Ikuo Mori

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene.

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.

Comparison of Contrast Agents for Atherosclerosis Imaging Using Cultured Macrophages: FDG Versus Ultrasmall Superparamagnetic Iron Oxide

Various noninvasive imaging methods have been developed to evaluate atherosclerotic plaques. Among them, 18F-FDG PET and MR imaging with ultrasmall superparamagnetic iron oxide particles (USPIO) have been used to quantify plaque inflammation. Both methods are based on the efficient uptake of FDG and USPIO by macrophages in atherosclerotic lesions. Differently polarized macrophages have been reported to have different characteristics that are involved in the pathologic development of atherosclerosis. M1 polarized macrophages are considered the more proatherogenic phenotype than M2 polarized macrophages. However, little is known regarding the association between macrophage polarization and FDG or USPIO accumulation. In this study, we investigated intracellular FDG and USPIO accumulation in M1 and M2 polarized macrophages. Methods: THP-1 macrophages were differentiated into M1 and M2 polarized macrophages. Under optimal glucose conditions, we investigated the 3H-labeled FDG uptake in M1 and M2 polarized macrophages. We then investigated intracellular USPIO uptake by M1 and M2 macrophages. Results: We found that M1 polarization, compared with M2 polarization, results in increased intracellular accumulation of FDG. To elucidate the mechanism by which FDG was preferentially accumulated in M1 macrophages, we examined messenger RNA expressions of glucose transporters (GLUTs) and hexokinases, which have pivotal roles in glucose uptake, and glucose-6-phosphatase (G6Pase), which catalyzes the reverse reaction of hexokinase. In M1 macrophages, GLUT-1, GLUT-3, hexokinase 1, and hexokinase 2 were upregulated and G6Pase was downregulated. In contrast to FDG, M1 polarization resulted in decreased intracellular accumulation of USPIO. We found that scavenger receptor A and CD11b, which are involved in USPIO binding and uptake, were significantly downregulated by M1 polarization. Conclusion: Compared with M2, proatherogenic M1 macrophages preferentially accumulated FDG but not USPIO, suggesting that FDG PET is a useful method for the detection of proinflammatory M1 macrophages.

Point Mutations of the c-H-ras Gene in Spontaneous Liver Tumors of Transgenic Mice Carrying the Human c-H-ras Gene

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversion, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosarcomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.

Relationship between in vitro phospholipidosis assay using HepG2 cells and 2-week toxicity studies in rats

Drug candidates under development by industry frequently show phospholipidosis as a side-effect in pre-clinical toxicity studies. This study sets up a cell-based assay for drug-induced phospholipidosis (PLD) and its performance was evaluated based on the in vivo PLD potential of compounds in 2-week toxicity studies in rats. When HepG2 cells were exposed simultaneously to PLD-inducing chemicals and a phospholipid having a fluorophore, an accumulation of phospholipids was detected as an increasing fluorescent intensity. Amiodarone, amitriptyline, fluoxetine, AY-9944, and perhexiline, which are common PLD-inducing chemicals, increased the fluorescent intensity, but acetaminophen, ampicillin, cimetidine, famotidine, or valproic acid, which are non-PLD-inducing chemicals, did not. The fluorescent intensity showed concordance with the pathological observations of phospholipid lamellar bodies in the cells. Then to confirm the predictive performance of the in vitro PLD assay, the 32 proprietary compounds characterized in 2-week toxicity studies in rats were evaluated with this in vitro assay. Because this in vitro assay was vulnerable to cytotoxicity, the innate PLD potential was calculated for each compound. A statistically significant increase in the in vitro PLD potential was seen for the compounds having in vivo PLD-inducing potential in the rat toxicity studies. The results suggest that the in vitro PLD potential could be appropriate to detect the appearance of PLD as a side effect in pre-clinical toxicity studies in rats.

Carcinogen dose-dependent variation in the transgene mutation spectrum in urethane-induced lung tumors in transgenic mice carrying the human prototype c-Ha-ras gene

Urethane-induced lung tumors and their genetic changes were investigated in transgenic (Tg) mice carrying a human prototype c-Ha-ras gene (rasH2 mice). Male and female rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1000 mg/kg of urethane once or three times at 2-day intervals. Hyperplasias and adenomas of the lung were observed in all animals of each group from week 10, and carcinomas were observed in male and female rasH2 mice of the triple injection group from week 10 and female non-Tg mice of the single injection group at 15/20 weeks. The multiplicities of lung proliferative lesions including hyperplasias, adenomas and carcinomas, in treated rasH2 mice were significantly higher than those in treated non-Tg mice. CAG to CTG transversions were observed in the c-Ha-ras gene in these lung proliferative lesions of rasH2 mice of the single injection group at high incidence (male: 58.3%, female: 62.5%), but no mutations of the mouse c-Ki-ras gene were evident in either rasH2 or non-Tg mice. In the triple injection group, transgene mutations were detected at a relatively low incidence, and mouse c-Ki-ras gene mutations(CAA to CGA) were observed in both rasH2 and non-Tg mice. These results suggest that the variation of the lesions induced by different doses of urethane was not the cause of the variation of the mutation spectrum and mutations of both transgene and mouse c-K-ras gene are not principal genetic events in urethane-induced lung proliferative lesions in rasH2 mice.

Essential similarities between spontaneous and MeIQx-promoted aberrant crypt foci in the F344 rat colon

Aberrant crypt foci (ACFs) in the Fischer 344 (F344) rat colon, of control or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-treated groups, were compared morphologically, immunohistochemically, and at the molecular biological level in order to elucidate their biological characteristics. Male 3-week-old rats were fed a diet supplemented with or without MeIQx at doses of 100 ppm or less for 16 weeks. The incidence of ACFs was the highest (90%) in animals given 100 ppm MeIQx but that in untreated rats was also surprisingly high (57%). Nine ACFs from nine MeIQx-treated rats and ten ACFs from ten untreated control rats were selected for detailed examination for their large size. There were no morphological differences in macroscopic and microscopic features between MeIQx-promoted and spontaneous ACFs. There were also no differences in immunohistochemical labeling for proliferating cell nuclear antigen (PCNA) and p53 protein between these ACFs although in both cases labeling was higher than in normal crypts. Dot blot hybridization revealed no c-K-ras mutations in codon 12 except in one ACF (11.1%) developing in a rat treated with 100 ppm MeIQx, in which a GGT-->GAT single base substitution was detected. Our results thus suggest that in terms of morphology, cell proliferation, P53 expression and c-K-ras mutation, most ACFs found in rats given 100 ppm MeIQx are essentially identical to their spontaneous counterparts.

Pulmonary fibrosis caused by N-methyl-N-nitrosourethane inhibits lung tumorigenesis by urethane in transgenic mice carrying the human prototype c-Ha-ras gene.

Male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and their wild littermates (non-Tg mice) received three subcutaneous injections of 0.3 mg N-methyl-N-nitrosourethane (MNUR) once every 2 weeks for the first 4 weeks followed by a single intraperitoneal injection of 1000 or 0 mg/kg urethane (UR) 2 weeks later. They were then maintained without any other treatment for a further 13 weeks and sacrificed for assessment of pulmonary pathology. Inflammatory lesions, such as macrophage infiltration, alveolar bronchiolization and/or fibrosis, were induced in both rasH2 and non-Tg mice treated with MNUR or MNUR + UR. Lung proliferative lesions were induced in 100% of the UR-treated rasH2 mice but to a significantly lesser extent in the MNUR + UR case. The incidences of lung tumors in non-Tg mice treated with UR or MNUR + UR were relatively low. Point mutations of the transgene were detected in approximately 80% of lung tumors in rasH2 mice treated with UR and MNUR + UR, but murine Ki-ras mutations were rare. No marked difference in the mutation pattern was found between the UR-treated and the MNUR + UR-treated rasH2 mice. In non-Tg mice treated with UR or MNUR + UR, point mutations of the murine c-Ki-ras gene were observed in about 50% of the lung tumors examined. The present study confirmed that rasH2 mice are very sensitive to lung tumor induction by UR and suggested that alveolar epithelial cells in the reparative stage during pulmonary fibrosis are resistant to DNA damage by this carcinogen.

Spontaneous Proliferative Lesions in the Nasopharyngeal Meatus of F344 Rats

Spontaneous proliferative lesions in the nasopharyngeal meatus were identified as the cause of death in 12 of 1,600 male and 5 of 1,600 female Fischer 344 (F344) rats used in 2-yr carcinogenicity studies; none of the lesions were considered treatment related. All the rats showed dyspnea, abdominal distension, and clinical deterioration. Gross features were characterized by simultaneous occurrence of conspicuous gaseous distension of the intestinal tract, especially in the ileum and cecum, and focal nodular lesions in the nasopharyngeal meatus. Histopathologically, the nasopharyngeal meatus was partially obstructed by the following proliferative lesions: 3 areas of hyperplasia of the ectopic sebaceous glands in the soft and hard palate, 4 areas of squamous metaplasia (SM) with massive hyperkeratosis, 5 squamous cell papillomas (SCPs), and 5 squamous cell carcinomas (SCCs). No pathological changes were found in the distended portion of the intestinal tract. Formalin-fixed, paraffin-embedded samples of the proliferative lesions from the nasopharyngeal meatus were examined for the presence of mutations in the c-H-ras and c-K-ras genes. In vitro amplification of DNA using a polymerase chain reaction was combined with a nonisotopic method for selective oligonucleotide hybridization. Two of the 4 SM lesions, 3 of the 5 SCPs, and 5 of the 5 SCCs contained 1-3 point mutations in the c-H-ras and/or c-K-ras gene. Immunohistochem-ically, overexpression of p53 protein was found in 1 area of SM with a dysplastic lesion and 2 SCCs. These findings indicate that detailed examination of the upper respiratory system, including the nasopharyngeal meatus, may be particularly helpful for identifying primary occult lesions in F344 rats that show only gut distension at necropsy in long-term toxicity studies. In addition, mutations of the ras genes may be an important step in the early stages of carcinogenesis in the rat nasopharyngeal meatus, whereas p53 mutations could occur relatively late.

Spontaneous synovitis in Wistar rats.

Spontaneous synovitis has been detected in the stifle joints of control Wistar rats in 4-wk and 2-yr toxicity studies, and a specific site was found to be predisposed to synovitis. The incidence of synovitis was 27.5% for males in the 4-wk study and 40.0% for males and 16.0% for females in the 2-yr study. The severity was not increased with aging. The degenerative changes, including chondroid metaplasia of the cruciate ligaments of the stifle and/or the surrounding connective tissue at the border of the cruciate ligaments of the stifle, were present even in younger animals, and moreover there was a good correlation between synovitis and chondroid metaplasia of the cruciate ligaments of the stifle and the surrounding connective tissue at the border of the cruciate ligaments of the stifle. Vascular changes, including the development of fibrin-containing thrombi, were observed in the surrounding connective tissue at the border of the cruciate ligaments of the stifle. It is speculated that the pathogenesis of the observed synovitis reflects joint instability that induces mechanical damage of the synovium.

Glucose uptake of the muscle and adipose tissues in diabetes and obesity disease models: evaluation of insulin and β3-adrenergic receptor agonist effects by 18F-FDG

ObjectiveOne of the major causes of diabetes and obesity is abnormality in glucose metabolism and glucose uptake in the muscle and adipose tissue based on an insufficient action of insulin. Therefore, many of the drug discovery programs are based on the concept of stimulating glucose uptake in these tissues. Improvement of glucose metabolism has been assessed based on blood parameters, but these merely reflect the systemic reaction to the drug administered. We have conducted basic studies to investigate the usefulness of glucose uptake measurement in various muscle and adipose tissues in pharmacological tests using disease-model animals.MethodsA radiotracer for glucose, 18F-2-deoxy-2-fluoro-d-glucose (18F-FDG), was administered to Wistar fatty rats (type 2 diabetes model), DIO mouse (obese model), and the corresponding control animals, and the basal glucose uptake in the muscle and adipose (white and brown) tissues were compared using biodistribution method. Moreover, insulin and a β3 agonist (CL316,243), which are known to stimulate glucose uptake in the muscle and adipose tissues, were administered to assess their effect. 18F-FDG uptake in each tissue was measured as the radioactivity and the distribution was confirmed by autoradiography.ResultsIn Wistar fatty rats, all the tissues measured showed a decrease in the basal level of glucose uptake when compared to Wistar lean rats. On the other hand, the same trend was observed only in the white adipose tissue in DIO mice, while brown adipose tissue showed increments in the basal glucose uptake in this model. Insulin administration stimulated glucose uptake in both Wistar lean and fatty rats, although the responses were inhibited in Wistar fatty rats. The same tendency was shown also in control mice, but clear increments in glucose uptake were not observed in the muscle and brown adipose tissue of DIO mice after insulin administration. β3 agonist administration showed the similar trend in Wistar lean and fatty rats as insulin, while the responses were inhibited in the adipose tissues of Wistar fatty rats.ConclusionA system to monitor tissue glucose uptake with 18F-FDG enabled us to detect clear differences in basal glucose uptake between disease-model animals and their corresponding controls. The responses in the tissues to insulin or β3 agonist could be identified. Taken as a whole, the biodistribution method with 18F-FDG was confirmed to be useful for pharmacological evaluation of anti-diabetic or anti-obesity drugs using disease-model animals.