Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model.

Bifidobacterium is a nonpathogenic strain of anaerobic bacteria that selectively localizes and proliferates in tumors. It has emerged as a specific carrier of anticancer proteins against malignant tumors. Claudins are tetraspanin transmembrane proteins that form tight junctions. Claudin-4 is overexpressed in certain epithelial malignant cancers. The C-terminal fragment of the Clostridium perfringens enterotoxin (C-CPE), an exotoxin without the cytotoxic domain, strongly binds to claudin-4. The C-CPE fusion toxin (C-CPE-PE23), which targets claudin-4, strongly suppresses tumor growth; however, C-CPE fusion toxins exhibit hepatic toxicity. In this study, we successfully generated a strain of Bifidobacterium longum that secreted C-CPE-PE23 (B. longum-C-CPE-PE23) and was specific to and cross reactive with human and mouse claudin-4. We evaluated the therapeutic potential of this strain against triple-negative breast cancer using a mouse model. C-CPE-PE23 decreased cell viability in a dose-dependent manner in human and mouse breast cancer cell lines. After intravenous injection, Bifidobacterium was specifically distributed in the tumors of mice bearing breast cancer tumors. Moreover, B. longum-C-CPE-PE23 significantly suppressed tumor growth in mice with breast cancer without serious side effects, such as weight loss or hepatic and renal damage. We suggest that B. longum-C-CPE-PE23 is a good candidate for breast cancer treatment. Bifidobacterium could also be used as a drug delivery system for hepatotoxic agents.

Toxicity of Gold Nanoparticles in Mice due to Nanoparticle/Drug Interaction Induces Acute Kidney Damage.

Nanomaterials are innovative materials with many useful properties, but there is concern regarding their many unknown effects on living organisms. Gold nanoparticles are widely used as industrial materials because of their excellent properties. The potential biological hazards of gold nanoparticles are unknown, and thus, here we examined the in vivo effects of gold nanoparticles 10, 50, and 100 nm in diameter (GnP10, GnP50, and GnP100, respectively) and their interactions with drugs in mice to clarify their safety in mammals. Cisplatin, paraquat, and 5-aminosalicylic acid cause side-effect damage to the liver and kidney in mice. No hepatotoxicity or nephrotoxicity was observed when any of the gold nanoparticles alone were administered via the tail vein. In contrast, co-administration of GnP-10 with cisplatin, paraquat, or 5-aminosalicylic acid caused side-effect damage to the kidney. This suggests that gold nanoparticles with a particle size of 10 nm are potentially nephrotoxic due to their interaction with drugs.

Hepatotoxicity and Drug/Chemical Interaction Toxicity of Nanoclay Particles in Mice.

Nanomaterials are relatively new and unconventional materials with many useful properties, but their effects on biological systems are poorly understood. Nanoclay is a general term for layered mineral silicate nanoparticles that are ideally suited for use in clay-based nanocomposites. The potential biological hazards of nanoclays have not been addressed, however. Therefore, we investigated the in vivo effects and drug interactions of nanoclays. In mice, administration of nanoclay particles via the tail vein led to acute liver injury. Co-administration of nanoclay and carbon tetrachloride, paraquat, or cisplatin resulted in both liver and kidney injury. Our findings thus indicate that nanoclay particles are potentially hepato- and nephrotoxic.

Liver injury induced by thirty- and fifty-nanometer-diameter silica nanoparticles.

Nano-size silica material is a promising reagent for disease diagnosis, cosmetics, and the food industry. For the successful application of nanoparticle materials in bioscience, evaluation of nano-size material toxicity is important. We previously found that nano-size silica particles caused acute liver failure in mice. However, the hepatotoxicity of nanosilica particles with the diameter of 70 nm or less is unknown. Here, we investigated the relationship between particle size and toxicity using nanosilica particles with diameters of 30, 50, and 70 nm (SP30, SP50, and SP70, respectively). We observed dose-dependent increases in hepatic injury following administration of SP50 and SP30, with SP30 causing greater acute liver injury than that seen with SP50. Smaller silica nanoparticles induced liver injury even at proportionally lower dose levels. Furthermore, we investigated the combinatorial toxicity of SP30 in the presence of chemically induced liver injury (including that caused by carbon tetrachloride, paraquat, cisplatin, and acetaminophen). We observed that particles of the smallest size tested (SP30) synergized with chemical substances in causing liver injury. These data suggest that the size (diameter) of the silica nanoparticles affects the severity of nanoparticle-induced liver injury, a finding that will be useful for future investigations in nanotechnology and nanotoxicology.

Hepatoprotective effect of syringic acid and vanillic acid on CCl4-induced liver injury.

The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl(4)-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl(4)-induced chronic liver injury in mice. The injection of CCl(4) into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl(4) treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

[Development of novel culture system for regulation of hepatocyte function].

Cultured hepatocytes are expected to be used for drug screening and bioartificial liver. Since hepatocytes lose their functions very rapidly in vitro, many attempts have been made to maintain their viability and functions. First, we want to introduce the surface modification of culture substrate using a starburst dendrimer. Addition of fructose to the terminal of the dendrimer was shown to be effective in maintaining hepatocyte function. As the second topic, we will show results of the use of a three-dimensional carrier for hepatocyte cultivation. Hepatocytes and bone marrow stromal cells were cocultured in silane beads, and packed into a radial flow-type bioreactor. The perfusion culture showed the effectiveness of bone marrow stromal cells for the maintenance of hepatocyte function. The next topic will be the trial of adenoviral gene transfer into hepatocytes. Thioredoxin gene was chosen because the products play important roles in redox control and antiapoptosis. The introduction of the gene could inhibit apoptosis and maintain the hepatocyte viability. Finally, we want to introduce the results on differentiation of stem cells into hepatocytes, because it is very difficult to obtain sufficient number of human hepatocytes. Human mesenchymal stem cells were cultured in the presence of several protein factors and the hepatocyte-specific marker was expressed after 2 weeks of induction culture. The use of human stem cells could be an important strategy for the support of a drug development system.

Hepatoprotective effect of syringic acid and vanillic acid on concanavalin a-induced liver injury.

The edible mushroom Lentinula edodes (shiitake) contains many bioactive compounds. In the present study, we cultivated L. edodes mycelia in solid medium and examined the hot-water extract (L.E.M.) for its suppressive effect on concanavalin A (ConA)-induced liver injury in mice. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intraperitoneal administration of L.E.M. significantly decreased the levels of the transaminases. L.E.M. contains many bioactive substances, including polysaccharides and glucan, which could be immunomodulators. Since ConA-induced liver injury is caused by the activation of T cells, immunomodulating substances might be responsible for the suppressive effect of L.E.M. L.E.M. also contains phenolic compounds that are produced from lignocellulose by mycelia-derived enzymes. The major phenolics in L.E.M., syringic acid and vanillic acid, were intraperitoneally injected into mice shortly before the ConA treatment. Similar to L.E.M., the administration of syringic acid or vanillic acid significantly decreased the transaminase activity and suppressed the disorganization of the hepatic sinusoids. In addition, the inflammatory cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6 in the serum increased rapidly, within 3 h of the ConA administration, but the administration of syringic acid or vanillic acid significantly suppressed the cytokine levels. Together, these findings indicate that the phenolic compounds in L.E.M. are hepatoprotective through their suppression of immune-mediated liver inflammation.

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.

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.

Novel carotenoid-based biosensor for simple visual detection of arsenite: characterization and preliminary evaluation for environmental application.

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 microg/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.

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.

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.

Hepatoprotective effect of vitamin B12 on dimethylnitrosamine-induced liver injury.

Vitamin B(12) contains a cobalt complex and accumulates at high levels in the liver. Vitamin B(12) was examined for its hepatoprotective effect on dimethylnitrosamine-induced liver injury in mice. Vitamin B(12) decreased the blood levels of aspartate aminotransferase and alanine aminotransferase, and clearly inhibited the overaccumulation of collagen fibrils. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the liver showed that the gene expression of alpha-smooth muscle actin and heat-shock protein 47, which are markers of fibrosis, were suppressed by vitamin B(12) administration. Our findings indicate that vitamin B(12) could be an effective hepatoprotective agent.

[Application of mesenchymal stem cells to liver regenerative medicine].

Stem cell-based therapy has received attention as a possible alternative to organ transplantation, owing to the ability of stem cells to repopulate and differentiate at the engrafted site. We transplanted bone marrow-derived mesenchymal stem cells (BMSCs) into liver-injured rats to test the therapeutic effect. Rat bone marrow cells were cultured in the presence of hepatocyte growth factor (HGF). RT-PCR and immunocytochemical analysis indicated that the BMSCs expressed the albumin mRNA and the production of protein after cultivation with HGF for 2 weeks. The BMSCs appeared to differentiate into hepatocyte-like cells in response to the culture with HGF. After labeling with a fluorescent marker, the BMSCs were transplanted into CCl(4)-injured rats by injection through the caudal vein. The liver was excised and blood samples were collected 4 weeks later. Engraftment of the transplanted BMSCs was seen with significant fluorescence in the injured liver. Transplantation of the BMSCs into liver-injured rats restored their serum albumin level and suppressed transaminase activity and liver fibrosis. Therefore, BMSCs were shown to have a therapeutic effect on liver injury. Recently, we have been trying to use mesenchymal stem cells isolated from dental papilla of discarded human wisdom teeth. Autologous transplantation of mesenchymal stem cells from bone marrow and dental papilla could be ethically and functionally promising for stem cell-based therapy.

Fucoidan partly prevents CCl4-induced liver fibrosis.

Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl4-induced liver fibrosis. Administration of fucoidan reduced CCl4-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl4 was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl4-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation.

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Protection against CCl4-induced injury in liver by adenovirally introduced thioredoxin gene.

Antioxidation therapy is a promising strategy for treating or preventing oxidative stress-related liver diseases. The human thioredoxin (TRX) gene was inserted into an adenovirus vector (Adv-TRX), which was administered to mice. The mice were treated with 1 ml/kg CCl4 48 h after the infection. Blood samples were taken and the liver was excised 24 h after the CCl4 treatment. Serum ammonia, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined, and liver sections were stained with hematoxylin and eosin. RT-PCR analysis showed that the introduced TRX gene was expressed only in the liver. Adv-TRX decreased the serum ammonia, AST, and ALT levels. Hematoxylin-eosin staining indicated that the CCl4-induced injury was significantly prevented by the Adv-TRX infection. The gene delivery of TRX, which plays a central role in intracellular redox control, was shown to be effective in protecting the liver against oxidative stress-induced injury.

Protection against D-galactosamine-induced acute liver injury by oral administration of extracts from Lentinus edodes mycelia.

The development of oral medications to help prevent liver injury is desirable, and some mushrooms contain chemicals that show promise as such a treatment. Here, we tested whether a hot-water extract (L.E.M.) of the cultured mycelia of an edible mushroom, Lentinus edodes, could protect primary cultured hepatocytes from D-galactosamine (GalN)-induced injury. GalN induced cell death in the hepatocytes, and this effect was completely suppressed by the addition of 0.5 mg/ml L.E.M. Polyphenolic compounds contained in the L.E.M. seemed to be responsible for the protective effect. We next examined the protective effect of L.E.M. in a GalN-induced liver injury model in rats. In rats that had been treated with L.E.M. given orally or intraperitoneally, GalN caused less leakage of aspartate aminotransferase and alanine aminotransferase, markers for liver injury, and a lower decrease in serum protein content, than in non-L.E.M.-treated rats. Histological analysis of the liver also showed a protective effect of L.E.M. Our findings indicate that L.E.M. administration is a promising treatment for protecting the liver from acute injury.

Whole-cell arsenite biosensor using photosynthetic bacterium Rhodovulum sulfidophilum. Rhodovulum sulfidophilum as an arsenite biosensor.

An arsenite biosensor plasmid was constructed in Escherichia coli by inserting the operator/promoter region of the ars operon and the arsR gene from E. coli and the crtA gene, which is responsible for carotenoid synthesis in the photosynthetic bacterium, Rhodovulum sulfidophilum, into the broad-host-range plasmid vector, pRK415. The biosensor plasmid, pSENSE-As, was introduced into a crtA-deleted mutant strain of R. sulfidophilum (CDM2), which is yellow in culture due to its content of spheroiden (SE) and demethylspheroidene (DMSE). CDM2 containing pSENSE-As changed from yellow to red by the addition of arsenite, which caused enzymatic transformation of SE and DMSE to spheroidenone (SO) and demethylspheroidenone (DMSO). Reverse transcriptase PCR analysis showed that the color change depended on transcription of the crtA gene in pSENSE-As. The color change could be clearly recognized with the naked eye at 5 microg/l arsenite. The biosensor strain did not respond to other metals except for bismuth and antimony, which caused significant accumulation of SO and DMSO in the cells at 60 and 600 microg/l, respectively. This biosensor indicates the presence of arsenite with a bacterial color change without the need to add a special reagent or substrate for color development, enabling this pollutant to be monitored in samples by the naked eye in sunlight, even where electricity is not available.

Suppression of cell death in primary rat hepatocytes by alpha1-acid glycoprotein.

In screening for effective additives for the long-term culture of hepatocytes, the hepatoprotective effect of alpha1-acid glycoprotein (AGP) was observed. AGP prevented primary hepatocytes from undergoing cell death induced by the chemical toxin, bromobenzene. Moreover, AGP added to medium was found to maintain the number of viable hepatocytes for as long as 6 d. The hepatoprotective effect of AGP was lost by removing sialic acid groups at the N-glycan chain terminal of AGP. It is shown that the complete form of N-glycan chain is needed for the hepatoprotectivity of AGP.