Makio Takeda

Analysis of PrPc mRNA by in situ Hybridization in Brain, Placenta, Uterus and Testis of Rats

An amyloid-like isoform of a 33- to 34-kD glycoprotein, termed as the scrapie prion protein (PrPsc), plays a critical role in transmissible spongiform encephalopathies of animals and humans. It has even been suggested to present the responsible infectious agent. This protein is a posttranslationally modified form of the cellular isoform of prion protein (PrPc). Hitherto, little has been known about the functions of PrPc. In order to examine the localization of PrPc mRNA in rat tissues, the in situ hybridization technique was performed. In rat brain, PrPc mRNA was predominantly localized within pyramidal cells of the hippocampus, large neurons of the thalamus and neocortex, and Purkinje cells of the cerebellum. In the placenta, not only PrPc mRNA was localized to a subpopulation of decidual cells at the highest levels, it was also expressed in the amnion and mesodermal layer of the yolk sac. Furthermore, PrPc mRNA was also expressed in the myometrium of the uterus and seminiferous tubule in the testis. However, signals were not obtained in the lung, spleen, liver of prenatals and other fetus tissues. The distribution of rat PrPc mRNA portrayed the levels which were different among the various types of cells, suggesting that its expression may be regulated in a tissue-specific manner.

Liver Tumor Promoting Effects of Fenbendazole in Rats

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given ≥600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given ≥600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.

Thymic atrophy induced by methoxychlor in rat pups

The effect of Methoxychlor (MXC) on the thymus was examined in rat pups that were delivered from dams receiving MXC at a dietary concentration of 0 or 1500 ppm for a period from pregnancy to lactation. The pups of both sexes were euthanized on postnatal days (PNDs) 7, 14, and 21. Histologically, the thymus showed marked depletion of cortical lymphocytes on PND 7 and also had an increase in lymphophagocytosis in the cortical area on PNDs 14 and 21. Morphometrical analysis disclosed that both cortex and medulla of the thymus from treated pups were reduced in size, but the reduction was more evident in the cortex. A significant increase in transferase-mediated dUTP nick end labeling-positive cells was detected in the cortex area, corresponding to the presence of lymphophagocytosis. Flow cytometric analysis revealed a significant decrease in the double positive (CD3(int)CD4(+)CD8(+)) immature cells on PND 21. These results have suggested that MXC may impair maturation of thymic lymphocytes in rat pups, which results in enhancement of apoptosis leading to thymic atrophy during the postnatal period.

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice.

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 μg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 μg/kg of DDAC caused weakly induced inflammation, and exposure to 15μg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 μg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C-C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.

Relation between distribution of viral RNA and development of histopathological changes in encephalomyocarditis virus‐induced orchitis in mice

The relation between the distribution of viral RNA and the development of histopathological changes was investigated in the early stage of encephalomyocarditis (EMC) virus‐induced orchitis in mice. Signals of viral RNA were first detected by in situ hybridization in a few Sertoli cells in almost intact germinal epithelia at 2 days post‐inoculation (d.p.i.). The number of Sertoli cells bearing signals of viral RNA increased at 3 d.p.i. when mild degenerative changes were exceptionally found in germinal epithelia. Signals of viral RNA came to be detected not only in Sertoli cells but also in a small number of germinal cells and spermatogonia at 4 d.p.i. when mild to moderate degenerative changes developed in germinal epithelia, resulting in desquamation of degenerated cells. At the same time, virus‐like particles were observed by electron‐microscopy in the degenerated and desquamated germinal cells. At and after 5 d.p.i., luminal obstruction with cellular debris and inflammatory cells was generally found. These results suggest that EMC virus carried to seminiferous tubules via the blood first attacks Sertoli cells and then damages germinal cells and spermatogonia.

Toxicity and Carcinogenicity of Dichlorodiphenyltrichloroethane (DDT)

Dichlorodiphenyltrichloroethane (DDT) is still used in certain areas of tropics and subtropics to control malaria and other insect-transmitted diseases. DDT and its metabolites have been extensively studied for their toxicity and carcinogenicity in animals and humans and shown to have an endocrine disrupting potential affecting reproductive system although the effects may vary among animal species in correlation with exposure levels. Epidemiologic studies revealed either positive or negative associations between exposure to DDT and tumor development, but there has been no clear evidence that DDT causes cancer in humans. In experimental animals, tumor induction by DDT has been shown in the liver, lung, and adrenals. The mechanisms of hepatic tumor development by DDT have been studied in rats and mice. DDT is known as a non-genotoxic hepatocarcinogen and has been shown to induce microsomal enzymes through activation of constitutive androstane receptor (CAR) and to inhibit gap junctional intercellular communication (GJIC) in the rodent liver. The results from our previously conducted 4-week and 2-year feeding studies of p,p′-DDT in F344 rats indicate that DDT may induce hepatocellular eosinophilic foci as a result of oxidative DNA damage and leads them to hepatic neoplasia in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

Pulmonary injury and antioxidant response in mice exposed to arsenate and hexavalent chromium and their combination

Chromated copper arsenate, which is used worldwide as a wood preservative, can adversely affect human health. Accumulating evidence suggests that chromium (Cr) and arsenic (As) can potentially disrupt the redox balance and cause respiratory diseases and cancer in humans. The present study was designed to determine the combined toxic effects of these metals in the lungs and to clarify the specific molecules that are stimulated by combined exposure to both metals. Male C57BL/6J mice were intratracheally instilled with arsenate [As(V)], hexavalent chromium [Cr(VI)], or a combination of both metals. Mice were sacrificed 2 days after treatment to collect bronchoalveolar lavage fluid and lung tissue samples. Inflammation, cytotoxicity, apoptosis, and oxidative stress markers were measured. Our results indicated that administration of Cr(VI) alone or in combination with As(V) induced neutrophil-dominant inflammation as well as phosphorylation of mitogen-activated protein kinases; effects of treatment with As(V) alone were comparatively less potent. By analyzing the production of interleukin-6 and activity of lactate dehydrogenase and caspase, we confirmed that co-treatment intensified pulmonary injury and that it was accompanied by oxidative stress, as confirmed by marked increases in the production of reactive oxygen species, reduced glutathione content, and thioredoxin reductase (TRXRD) activity. Expressed mRNA levels of heme oxygenase-1, glutamylcysteine ligase, glutathione peroxidase 2, thioredoxin (TRX) 1, and TRXRD1 were also enhanced by co-treatment, whereas treatment with As(V) alone reduced the mRNA expression level of TRX2. Our data suggest that co-treatment with As(V) exacerbated Cr(VI)-induced pulmonary injury and that this effect may be exerted through a disruption in the balance among several antioxidant genes.

Galactosamine-induced apoptosis in the primary mouse hepatocyte cultures

Galactosamine (GalN)-induced apoptosis was investigated in cultured hepatocytes from mice. The percentage of fragmented DNA measured by the ELISA method increased in a concentration-dependent manner from the very early stage, i.e. 0.5 hrs, after GalN-exposure. In addition, a ladder-like fragmentation pattern by agarose gel electrophoresis appeared first at 3 hr-exposure to 20 mM GalN, at 6 hr-exposure to 10 mM GalN and at 12 hr-exposure to 5 mM GalN, respectively. On the other hand, cytotoxicity indicated by leakage of lactate dehydrogenase from cultured hepatocytes to culture medium was first detected at 24 hrs after GalN-treatment. Morphologically, formation of blebs and apoptotic bodies was observed from 12 hr-exposure to 20 mM GalN and from 24 hr-exposure to 10 mM GalN, respectively. Thus GalN could induce apoptosis in primary hepatocyte cultures from mice.

Transforming growth factor β production by spontaneous malignant mesothelioma cell lines derived from Fisher 344 rats

Abstract. We investigated whether transforming growth factor β (TGF-β) is involved in the growth of malignant mesothelioma (MM) cells in culture. TGF-β production was examined in two mesothelioma cell lines (MeET-4 and -6) that were established from rat spontaneous MM in our laboratory. TGF-β bioactivity in conditioned medium of these cell lines was analyzed using a CCL64 mink lung epithelial cell growth inhibition assay and found to be 30–70 times higher than that of normal rat mesothelial cells (MCs). The MM cell lines also showed considerably higher levels of TGF-β mRNA expression when compared with MCs. The bioactivity and mRNA expression level were greater in MeET-4 than MeET-6. When MeET-4 was treated with antisense TGF-β1 oligonucleotide (ODN), a significant decrease in both anchorage-dependent and -independent growth was observed. Treatment with exogenous TGF-β resulted in no effects on the growth pattern of the MM cell lines, while proliferation of the MCs was slightly induced. It is considered that TGF-β appears to be produced by rat spontaneous MM cells through an autocrine mechanism and could modulate the malignant growth of the tumor cells.

An age-related change in susceptibility of rat brain to encephalomyocarditis virus infection

Rats were inoculated intraperitoneally (i.p.) or intracerebrally (i.c.) with 1 × 104 plaque forming units (PFU)/animal of the D variant of encephalomyocarditis virus (EMC‐D) at 2, 4, 7, 14, 28 or 56 days of age for virological and histopathological examination. In the i.p.‐inoculation study, neither viral replication nor lesions were detected in the animals inoculated at 28 and 56 days of age. In the animals inoculated when younger than 14 days of age, lesions were restricted to the brain although viral replication was detected in the brain, heart and pancreas. The brain lesions were characterized by acute meningoencephalitis with neuronal necrosis in the cerebral cortex, hippocampus and thalamus, and viral RNA was detected in degenerated and/or intact neurons. In the i.c.‐inoculation study, similar age‐related changes in susceptibility of rat brain to EMC‐D infection were observed, but a minor difference was that viral replication and lesions were still detected in the hippocampus of some animals inoculated at 28 days of age. These results suggest that an age‐related decrease in the susceptibility of rat brain to EMC virus infection may reflect an age‐related change in the susceptibility of neurons themselves as well as in maturation of the immune system.