Both genotoxic and non-genotoxic carcinogens may cause changes in cell cycle regulatory mechanisms. One of the most frequent genetic alterations observed in human malignancies, including urinary bladder carcinomas, involves the p53 tumor suppressor gene. The H- and K-ras oncogenes also appear to be prime targets in man and experimental animals. In rat urinay bladder carcinogenesis models using N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), most transitional cell car-cinomas (TCCs) produced are of papillary type and invasion is rare. While p53 mutations are relatively frequent in animals continuously exposed to BBN, they are less common when tumors are generated by protocols with a no-treatment interval before terminal sacrifice. The ras gene alterations do not vary under these conditions. In two-stage (initiation-promotion) rat models, p53 mutations are rare but cyclin D1 overexpression is frequently observed in TCCs. No mutations are generally found in rat tumors due to the non-genotoxic carcinogen, uracil.
In contrast to rat tumors, mouse urinary bladder tumors induced by BBN aggressively invade and demonstrate occasionally metastasis. In one series, p53 mutations were found in 14 of 18 invasive tumors. Moreover, urinary bladder carcinomas with metastasis harbored more frequent alterations. Loss of heterozygosity of the p53 gene was also evident in about 14% of such carcinomas. In conclusion, genetic alterations differ according to the experimental protocol, the species, and the type of carcinogen used.

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The HST-1 gene, originally identified in our laboratory as a transforming gene, encodes HST-1 protein or fibroblast growth factor (FGF)-4, a member of the FGF family. In this study, to explore a novel function of HST-1 gene, gene knock-in (gain of function) and knock-out (loss of function) were performed.
To identify the target cells and tissues which are susceptible to the biological actions of HST-I protein in vivo, we gave mice a recombinant adenovirus carrying the HST-l gene (AdexHST-l). The results show that AdexHST-l caused a two-fold increase in peripheral platelet count for a period of 30 days, and also effectively prevented experimentally induced thrombocytopenia. We further elucidated that thrombopoietic action of HST-1 is achieved in that HST-1 can regulate megakaryocyte development as a megakaryocyte potentiator synergistically with IL-3 and TPO as well as potentiates magakaryocyte maturation.
As a means of exploring the role of HST- I in vertebrate embryogenesis, mice with distrupted HST-1 gene were produced by homologous recombination. Null mutant ES cells survived but homozygous HST-1-/- mutants showed severe develop-mental delay and died before 9 days of gestation, suggesting that HST-l plays essential roles in early phases of mouse development.
In addition to its early embryonic function, HST-1 may play a crucial role in the later stage of development. In fact, we and others have found that HST-1 is expressd in the cells of the apical ectodermal ridge of mouse limb bud at 11 and 12 days of gestation. In an effort to further understand the role of HST-l in mouse limb development, we initially established a novel limb culture system which provides us with a suitable model system to explore the key factors for limb development. Using this culture system, we have shown that exposure of embryonal limb bud explants to antisense oligonucleotides of HST-1 blocks limb outgrowth. Our results suggest direct evidence that HST-1 worked naturally as an inducer of mouse limb development.

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To study the response of human glutathione S-transferase (GST) to chemical inducers, we have developed a line of transgenic rats which harbor 4.5 kb of human GST alpha I promoter region in their genome. This promoter is linked to the chloramphenicol acetyltransferase (CAT) reporter gene which allows determination of the expression of human GST in rat tissues. Three chemical inducers, which show clearly different induction profiles, phenobarbital (PB), β-naphthoflavone (BNF), and butylated hydroxyanisole (BHA), were administered to the transgenic rats. Induction of constitutive rat liver enzymes by the inducers, which was evaluated in terms of the activities of P450, GST, and UDP-glucuronosyltransferase in the liver tissues, were in agreement with what has been reported for non-transgenic rats. Expression of CAT protein was detected in the liver of the transgenic rats, and an unequivocal increase in CAT protein was found in the transgenic rats treated with PB. No remarkable changes in CAT protein were observed in the transgenic rats treated with BNF or BHA. Moreover, immunohistochemical staining with anti-CAT antibody revealed that the expression and increase of CAT protein were localized in the central zone of the liver lobule. The results of this study suggest that human GST alpha 1 is induced by PB, in particular, in the central zone of the liver lobule. The transgenic rat is concluded to be a useful animal model for predicting metabolizing functions in humans.

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Female Syrian golden hamsters were given a subcutaneous injection of 0.6 mg of N-methyl-N-nitrosourethane (MNUR) every 2 weeks for 8 weeks (a total of 5 doses) and then maintained without any treatment for the next 26 weeks. The MNUR-induced bronchiolo-alveolar cell tumors were all adenomas composed of basophilic or clear cells in a papillary growth pattern or papillary/solid tumors consisting of pleomorphic cells. Ultrastructurally, most of the tumor cells had the lamellar bodies characteristic of alveolar type II cells in their cytoplasm. Positive cyclin D1 immunostaining was associated with pleomorphic cell type adenomas, whereas no overexpression of p53 protein, hsp70 or mdm2 gene protein was detected in any of the tumors. The lung tumor samples were examined for the presence of mutations in the K-ras gene in codon 12, 13, and 61 by a non-isotopic method for selective oligonucleotide hybridization after PCR amplification of DNA from formalin fixed tissue embedded in paraffin. Lung tumors from 6 of 18 (33%) tumor bearers contained a point mutation in codon 13 or 61, 3 involving GGC→GAC transitions at the second base of codon 13 and 3 affecting the 61st codon, one CAA→CTA, and two CAA→CGA. No mutations were detected in codon 12.
These findings indicate that, at least, a point mutation of the K-ras gene was related to the development of bronchiolo-alveolar cell adenomas induced by MNUR and may suggest that cyclin DI overexpression in pleomorphic cell type tumors plays a role in the process of malignant transformation.

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Male and female F344 rats were exposed to 0 to 100ppm methyl bromide for 6 hr/day×5 days/week×104 weeks. Dose-dependent lesions were produced in the nasal passage of male rats, characterized by enhanced foreign body rhinitis mostly due to inhaled hairs as well as necrosis and respiratory metaplasia of the olfactory epithelium.

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Acute toxicity of aflatoxin B₁ (AFB1) by AFB1-diet (3 ppm) feeding for 7 days was simultaneously examined in infant and adult domestic fowls of 3 species (ducks, chickens, and Japanese quails) from the viewpoint of comparative pathology. As a result, AFB1 brought about high mortality in 5-day-old ducklings. In addition, AFB1 showed a significant depressive effect on egg production in laying ducks, hens, and quails. Moreover, in correspondence to blood biochemical findings such as significant increases in alkaline phosphatase, glutamic oxaloacetic transaminase, and/or glutamic pyruvic transaminase activities, such hepatic changes as bile duct hyperplasia and fatty degeneration of hepatocytes were common to infant and adult fowls, and the hepatic damage was most frequent and marked in 5-day-old ducklings. In the markedly affected liver, cells showing an intermediate morphology between hepatocytes and bile duct epithelial cells were sometimes observed. Judging from the present study, ducks were considered to be most susceptible to AFB1 as a whole.

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Variation and location of cytochrome P450 isoenzyme expression in various organs of male F344 rats treated with 1, 2-dimethylhydrazine (DMH), N, N-dibutylnitrosamine (DBN), and methylbenzylnitrosamine (MBNA) were immunohisto-chemically and biochemically investigated. The animals received MBNA (6mg/6ml/kg b.w. i.p.) in corn oil, DBN (400mg/4ml/kg b.w. i.p.) in distilled water with Tween 80, or DMH (40mg/4ml/kg b.w. i.p.) dissolved in saline. All animals were sacrificed at hours 15 after administration of the test compound. CYP2C11 was immunohistochemically observed in the centrilobular areas of the liver in rats treated with both DBN and DMH. Western blotting analysis, however, showed significantly decreased level of CYP2C11 protein in the liver of rats treated with DBN. CYP2D1 was immunohistochemically observed in periportal areas of the liver in rats treated with MBNA. The present investigation demonstrated that expression of cytochrome P450 isoenzymes can modulate exogenous agents in the liver.

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The effect of hypertriglyceridemia was examined on streptozotocin (STZ)-induced diabetic nephropathy in rats. After repeated intraperitoneal injection with Triton WR- 1339 (Triton) in STZ-diabetic rats, food and water consumption and urine volume were significantly decreased and albumin/globulin ratio lowered compared with non-injected Triton and STZ-diabetic rats. Between STZ-diabetic rats with Triton injection and those without, no significant difference was observed in the extent of mesangial matrix and PAS, type IV collagen, fibronectin, and laminin positive areas. The results suggested that the proliferation of mesangial cells might be inhibited by the high levels of low and very low density lipoproteins, and diabetic nephropathy was not affected by the inducing of hypertriglyceridemia.

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The testicular toxicity of cyclophosphamide (CP) in rats and recovery of normal spermatogenesis over an 8-week period were assessed. Male rats were administered 40mg/kg CP by gavage daily for 1 week and sacrificed at 1 day, 3 weeks, and 8 weeks after the cessation of treatment. The numbers of seminiferous epithelial cells were counted in the seminiferous tubules in stages II-III, V, VII, and XII of the spermatogenic cycle. Spermatogonia were decreased in number at 1 day after the last treatment, but most of the surviving spermatogonia including type A had proliferative activity, as demonstrated by PCNA staining. After 3-week recovery, the numbers of spermatogonia were improved, but the numbers of spermatocytes and round spermatids were reduced in some stages. Furthermore, focal substantial changes such as atrophy of seminiferous tubules were present in the testis. After 8-week recovery, the numbers of spermatogenic cells up to round spermatids were comparable to those in control specimens, and histopathologically no remarkable changes were observed in the testis. However, lower weights and sperm counts for testis and epididymis were still found at this time. These findings suggested that histopathological changes as subtle as decreased number of spermatogonia should be detected in evaluations of male chemical reproductive toxicity. Our findings also suggested that PCNA staining may be useful for predicting reversibility of testicular toxicity.

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Rats of Jcl : Wistar TGN (ARGHGEN) 1 Nts strain (Mini rats) are transgenic animals in which the expression of growth hormone (GH) gene is suppressed by the presence of an antisense transgene. In the present study, we examined hepatocyte regeneration after 2/3 partial hepatectomy (PH) in male Mini rats, using Wistar rats as controls. As a result, the restoration rate of liver weight was similar between the 2 strains, though bromodeoxyuridine (BrdU)- and proliferating cell nuclear antigen (PCNA)-labeling indices (LIs) of hepatocytes were generally higher in Wistar rats than in Mini rats at 1 day after PH. At 2 days, on the contrary to BrdU-LI, PCNA-LI was higher in Mini rats than in Wistar rats, and the difference in the total PCNA-LIs at 1 and 2 days between the 2 strains was not so large. This suggests that hepatocyte proliferation may start more slowly in Mini rats than in Wistar rats but the total proliferation rate during the regenerating process may not be significantly different between the 2 strains as a whole.

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