Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of ß- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

Effects of aging and uninephrectomy on renal changes in Tsukuba hypertensive mice.

Renal dysfunction is accelerated by various factors such as hypertension, aging and diabetes. Glomerular hyper-filtration, considered one of the major risk factors leading to diabetic nephropathy, is often encountered in diabetic patients. However, the interrelationship of these risk factors during the course and development of renal dysfunction has not been fully elucidated. In this study, the effects of aging and uninephrectomy (UNx)-induced hyperfiltration on renal changes were investigated in Tsukuba hypertensive mice (THM) carrying both human renin and angiotensinogen genes. In THM, the urinary albumin/creatinine (Alb/Cr) ratio was elevated with age without a concomitant increase in the plasma Cr concentration. Moreover, the urinary neutrophil gelatinase-associated lipocalin/Cr (NGAL/Cr) ratio, the renal monocyte chemoattractant protein-1 (MCP-1) mRNA expression and the renal collagen type I α 2 (COL1A2) mRNA expression were also increased with age. Age-related albuminuria in THM is likely caused by renal tubular damage, enhanced inflammatory response and tubulointerstitial fibrosis. Furthermore, following UNx, the urinary Alb/Cr ratio and the plasma Cr concentration were increased in THM. The urinary NGAL/Cr ratio and the renal MCP-1 and COL1A2 mRNA expression were not affected by UNx. These results suggested that UNx-induced albuminuria in THM was caused by glomerular dysfunction, rather than renal tubular injury. In conclusion, this study demonstrated for the first time the effects of aging and UNx on renal changes in THM. These findings strongly reinforce the significance of applying a diversity of therapeutic approaches to the management of renal dysfunction.

Gene expression in livers of BALB/C and C57BL/6J mice fed a high-fat diet.

We previously demonstrated that high-fat diet (HFD)-induced hepatic lipid accumulation is more severe in BALB/c mice than in C57BL/6J (B6) mice. To understand the changes in liver metabolism, we studied blood chemistry, gene expression, and histopathological changes of the liver in nine-week HFD-fed BALB/c and B6 mice and one- or four-week HFD-fed BALB/c mice. Serum total cholesterol and triglyceride levels were significantly increased in all HFD-fed groups, and one- and four-week HFD-fed BALB/c groups, respectively. Histopathology revealed that vacuolation of hepatocytes was severe in nine-week HFD-fed BALB/c mice, although it was less severe in the other groups. Microarray analysis of mRNA expression of nine-week HFD-fed BALB/c mice showed up-regulation of genes involved in fatty acid uptake and biosynthesis, such as Cd36, Acaca, Acly, and Fasn. Some changes were observed in the one- and four-week HFD-fed BALB/c groups and the nine-week HFD-fed B6 group, however these changes in mRNA expression were not so marked. In conclusion, the fatty accumulation observed in BALB/c mice may be caused, at least in part, by up-regulation of fatty acid uptake and biosynthesis. Cd36, Acaca, Acly and Fasn may be involved in these metabolic processes.

Spontaneous poorly differentiated carcinoma with cells positive for vimentin in a salivary gland of a young rat.

Spontaneous salivary gland tumors in rats are rare. The authors report a poorly differentiated carcinoma of a submandibular gland in a ten-week-old rat that was positive for vimentin. Microscopically, the neoplastic cells showed a diffuse growth pattern in most areas of the tumor mass and a nestlike structure in a part of the peripheral area. Immunohistochemically, the cells were positive for keratin and vimentin but not for alpha-smooth muscle actin. Ultrastructurally, desmosome-like structures were observed. Based on these findings, the tumor was diagnosed as a poorly differentiated carcinoma. The origin of the neoplastic cells would be either acinar or ductal cells. This suggests that acinar or ductal cells have the potential to transform into vimentin-expressing cells.

Fenofibrate-induced muscular toxicity is associated with a metabolic shift limited to type-1 muscles in rats.

Morphological changes and mRNA expression levels in type-1 predominant soleus and type-2 predominant tensor fasciae latae muscles of rats treated with fenofibrate were investigated. After fenofibrate by oral gavage at 300 mg/kg/day for 28 days, degeneration/necrosis and regeneration of muscle fibers, cellular infiltration, and fibrosis were seen in soleus muscle. Additionally, expression of PDK4, CPT1-M, CPT2, and FACO mRNAs was increased. In contrast, no morphological changes or mRNA induction were apparent in tensor fasciae latae muscle. These data suggest that sensitivity to fenofibrate-induced muscle toxicity differs among muscles, with only type-1 fibers being susceptible. The up-regulation of PDK4, CPTs and FACO mRNA expression in soleus muscle indicates that the energy source is switched from glucose to fatty acids, and this might be related to the observed fenofibrate-induced muscular toxicity.

Collaborative work on evaluation of ovarian toxicity. 17) Two- or four-week repeated-dose studies and fertility study of sulpiride in female rats.

To find the appropriate dosing period to detect ovarian toxicity, sulpiride, a D2 antagonist was orally dosed to female rats at dose levels of 1, 10, and 100 mg/kg/day daily for 2 or 4 weeks in repeated-dose toxicity studies. In addition, sulpiride at the same dose levels was given to female rats daily during the pre-mating period, mating period, and Days 0-7 of gestation to assess its effect on fertility. In ovarian histology in the 2-week study, increases in atretic follicle were seen at 1 mg/kg or more and increases in follicular cysts at 10 mg/kg or more. In the 4-week study, these findings were seen at 1 mg/kg or more, and a decrease in large follicles was seen at 10 mg/kg or more. Increased body weight gain was observed at 10 mg/kg or more in the 2- and 4-week studies. The females in these groups exhibited development of mammary alveolus by sulpiride-induced hyperprolactinemia. In the fertility study, sulpiride-treated females showing persistent diestrus resulted in successful mating, and almost all females got pregnant. However, increased implantation loss was observed at 10 mg/kg or more, which was considered to be caused by the adverse effect of sulpiride on oocyte development. From these results, sulpiride-induced ovarian toxicity was seen at 1 mg/kg or more in the 2- and 4-week repeated-dose toxicity studies, and the observed ovarian changes were considered to be related to adverse effects on female fertility.

Evaluation of gene expression related to hepatic cell maturation and differentiation in a chemically induced mouse hepatoblastoma cell line.

The MHB-2 cell line, established from a mouse hepatoblastoma (HB), was subjected to the reverse transcriptase-polymerase chain reaction (RT-PCR) for evaluation of gene expression related to cell differentiation. RNAs for c-kit, CD34, thy-1, albumin, cytokeratin (CK) 8, 18 and 19 could be detected, but expression of alpha-fetoprotein, glucose-6-phosphatase, tyrosine aminotransferase and CK7 was not observed. MHB-2 cells were positive for CK8/18 but negative for c-kit, CD34, thy-1 and albumin on protein level. Immunohistochemical staining of the HB in vivo revealed diffusely expressed c-kit. Thy-1-positive HB cells were sparsely observed, but the tumor was negative for CD34 and rarely positive for CK8/18. By in situ hybridization, the HB was positive for CK18 but negative for CK19. Slight expression of albumin, but the lack of immature hepatocytic marker suggested some heterogeneous hepatocyte or an undifferentiated cell from other origin. Furthermore, positive expression of CK19 as well as CK8 and CK18 in culture strongly suggested the differentiation into a biliary lineage or the bidirectional state. In conclusion, the present study indicated the mouse HB to have de-differentiated, bipotent, or biliary-like cell characteristics, and considering the histological difference between HB and biliary tumors, it suggests the mouse HB cells are closely like some sort of hepatic undifferentiated cells.

Skeletal muscle susceptibility to clofibrate induction of lesions in rats.

Morphological changes induced by clofibrate in type-1 predominant soleus, type-2 predominant tensor fasciae latae, and type-1 and -2 mixed biceps femoris muscles and diaphragm in rats were investigated. Administration of the agent at 500 or 750 mg/kg/day by oral gavage for 14 or 28 days caused lesions in the soleus muscle and diaphragm, bur no changes in the tensor fasciae latae and biceps femoris muscles. In soleus muscle, vacuolation of muscle fibers was observed in all animals treated with clofibrate, and degeneration of muscle fibers and infiltration of leukocytes were noted at 750 mg/kg/day. In diaphragm, vacuolation of muscle fibers was also observed in all animals treated with clofibrate, and these lesions were located in type-1 skeletal muscles densely stained with NADH-TR. The vacuoles seen in soleus muscle and diaphragm were positive for oil red O staining. In addition, increase of lipid droplets and mitochondrial hypertrophy was seen in soleus muscle, ultrastructurally. These data suggest that sensitivity to clofibrate-induced muscle toxicity differs among muscles, with type-1 fibers being susceptible.

Histopathological characterization of the skeletal myopathy in rasH2 mice carrying human prototype c-Ha-ras gene.

A skeletal myopathy is found in approximately 100% of rasH2 mice. To confirm detailed features of the rasH2 skeletal myopathy, the biceps femoris, diaphragm, triceps brachii, gastrocnemial (types I and II fiber-mixed muscles) and soleus muscle (type I fiber-dominant muscle) obtained from male rasH2 and non-transgenic littermates aged 10-13 and 34 weeks were examined. Variations in the muscle fiber size, early-scattered degeneration/necrosis and regeneration of muscle fibers were detected in 10-13-week-old rasH2 mice. The severity of the above muscular lesions was more prominent in older rasH2 mice. These lesions were noted in the type II myofiber dominant muscles (biceps femoris, triceps brachii and gastrocnemial). NADH-TR stain clearly demonstrated a disorganized intermyofibrillar network and necrotic change in muscle fibers. No specific morphological changes, like rod structure or tubular aggregation seen in some types of myopathy, were noted in Gomori trichrome and NADH-TR stains in the rasH2 mouse like in many types of muscular dystrophy. Electronmicroscopically, occasional muscle fiber degeneration/regeneration, invaded phagocytic cells, indistinct Z-band suggesting excessive contraction and dilatation of the sarcoplasmic reticulum were observed. In summary, the skeletal myopathy occurring in rasH2 mice is consistent with muscular dystrophy characterized morphologically by progressive degeneration and regeneration of myofibers. The myopathy is confined to the type II myofiber predominant muscles and is not associated with any pathognomonic lesions. These characteristics will provide us with a useful model for research in muscular dystrophy of diverse myofibers.

One-step RT-PCR without initial RNA isolation sStep for laser-microdissected tissue sample.

One-step RT-PCR procedure without initial RNA extraction step is tested for laser microdissected tissue sample. Unfixed cryosections of liver and kidney tissue of male SD rats were cut using laser microdissection system and directly used as templates for RT-PCR study. To check the sensitivity, 5, 25, 125, and 625 hepatocytes were cut and put in PCR-tube. After DNase treatment and cDNA synthesis with pd(N)6 random primer, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified by 60 thermal cycles. GAPDH-specific bands were observed at as few as 25 hepatocytes. Specificity of this procedure was tested for hepatocytes, renal tubular epithelium and glomerular tissue using albumin PCR primers. Approximately 250 cells were cut and albumin cDNA was amplified as described above. Albumin specific band was observed only in hepatocytes sample. To apply this approach to quantitative PCR, various numbers of hepatocytes were cut and put in 0.2 mL PCR tube. After reverse transcription and 10 cycles of GAPDH cDNA amplification by regular thermal-cycler, PCR solution was transferred to 96-well plate designed for real-time PCR system, and further 40 cycles were performed. As a result, GAPDH cDNAs were successfully amplified with a good correlation between the number of template hepatocytes and the intensity of PCR signal. From these results, we concluded this approach would be very useful for the expression analysis of microdissected pathology samples.

Immunohistochemical localization of transforming growth factor alpha in regenerating rat liver.

Immunohistochemical localization of TGF-alpha and cell proliferation kinetics during liver regeneration after two-thirds partial hepatectomy (PH) were investigated. Twenty-four to 72 hr after PH, appreciable increase in the number of TGF-alpha-positive hepatocytes was observed in zones 1 and 2. At the peak at 36 hr, almost all positive cells were stained in their nuclei. Considerable increase in the BrdU labeling index was observed 24-36 hr after PH with a peak at 24 hr in zones 1 and 2. These results indicated an association between TGF-alpha expression and hepatocyte regeneration. It is suggested that immunohistochemical localization of TGF-alpha may be a useful marker of cell proliferation activity in rat liver.

Skeletal myopathy in transgenic mice carrying human prototype c-Ha-ras gene.

Skeletal myopathy was found in almost all-transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mouse). Microscopically, variation of the muscle fiber size, centrally placed nuclei, regenerating fibers, and interstitial fibrosis were evident; hyalinization and necrosis were sometimes observed in the skeletal muscle (femoralis and pectoralis) of the rasH2 mice. Inflammatory changes in the skeletal muscle or abnormality of adjacent peripheral nerve were not observed. The features were essentially similar to those of muscular dystrophy. Although the severity was relatively mild compared to 34-week-old rasH2 mice, the skeletal myopathy was also observed in younger male (10 weeks of age) rasH2 mice. In nontransgenic littermates, skeletal myopathy was not observed. The mRNA of human c-Ha-ras product was detected in femoral muscle from the rasH2 mice by RT-PCR. In conclusion, these data suggest that skeletal myopathy is occurring in almost all rasH2 mice. Integration of c-Ha-ras gene is thought to be crucial to pathogenesis of skeletal myopathy in the rasH2 mice. Further characterization of the muscular lesion and its pathogenesis are needed to explore the possibility of rasH2 mouse becoming a new model for muscular dystrophy.