Katsuhiro Isoda

Silica nanoparticles as hepatotoxicants.

Nano-size materials are increasingly used in cosmetics, diagnosis, imaging and drug delivery, but the toxicity of the nano-size materials has never been fully investigated. Here, we investigated the relationship between particle size and toxicity using silica particles with diameters of 70, 300 and 1000 nm (SP70, SP300, and SP1000) as a model material. To evaluate acute toxicity, we first performed histological analysis of liver, spleen, kidney and lung by intravenous administration of silica particles. SP70-induced liver injury at 30 mg/kg body weight, while SP300 or 1000 had no effect even at 100 mg/kg. Administration of SP70 dose-dependently increased serum markers of liver injury, serum aminotransferase and inflammatory cytokines. Repeated administration of SP70 twice a week for 4 weeks, even at 10mg/kg, caused hepatic fibrosis. Taken together, nano-size materials may be hepatotoxic, and these findings will be useful for future development in nanotechnology-based drug delivery system.

Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis.

A C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a modulator of claudin-4. We previously found that upon deletion of the C-terminal 16 amino acids, C-CPE lost its ability to modulate claudin-4. Tyrosine residues in the 16 amino acids were involved in the modulation of claudin-4. In the present study, we performed functional domain mapping of the 16-amino acid region of C-CPE by replacing individual amino acids with alanine. To evaluate the ability of the alanine-substituted mutants to interact with claudin-4, we carried out a competition analysis using claudin-4-targeting protein synthesis inhibitory factor. We found that Tyr306Ala, Tyr310Ala, Tyr312Ala, and Leu315Ala mutants had reduced binding to claudin-4 compared to C-CPE. Next, we investigated effects of each alanine-substituted mutant on the TJ-barrier function in Caco-2 monolayer cells. The TJ-disrupting activity of C-CPE was reduced by the Tyr306Ala and Leu315Ala substitutions. Enhancement of rat jejunal absorption was also decreased by each of these mutations. The double mutant Tyr306Ala/Leu315Ala lost the ability to interact with claudin-4, modulate TJ-barrier function, and enhance jejunal absorption. These data indicate that Tyr306 and Leu315 are key residues in the modulation of claudin-4 by C-CPE. This information should be useful for the development of a novel claudin modulator based on C-CPE.

Histological analysis of 70-nm silica particles-induced chronic toxicity in mice.

Nano-sized silica is a promising material for disease diagnosis, cosmetics and drugs. For the successful application of nano-sized material in bioscience, evaluation of nano-sized material toxicity is important. We previously found that nano-sized silica particles with a diameter of 70 nm showed acute liver failure in mice. Here, we performed histological analysis of major organs such as the liver, spleen, lung, kidney, brain and heart in mice, chronically injected with 70-nm silica particles for 4 weeks. Histological analysis revealed hepatic microgranulation and splenic megakaryocyte accumulation in these 70-nm silica particles treated mice, while the kidney, lung, brain and heart remained unaffected. Thus, liver and spleen appear to be the major target organs for toxicity by the chronic administration of the 70-nm silica particles.

Novel Carotenoid-Based Biosensor for Simple Visual Detection of Arsenite: Characterization and Preliminary Evaluation for Environmental Application

ABSTRACT A novel whole-cell arsenite biosensor was developed using the photosynthetic bacterium Rhodopseudomonas palustris no. 7 and characterized. A sensor plasmid containing the operator-promoter region of the ars operon and arsR gene from Escherichia coli and the crtI gene from R. palustris no. 7 was introduced into a blue-green mutant with crtI deleted, R. palustris no. 711. The biosensor changed color in response to arsenite, and the change was obvious to the naked eye after 24 h without further manipulation. Real-time reverse transcription-PCR showed that the crtI mRNA was induced 3-fold at 3 h and 2.5-fold at 6 h after addition of 50 μg/liter arsenite compared with the no-arsenite control, and consistent with this, the relative levels of lycopene and rhodopin also increased compared with the control. Colorimetric analysis of the bacteria showed that the hue angle had clearly shifted from green-yellow toward red in an arsenic dose-dependent manner at 24 h after arsenite addition. This obvious shift occurred irrespective of the culture conditions before arsenite was added, indicating that the color change of the biosensor is stable in water samples containing various concentrations of dissolved oxygen. Finally, assays using samples prepared in various types of mineral water indicated that this biosensor could be used to screen groundwater samples for the presence of arsenite in a variety of locations, even where electricity is not available.

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis.

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.

Dendrimer-Immobilized Culture Surface as a Tool to Evaluate Formation of Cellular Cytoskeleton of Anchorage-Dependent Cells

Anchorage-dependent cultivation of human epithelial and keratinocyte cells was carried out on surfaces modified with synthesized dendrimers. Notable elongation of the epithelial cells was recognized on the culture surface immobilized with a dendrimer having D-glucose as a functional ligand, but not when a dendrimer having L-glucose was used or when the dendrimer was ligand-free. This morphological change was attributable to a temporary grasping of the cells at the D-glucose moiety via a glucose transporter-mediated mechanism present in the cell membrane. Following visualization of the actin filaments of the cells, it was considered that the cellular elongation on the D-glucose-bound dendrimer surface reflected the degree of formation of the cellular cytoskeleton. The cellular roundness was calculated by means of image analysis of the individual cells and employed as a parameter to evaluate the formation of the cellular cytoskeleton. In the culture of keratinocytes on the D-glucose-bound dendrimer surface, it was demonstrated that the decrease in the ratio of elongated cells (i.e., cells with a low roundness value) was correlated with the deterioration in the growth potential associated with cellular senescence.

Evaluation of promoter strength in mouse and rat primary hepatocytes using adenovirus vectors

Primary cultured hepatocytes are widely used in the studies of basic and clinical hepatology. Finding an efficient method for gene transfer into primary hepatocytes will be an important advance for these studies. In the present study, we evaluated the activity of an adenovirus vector including promoters for the Rous sarcoma virus (RSV), elongation factor 1alpha, and cytomegalovirus (CMV) as well as the beta-actin promoter/CMV enhancer (CA) using beta-galactosidase as a reporter gene. Although RSV and elongation factor 1alpha promoters had low transcriptional activity in hepatocytes, the CA and CMV promoters had high activity. The CA promoter was the most active, mediating 50.3- and 204.4-fold more activity than the RSV promoter in mouse and rat hepatocytes, respectively. Dose-response studies revealed that transgene activity can be controlled by as much as 1000-fold, by selection of the promoter and the number of infectious particles per cell. These findings should help in the construction of adenovirus vectors for expressing genes of interest in rodent primary cultured hepatocytes.