Masami Kawagoe

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells.

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against alpha-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.

Urinary copper excretion in type 2 diabetic patients with nephropathy.

Background/Aims: The aim of this study was to examine the relationship between the degree of urinary copper excretion and stages of diabetic nephropathy. Methods: Copper, ceruloplasmin and albumin concentrations were measured in serum and urine samples from 41 type 2 diabetic outpatients with different stages of nephropathy and from 10 healthy controls. The copper/albumin and copper/ceruloplasmin ratios in serum and urine were determined. Furthermore, we examined whether free copper ions are dissociated from ceruloplasmin under various pH conditions. Results: Urinary copper concentrations significantly increased only in macroalbuminuric patients. The copper/ceruloplasmin and copper/albumin ratios in urine were consistently greater than those in serum which were not different between patients and healthy controls except the copper/albumin ratio in macroalbuminuric patients. The ratios in urine decreased in parallel with the progression of nephropathy. Copper was found to be released from ceruloplasmin under acidic conditions. Conclusion: Urinary copper excretion in healthy controls may be the result of dissociation from the albumin-copper complex of serum during its passage through the kidney. In diabetic patients with advanced nephropathy, urinary copper excretion may be due to dissociations from both copper-albumin and ceruloplasmin-copper complexes filtered through the damaged glomerulus. Overloading of urinary copper to damaged renal tubules may play some roles in the progression of nephropathy in patients with advanced nephropathy.

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.

Application of spontaneously immortalized odontoblast cells in tooth regeneration

Here, we report on the first attempt to bioengineer tooth using a spontaneously immortalized mesenchymal cell line. To assess the odontogenic potential of this cell line, odontoblast-lineage cells (OLC) were re-associated with competent dental epithelium isolated from E14.5 mice. A novel three-dimensional organ germ culture method was applied to nurture the constructs in vitro. Additionally, recombinants were transplanted under the kidney capsule in host animals for 2 weeks. Transplants developed into tooth tissues in one-third of the cases. OLC-derived GFP-positive cells could be identified in mineralizing tooth germs by immunohistochemistry. OLCs were capable of intercellular and cell-matrix communication, thus they eventually differentiated into functional odontoblasts. In summary, we managed to utilize OLCs for dental mesenchyme substitution in tooth regeneration experiments. Therefore, our spontaneously transformed cell line proved its potential for future complex, tooth developmental and bioengineering studies.

Styrene monomer primarily induces CYP2B1 mRNA in rat liver

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600 mg kg−1 styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.

Expression and localization of regenerating gene I in a rat liver regeneration model.

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.