 rats) were given i.p. injections of MAM acetate (20 mg/kg body weight) once a week for 3 weeks. Sixteen weeks after the first injection of MAM acetate, the rats of Groups 3, 4, 5 (20 rats), and 6 (20 rats) received s.c. injections of DMH (20 mg/kg body weight) once a week for 10 weeks. Fifteen weeks after the first treatment of MAN acetate, the rats of Groups 2, 4, 6, and 7 (20 rats) were exposed to DFMO in drinking water at a concentration of 0.1%. Group 8 (20 rats) was served as an untreated control. The incidence of colon cancer of Group 3 (MAM→DMH, 62%) was higher than that of Group 1 (MAM alone, 31%, P<0.05). The numbers of intestinal cancer with a high grade of atypia per rat (0.69 ± 0.79) and a deeper invasion in the wall per rat (0.65 ± 0.69) of Group 3 were significantly larger than those of Group 1 (0.31 ± 0.47, P<0.05; 0.24 ± 0.44, P<0.01). The incidence and multiplicity of colon cancer of Groups 2 (MAM→DFMO, 21%, 0.25 ± 0.52) and 4 (MAM→DMH + DFMO, 39%, 0.39 ± 0.50) were less than those of Groups 1 (31%, 0.45 ± 0.87) and 3 (62%, 0.77 ± 0.76). Concerning the number of intestinal cancer with a high grade of atypia or a deeper invasion in the gut wall, however, no significant differences were recognized. Only the rats of Groups 3 and 4 had metastatic cancer to lymph nodes or other organs (Group 3, 31%; Group 4, 29%). These results suggest that the combination of MAM acetate and DMH could be effective as a progression model for colorectal carcinogenesis. DFMO did not show clear effects on progression in colon carcinogenesis, although the agent had an inhibitory potential on the development of tumors.

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To investigate the modifying effects of repeated doses of NNK on the development of lung tumors in hamsters with pulmonary fibrosis, female Syrian golden hamsters received 5 subcutaneous (s.c.) injections of 0.6 mg MNUR (Groups I and II) or 0.5% ethanol solution (Groups III and IV), at 2-week intervals, and were then given distilled water containing 3 ppm NNK (Groups I and III) or water alone (Groups II and IV) for 27 weeks. Glandular metaplasia, papillary hyperplasia and bronchiolo-alveolar adenomas were induced in the lungs of all the animals of Groups I and II without any significant differences in data for multiplicity and PCNA labeling indices of these between Groups I and II. In PCR-dot blod hybridization analysis on lung proliferative lesions, point mutations of c-K-ras gene at codon 12 or 61 were seen in bronchiolo-alveolar adenomas and carcinomas from animals of Group I. The mutation incidences were 4/13 in adenomas and 3/4 in carcinomas, involving changes from GGT-to GAT at codon 12, or CAA-to-CGA at codon 61. The results indicate that NNK treatment with the present dose regimen exerts no modifying effects on the development of lung proliferative lesions in hamsters with pulmonary fibrosis induced by MNUR.

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The mechanisms of apoptosis induction and mitotic inhibition in the intestinal epithelium were investigated in male BDF1 mice treated with a single oral dose of 5-fluorouracil (5-FU). We measured the concentrations of 5-FU in the plasma and intestinal tissues and followed the detailed incidental time courses of both apoptotic index (AI) and mitotic index (MI) in the five intestinal compartments. Although the plasma concentration of 5-FU decreased to a value lower than the minimal effective concentration (MEC) within 8 hr after administration, the intestinal tissue concentrations remained remarkably higher than the MEC until 48 hr. 5-FU induced an increases of AI and decrease of MI for more than 48 hr with time courses that differed among the intestinal compartments. We also determined the AI and MI after administration of two 5-FU derivatives, 5'-DFUR and Capecitabine. The derivatives were administered orally at the equivalent dose of 5-FU; nevertheless the observed changes of AI and MI were far less significant than those for 5-FU. This finding may reflect the number of enzymatic activation steps each compound undergoes and explain why the toxicities of the 5-FU derivatives in the intestinal epithelium are less than that of 5-FU itself. The present study suggests that the prolonged effect of 5-FU would be due to retention of 5-FU within the intestinal epithelium, and that the different time courses for AI and MI in each intestinal compartment would depend on the stage that the cell-cycle was in when 5-FU uptake occurred.

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tert-Butylated hydroxyanisole (BHA) and 1, 2-bis(2-pyridyl)ethylene (2PY-e) are known to induce phase II drug metabolizing enzymes without inducing phase I enzymes. In this study, male F344 rats were treated with BHA, 2PY-e, or phenobarbital (PB) that is known to induce both phase I and phase II enzymes, for 7 or 28 days, and the effects of these agents on livers, in particular morphological changes, were compared. An increase in relative liver weight was observed in all treated groups. The BHA- and 2PY-e-treated groups showed significant increases in glutathione S-transferase (GST) and UDP-glucuronosyltransferase activities, while cytochrome P450 (P450) contents or 7-alkoxycoumarin O-dealkylase activities showed no change. The PB-treated group showed significant increases in both phase I and II enzyme activities. Electron microscopic examination revealed that BHA and 2PY-e did not induce apparent SER proliferation which was observed in the PB treated liver. Immunohistochemical examination revealed that BHA and 2PY-e induced GST Yp in hepatocytes of the periportal and the centrilobular area, respectively. PB did not induce GST Yp in hepatocytes. The proliferating activities of the hepatocytes treated with these agents were also evaluated using the BrdU labeling index. In the PB-and 2PY-e-treated groups, significant increases in labeling indices were found and the index was higher in the centrilobular area than in the periportal area. The labeling index of the BHA-treated group was comparable to that of the control group. The present study clarified that there were different responses in SER proliferation, inducing pattern of GST Yp and proliferating activities of hepatocytes between the PB-, 2PY-e-, and BHA-treated groups. However, mechanisms which underlie the differences found in the present study remain to be determined.

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The cyclin-dependent kinase inhibitor p21^wafl/cipl plays major role in regulation of the cell cycle arrest following DNA damage in a p53-dependent and -independent manner. To elucidate the roles of p21^wafl/cipl in selective growth of preneoplastic lesions during chemical hepatocarcinogenesis, p21^wafl/cipl expression was examined in the livers of rats fed a 0.02% 2-acetylaminofluorene (2-AAF)-containing diet for 4 weeks or subjected to the protocol of Solt and Farber consisting of a single dose of diethylnitrosamine (DEN) followed 2 weeks later by 2-AAF feeding with two-thirds hepatectomy. By feeding of 2-AAF having a strong mitoinhibitory effect, p21^wafl/cipl expression was significantly induced in rat hepatocytes. The proliferating cell nuclear antigen (PCNA) labeling index of hepatocytes was consistently and significantly decreased by 2-AAF feeding. Immunohistochemically, both p21^wafl/cipl and p53 were detected in the livers of 2-AAF-fed rats more intensely than in those of control rats. In the livers of rats subjected to the protocol of Solt and Farber, most enzyme-altered foci (EAF) were immunohistochemically negative for p21^wafl/cipl, while hepatocytes surrounding the foci were positive. These results clearly show that 2-AAF acts on hepatocytes as a mitoinhibitor by inducing p21^wafl/cipl expression, presumably in a p53-dependent manner. Furthermore, the marked difference in p21^wafl/cipl expression between non-neoplastic and preneoplastic hepatocytes led to selective growth of preneoplastic lesions during 2-AFF feeding using the protocol of Solt and Farber.

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It has been known that monosodium glutamate (MSG) treatment to neonatal rodents affects hypothalamic arcuate nucleus, the primary site of growth hormone releasing hormone, and consequently inhibits growth hormone release. However, there have been few reports on bone changes with aging after MSG treatment to neonatal rodents. Male and female neonatal F344 rats were subcutaneously given 4,000 mg/kg of MSG on days 0, 2, 4, 6, and 8 after birth, and the changes of the femurs were examined at 12 months of age. As a result, the bone length, diameters of the femur diaphysis and bone mineral density, especially the metaphysis region, were decreased by the treatment. Histologically, the amount of trabecular bone in the epiphysis and metaphysis regions also decreased in MSG-treated rats, together with an increase of adipose tissue in the bone marrow.

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A spontaneous yolk sac carcinoma derived from the testis of an old male Sprague-Dawley rat was examined morphologically. The tumor was composed of cuboidal cells arranged into small groups or forming single cell rows. The tumor cells had round to oval, single central or polar nuclei with amphophilic, granular, finely vacuolated cytoplasms. The intercellular spaces contained large amounts of basement-membrane-like, eosinophilic matrix. This matrix was stained positively for laminin and periodic acid-Schiff (PAS). Some visceral cysts were lined with well differentiated cells, flat or cuboidal vacuolated epithelium. An ultrastructural examination showed that the hyaline matrix of the tumor had a fine-lamellar structure. These morphological characteristics in our case strongly suggest that this tumor is a yolk sac carcinoma, being similar to experimentally induced ones. No report of spontaneously occurring yolk sac carcinoma derived from rat testis was found in the literature.

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The dorsal skin topically treated wiht 0.5 and 30% solution of manganese ethylene bis (Maneb), a kind of dithiocarbamate fungicides, for 7 consecutive days was examined histopathologically in Wistar-derived inbred hypotrichotic WBN/ILA-Ht rats (HtRs). At day 1 after cessation of the treatment, except for slight inflammatory cell infiltration around hair follicles, no prominent histopathological changes were found. At 2 weeks after cessation of the treatment, prominent degeneration of epithelial cells characterized by pyknosis or karyorrhexis was found in the epithelial root sheath of hair follicles, and the nuclei of such epithelial cells were strongly stained by the TUNEL method. Thus, Maneb caused delayed-type lesions in the epithelial root sheath of hair follicles due to apoptosis.

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The incidence, distribution, and histopathology of respiratory metaplasia of the nasal gland epithelium were examined in F344 rats and BDF1 mice used as untreated control animals in 2-year carcinogenicity studies. Lesions were detected in almost all the male and female rats, while they were only detected in 67.8% and 46.6% of the male and female mice, respectively. In rats, most lesions were located in the septal and/or nasoturbinate subepithelial glands at the incisive papilla level, whereas the lesions varied in location and were histopathologically more severe in mice.

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