The present study was designed to compare the susceptibility to carbon tetrachloride (CCl₄)-induced hepatotoxicities of the different liver lobes of rats which had been subjected to pretreatment with phenobarbital sodium (PB) or β-naphthoflavone (BNF). A detailed investigation was done to determine whether the susceptibility to CCl₄ hepatotoxicities was correlated with hepatic cytochrome P450 (CYP) activities in the different liver lobes. Male Wistar rats were given a single oral administration of CCl₄ (0.1 mL/kg, dissolved in liquid paraffin) or vehicle alone following three daily intraperitoneal injections of PB (40 mg/kg, dissolved in sterile saline) or BNF (40 mg/kg, suspended in sesame oil). One day after the CCl₄ administration, all animals were necropsied, and liver samples were obtained from the left and median lobes. Each sample was used for histopathological examination and 7-alkoxycoumarin O-dealkylase activity assay to evaluate the CYP activity. All the CCl₄-treated animals revealed typical liver damage, such as the presence of ballooning degeneration and necrosis in centrilobular hepatocytes. PB pretreatment remarkably enhanced the extent of the liver damage, which was greater in the median lobe than the left lobe. BNF pretreatment only slightly enhanced the extent of the liver damage, which was also greater in the median lobe. These results indicate the different susceptibilities of rat liver lobes to CCl₄-induced hepatotoxicities enhanced by pretreatment with PB or BNF. Moreover, the heterogeneity of the CYP activities enhanced by PB pretreatment was closely correlated with the susceptibility to CCl₄-induced hepatotoxicities in the different liver lobes, although that by BNF pretreatment was not.

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Myofibroblasts play important roles in hepatic fibrosis through the production of extracellular matrix. The cells are derived mainly from hepatic stellate cells (HSCs). Immunoexpression of cytoskeletons such as vimentin, desmin, α-smooth muscle action (α-SMA) and glial fibrillary acidic protein (GFAP) was analyzed in myofibroblasts appearing in acute type hepatic fibrosis (AHF; 300 mg/kg, a single injection) and chronic type hepatic fibrosis (CHF; 100 mg/kg, twice a week for 10 weeks), which were both induced in F344 rats by thioacetamide. In AHF, the number of vimentin-and desmin-positive cells showed the highest levels on day 3 after the injection, in conformity with an increased number of α-SMA-positive myofibroblasts, whereas that of GFAP-positive cells reached a plateau as early as day 1. In CHF, α-SMA- and desmin-positive myofibroblasts showed similar kinetics to each other. The numbers gradually increased from weeks 1 to 7 and then expanded quickly at week 10 when pseudolobules were completely developed. On the other hand, the number of vimentin- and GFAP-positive cells in CHF slightly increased at weeks 3 and 4, respectively, and then decreased until week 10. Interestingly, the greatest expression number among these cytoskeletons was vimentin in AHF, whereas it was desmin in CHF. The present study showed that myofibroblasts express various cytoskeletons, and that there is a difference in the expression patterns between AHF and CHF. By RT-PCR, nerve growth factor (NGF) mRNA expression markedly increased with time in both AHF and CHF, suggesting that NGF might participate in development of hepatic fibrosis by regulating HSCs with its receptor.

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The present study was designed to evaluate dopaminergic neuronal loss in the substantia nigra pars compact (SNpc) with immunohistochemical staining. C57BL/6 mice were intraperitoneally injected four times with 15 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), at 2 h intervals on 10 and 21 days, and 6, 12, 24 and 48 weeks of age. Animals were sacrificed 48 hours after the last injection. No change in the number of tyrosine hydroxylase (TH)-positive neurons was observed in 10- and 21-day-old mice after MPTP treatment compared with their corresponding controls. In contrast, MPTP produced a loss of 20.3% of TH-positive neurons in 6 week-old mice, and further decreases with advancing age, i.e., 35.8%, 39.9% and 56.2% TH-positive neuronal loss at 12, 24 and 48 weeks of age, respectively. These results provide evidence of age-related susceptibility of C57BL/6 mice to MPTP using TH immunohistochemistry. However, we failed to observe apoptosis of neurons in SNpc of mice of all ages after a subacute protocol of MPTP treatment (30 mg/kg/day × 5days).

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F₁(AWE × WE) strain of Japanese quail (Corturnix japonica) is produced from a mating between male AWE (albino plumage color) and female WE (wild plumage color) strains of Japanese quail. Male and female offspring exhibit wild and albino plumage colors, respectively, ruled by a criss-cross inheritance. F₁(AWE × WE) eggs received 17 beta-estradiol (E₂) or methyltestosterone (MT) at 0, 20, 200, 2000, and 20000 ng/egg just before incubation. At 16 days of incubation, embryos were subjected to a complete necropsy and their gonads were grossly observed and examined histopathologically. Viabilities of the embryos at 16 days of incubation were not significantly different between the control and all E₂ groups and between the control and the MT 20, 200 and 2000 ng groups, whereas viability of the MT 20000 ng group was significantly lower than that of the control group. Grossly, genetic sex confirmed by plumage colors coincided completely with sex phenotype of the gonads in all embryos of the control and treated groups. Histopathologically, E₂ exposure induced a dose-dependent feminization such as ovotestis of the left testis. No abnormalities were detected in any male embryos of all the MT groups. E₂ and MT exposures induced no noticeable changes in the ovaries of any embryos. The present study suggests that the sex reversal test using F₁(AWE × WE) Japanese quail embryos may be a rapid and cost-effective tool to evaluate screening feminization effects of the estrogenic endocrine disrupters.

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To investigate the mechanism of bone formation by EP4 activation, an osmotic pump was implanted subcutaneously into the backs of rats and an EP4 receptor agonist was administered at a dose of 100 ng/kg/min for up to 28 days. The histology of the femur (including bone marrow) and the serum and/or urinary bone metabolism parameters were examined on Day 0, 1, 3, 5, 7, 14, and Day 28. In EP4 receptor agonist treated-rats, increase of osteoclasts in the metaphysis was observed on Day 1 and the number of osteoblast showed an increase from Day 3. In addition, cancellous bone and endosteal bone formation were observed in the metaphysis and diaphysis from Day 5 and this peaked on Day 28. Serum alkaline phosphatase activity showed a transient decrease on Day 1, but thereafter showed an increase. The Gla-type osteocalcin level showed an increase from Day 1. Moreover, Gla/Glu osteocalcin ratio showed an increase on Day 5. The urinary excretion of deoxypyridinoline increased on Day 3, showed a transient decrease on Day 5, and increased once again from Day 7. These results indicate that EP4 receptor agonist-induced bone formation is related to an increase of osteoclasts at the initial stage and a subsequent increase of osteoblasts.

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Abnormal β-catenin protein accumulation in cytoplasm or nucleus with somatic mutation of β-catenin has been reported to be very frequent in hepatoblastomas (HBs) both in humans and in experimental animals. Recently we noted HBs developing in an evaluation of the carcinogenicity of N, N-dimethylformamide (DMF) in a 104 week mouse inhalation study¹ performed by the Japan Bioassay Research Center. In the present study we evaluated β-catenin mutations in mouse HBs induced by DMF, which is considered to be a non-genotoxic carcinogen. Immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses were performed using formalin-fixed and paraffin-embedded sections. No abnormal β-catenin accumulation or no apparent mutation was detected, and these findings point to functional normality of the Wnt signal pathway in these HBs induced by DMF. Our results raise questions as to the general nature of any relationship between HB and abnormal β-catenin accumulation.

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The rat liver cirrhosis induced by carbon tetrachloride (CCl₄) animal model is widely used in efficacy studies. In the present study, we examined the difference among hepatic lobes of fibrotic lesions in CCl₄-induced liver cirrhosis rat models. Male Wistar rats were given phenobarbital which was mixed into their drinking water. CCl₄ was orally administered to rats weekly for 9 weeks. The total dosage of CCl₄ was 1.48 ml to 1.68 ml per animal. At necropsy, the liver was divided into lobes: left lobe, median lobe, right lobe, caudate lobe, and papillary process, and the weight of each lobe was measured. Hepatic lobes were submitted to histopathological examination, morphometric analysis of fiber composition content and hydroxyproline content. The weight ratio of lobe to whole liver markedly decreased in the median lobe and right lobe, whereas the weight ratio of lobe to whole liver of the left lobe, papillary process, and caudate lobe increased. In the median and right lobes, strong fibrotic lesions and distinct micronodular formation were observed. In the other lobes, only weak fibrotic lesions were observed. Additionally, high values in fiber composition content were observed in the median and right lobes compared with the other lobes. Hydroxyproline was also markedly higher in the median lobe than in the left lobe. These findings suggest that lobular differences in fibrotic lesions are present in CCl₄-induced liver cirrhosis rat models, and that severe fibrotic lesions are induced in the median and right lobes.

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Signs of glaucoma such as greenish eyes were observed clinically in a 9-month-old male beagle dog. Histopathologically, the glaucomatous eye was characterized by goniodysgenesis (the absence of a trabecular meshwork) in the filtration angle. Spheroid bodies suggestive of axonal degeneration were observed in the axons posterior to the lamina cribrosa, although cupping of the optic nerve head was not seen. The diagnosis of early-stage glaucoma due to goniodysgenesis was made based on the animal's clinical course and histopathological findings. In addition, narrowing of the episcleral veins, fluid retention in the ciliary processes, and retinal edema were noted. The above findings support the concept that glaucomatous optic neuropathy may be caused by a reduction in blood flow rather than an increase in intraocular pressure.

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Naturally occurring collagenofibrotic glomerulonephropathy was found in an intact male cynomolgus monkey at 4 years and 5 months of age. Clinical examination showed mild proteinuria and hypoproteinemia, and the kidneys were macroscopically pale and granular on the surface. Histopathology revealed diffuse panglomerular swelling with the accumulation of eosinophilic amorphous material that was negative for amyloid staining and periodic acid-Schiff, but positive for collagen fibers by Mallory-azan staining in the glomerular capillary wall and mesangial area. Immunohistochemistry revealed a strong positive reaction with anti-type III collagen antibody in the glomeruli. The mesangial area was widened, but there was no increase in the number of mesangial cells. No lesion was observed in the efferent/afferent arterioles or other arteries. Mild tubular degeneration was observed in the cortex, occasionally with cholesterin crystals and interstitial lymphocytic infiltration. Electron microscopy revealed various-sized collagen fibrils with typical banding periodicity in the subendothelial space and the mesangial area. The present case offers information on spontaneous and life-threatening primary glomerular fibrosis in a young monkey suggesting an idiopathic familial disease similar to those observed in humans.

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