A chronic toxicity study of diphenylarsinic acid in the drinking water of C57BL/6J mice for 52 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping. The purpose of the present study was to evaluate the potential toxicity of DPAA when administered to mice in their drinking water for 52 weeks. DPAA was administered to mice at concentrations of 0, 6.25, 12.5, and 25 ppm in their drinking water for 52 weeks. There were no significant differences in final body weights between the control groups and the DPAA treatment groups in male or female mice. Relative liver weights were significantly increased in males treated with 25 ppm DPAA, and absolute liver weights were significantly decreased in female mice treated with 25 ppm DPAA. In female mice, cholangitis and simple bile duct hyperplasia were observed in the 12.5 and 25 ppm DPAA groups, and focal necrosis of hepatocytes was observed in the 25 ppm DPAA group. Proteomic analysis and Ingenuity Pathway Analysis identified 18 proteins related to hepatotoxicity that were overexpressed in the female 25 ppm group. The phase I metabolic enzyme CYP2E1 was one of these overexpressed proteins. Immunostaining confirmed high expression of CYP2E1 in the livers of females in the 25 ppm group. These results suggest that DPAA is toxic to the intrahepatic bile duct epithelium and hepatocytes in female mice and that CYP2E1 might be involved in DPAA-associated toxicity. The no-observed-adverse-effect levels of DPAA were 12.5 ppm (1.6 mg/kg bw/day) for males and 6.25 ppm (1.1 mg/kg bw/day) for females under the conditions of this study.

Chronic dietary toxicity and carcinogenicity studies of dammar resin in F344 rats.

Dammar resin is a natural food additive and flavoring substance present in many foods and drinks. The present study evaluates the chronic toxicity and carcinogenicity of dietary dammar resin in F344 rats. Dietary concentrations in the 52-week chronic toxicity study were 0, 0.03, 0.125, 0.5, or 2%. The major treatment-related deleterious effects were body weight suppression, increased relative liver weight, and low hemoglobin levels in males and females. Foci of cellular alteration in the liver were observed in the male 2% group, but not in any other group. The no-observed-adverse-effect level for chronic toxicity was 0.125% for males (200.4 mg/kg b.w./day) and females (241.9 mg/kg b.w./day). Dietary concentrations in the 104-week carcinogenicity study were 0, 0.03, 0.5, or 2%. Dammar resin induced hemorrhagic diathesis in males and females, possibly via the inhibition of extrinsic and intrinsic coagulation pathways. Incidences of hepatocellular adenomas and carcinomas were significantly increased in the male 2% group, but not in any other group. In the 4-week subacute toxicity study, the livers of male rat-fed diet-containing 2% dammar resin had increased levels of protein oxidation and increased the expression of two anti-apoptotic and seven cytochrome P450 (CYP) genes. There was also an increased tendency of oxidative DNA damage. These findings demonstrate that dammar resin is hepatocarcinogenic in male F344 rats and underlines the roles of inhibition of apoptosis, induction of CYP enzymes, and oxidative stress in dammar resin-induced hepatocarcinogenesis.

Role of deltaNp63(pos)CD44v(pos) cells in the development of N-nitroso-tris-chloroethylurea-induced peripheral-type mouse lung squamous cell carcinomas.

The role of cells expressing stem cell markers deltaNp63 and CD44v has not yet been elucidated in peripheral-type lung squamous cell carcinoma (pLSCC) carcinogenesis. Female A/J mice were painted topically with N-nitroso-tris-chloroethylurea (NTCU) for induction of pLSCC, and the histopathological and molecular characteristics of NTCU-induced lung lesions were examined. Histopathologically, we found atypical bronchiolar hyperplasia, squamous metaplasia, squamous dysplasia, and pLSCCs in the treated mice. Furthermore, we identified deltaNp63(pos)CD44v(pos)CK5/6(pos)CC10(pos) clara cells as key constituents of early precancerous atypical bronchiolar hyperplasia. In addition, deltaNp63(pos)CD44v(pos) cells existed throughout the atypical bronchiolar hyperplasias, squamous metaplasias, squamous dysplasias, and pLSCCs. Overall, our findings suggest that NTCU induces pLSCC through an atypical bronchiolar hyperplasia-metaplasia-dysplasia-SCC sequence in mouse lung bronchioles. Notably, Ki67-positive deltaNp63(pos)CD44v(pos) cancer cells, cancer cells overexpressing phosphorylated epidermal growth factor receptor and signal transducer and activator of transcription 3, and tumor-associated macrophages were all present in far greater numbers in the peripheral area of the pLSCCs compared with the central area. These findings suggest that deltaNp63(pos)CD44v(pos) clara cells in mouse lung bronchioles might be the origin of the NTCU-induced pLSCCs. Our findings also suggest that tumor-associated macrophages may contribute to creating a tumor microenvironment in the peripheral area of pLSCCs that allows deltaNp63(pos)CD44v(pos) cancer cell expansion through activation of epidermal growth factor receptor signaling, and that exerts an immunosuppressive effect through activation of signal transducer and activator of transcription 3 signaling.

Novel medium-term carcinogenesis model for lung squamous cell carcinoma induced by N-nitroso-tris-chloroethylurea in mice.

Targeted treatments for lung cancer based on pathological diagnoses are required to enhance therapeutic efficacy. There are few well-established animal models for lung squamous cell carcinoma although several highly reproducible mouse models for lung adenoma and adenocarcinoma are available. This study was carried out to establish a new lung squamous cell carcinoma mouse model. In the first experiment, female A/J mice were painted topically on back skin twice weekly with 75 µL 0.013 M N-nitroso-tris-chloroethylurea for 2, 4, and 8 weeks (n = 15-20 per group) as initiation of lung lesions, and surviving mice were killed at 18 weeks. In the second experiment, mice were treated as above for 4 weeks and killed at 6, 12, or 18 weeks (n = 3 per group). Lung lobes were subjected to histopathological, immunohistochemical, immunoblotting, and ultrastructural analyses. In the case of treatment for 2, 4, and 8 weeks, incidences of lung squamous cell carcinoma were 25, 54, and 71%, respectively. Cytokeratin 5/6 and epidermal growth factor receptor were clearly expressed in dysplasia and squamous cell carcinoma. Desmosomes and tonofilaments developed in the squamous cell carcinoma. Considering the carcinogenesis model, we conclude that 2 or 4 weeks of N-nitroso-tris-chloroethylurea treatment may be suitable for investigating new chemicals for promotional or suppressive effects on lung squamous cell carcinoma.

Diphenylarsinic acid, a chemical warfare-related neurotoxicant, promotes liver carcinogenesis via activation of aryl hydrocarbon receptor signaling and consequent induction of oxidative DNA damage in rats.

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies.

Fabrication of contrast agents for magnetic resonance imaging from polymer-brush-afforded iron oxide magnetic nanoparticles prepared by surface-initiated living radical polymerization.

The aim of this study is to fabricate a contrast agent for magnetic resonance imaging (MRI) by using hybrid particles composed of a core of iron oxide magnetite (Fe3O4) nanoparticles and a shell of hydrophilic polymer brush synthesized by surface-initiated (SI) living radical polymerization. To achieve this, Fe3O4 nanoparticles were surface-modified with initiating groups for atom transfer radical polymerization (ATRP) via a ligand-exchange reaction in the presence of a triethoxysilane derivative having an ATRP initiation site. The ATRP-initiator-functionalized Fe3O4 nanoparticles were used for performing the SI-ATRP of methyl methacrylate to demonstrate the ability of the synthesized nanoparticles to produce well-defined polymer brushes on their surfaces. The polymerization proceeded in a living fashion so as to produce graft polymers with targeted molecular weights and narrow molecular weight distribution. The average graft density was estimated to be as high as 0.7 chains/nm(2), which indicates the formation of so-called concentrated polymer brushes on the Fe3O4 nanoparticles. Dynamic light scattering and transmission electron microscope observations of the hybrid nanoparticles revealed their uniformity and dispersibility in solvents to be excellent. A similar polymerization process was conducted using a hydrophilic monomer, poly(ethylene glycol) methyl ether methacrylate (PEGMA), to prepare Fe3O4 nanoparticles grafted with poly(PEGMA) brushes. The resultant hybrid nanoparticles showed excellent dispersibility in aqueous media including physiological conditions without causing any aggregations. The blood clearance and biodistribution of the hybrid particles were investigated by intravenously injecting particles labeled with a radio isotope, (125)I, into mice. It was found that some hybrid particles exhibited an excellently prolonged circulation lifetime in the blood with a half-life of about 24 h. When such hybrid particles were injected intravenously into a tumor-bearing mouse, they preferentially accumulated in the tumor tissues owing to the so-called enhanced permeability and retention effect. The tumor-targeted delivery was visualized by a T2-enhaced MRI measurement.

Human amnion-derived mesenchymal stem cells attenuate xenogeneic graft-versus-host disease by preventing T cell activation and proliferation.

Acute graft-versus-host disease (GVHD) is characterized by severe tissue damage that is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been increasingly examined for the treatment of immune-related diseases. We aimed to assess the immunosuppressive effects of human amnion-derived MSC (AMSC) in a xenogeneic GVHD NOD/Shi-scid IL2rγnull mouse model using human peripheral blood mononuclear cells (PBMC). Additionally, we used human bone marrow-derived MSC (BMSC) as comparative controls to determine differences in immunomodulatory functions depending on the MSC origin. Administration of AMSC significantly prolonged survival, and reduced human tumor necrosis factor-α (TNF-α) concentration and percentage of programmed cell death protein-1 receptor (PD-1)+CD8+ T cell populations compared with in GVHD control mice. Furthermore, colonic inflammation score and percentage of human CD8+ T cell populations in AMSC-treated mice were significantly lower than in GVHD control and BMSC-treated mice. Interestingly, gene expression and protein secretion of the PD-1 ligands were higher in AMSC than in BMSC. These findings are the first to demonstrate that AMSC exhibit marked immunosuppression and delay acute GVHD progression by preventing T cell activation and proliferation via the PD-1 pathway.

On-site fabrication of Bi-layered adhesive mesenchymal stromal cell-dressings for the treatment of heart failure.

Mesenchymal stromal/stem cell (MSC)-based therapy is a promising approach for the treatment of heart failure. However, current MSC-delivery methods result in poor donor cell engraftment, limiting the therapeutic efficacy. To address this issue, we introduce here a novel technique, epicardial placement of bi-layered, adhesive dressings incorporating MSCs (MSC-dressing), which can be easily fabricated from a fibrin sealant film and MSC suspension at the site of treatment. The inner layer of the MSC dressing, an MSC-fibrin complex, promptly and firmly adheres to the heart surface without sutures or extra glues. We revealed that fibrin improves the potential of integrated MSCs through amplifying their tissue-repair abilities and activating the Akt/PI3K self-protection pathway. Outer collagen-sheets protect the MSC-fibrin complex from abrasion by surrounding tissues and also facilitates easy handling. As such, the MSC-dressing technique not only improves initial retention and subsequent maintenance of donor MSCs but also augment MSC's reparative functions. As a result, this technique results in enhanced cardiac function recovery with improved myocardial tissue repair in a rat ischemic cardiomyopathy model, compared to the current method. Dose-dependent therapeutic effects by this therapy is also exhibited. This user-friendly, highly-effective bioengineering technique will contribute to future success of MSC-based therapy.

Polymer-brush-afforded SPIO Nanoparticles Show a Unique Biodistribution and MR Imaging Contrast in Mouse Organs.

INTRODUCTION: To investigate the biodistribution and retention properties of the new super paramagnetic iron oxide (new SPIO: mean hydrodynamic diameter, 100 nm) nanoparticles, which have concentrated polymer brushes in the outer shell and are difficult for phagocytes to absorb, and to compare the new SPIO with clinically approved SPIO (Resovist: mean hydrodynamic diameter, 57 nm). MATERIALS AND METHODS: 16 male C57BL/6N mice were divided in two groups according to the administered SPIO (n = 8 for each group; intravenous injection does, 0.1 ml). In vivo magnetic resonance imaging (MRI) was performed before and one hour, one day, one week and four weeks after SPIO administration by two dimensional-the fast low angle shot (2D-FLASH) sequence at 11.7T. Ex vivo high-resolution images of fixed organs were also obtained by (2D-FLASH). After the ex vivo MRI, organs were sectioned and evaluated histologically to confirm the biodistribution of each particle precisely. RESULTS: The new SPIO was taken up in small amounts by liver Kupffer cells and showed a unique in vivo MRI contrast pattern in the kidneys, where the signal intensity decreased substantially in the boundaries between cortex and outer medulla and between outer and inner medulla. We found many round dark spots in the cortex by ex vivo MRI in both groups. Resovist could be detected almost in the cortex. The shapes of the dark spots were similar to those observed in the new SPIO group. Transmission electron microscopy revealed that Resovist and the new SPIO accumulated in different cells of glomeruli, that is, endothelial and mesangial cells, respectively. CONCLUSION: The new SPIO was taken up in small amounts by liver tissue and showed a unique MRI contrast pattern in the kidney. The SPIO were found in the mesangial cells of renal corpuscles. Our results indicate that the new SPIO may be potentially be used as a new contrast agent for evaluation of kidney function as well as immunune function.

Pueraria mirifica Exerts Estrogenic Effects in the Mammary Gland and Uterus and Promotes Mammary Carcinogenesis in Donryu Rats.

Pueraria mirifica (PM), a plant whose dried and powdered tuberous roots are now widely used in rejuvenating preparations to promote youthfulness in both men and women, may have major estrogenic influence. In this study, we investigated modifying effects of PM at various doses on mammary and endometrial carcinogenesis in female Donryu rats. Firstly, PM administered to ovariectomized animals at doses of 0.03%, 0.3%, and 3% in a phytoestrogen-low diet for 2 weeks caused significant increase in uterus weight. Secondly, a 4 week PM application to non-operated rats at a dose of 3% after 7,12-dimethylbenz[a]anthracene (DMBA) initiation resulted in significant elevation of cell proliferation in the mammary glands. In a third experiment, postpubertal administration of 0.3% (200 mg/kg body weight (b.w.)/day) PM to 5-week-old non-operated animals for 36 weeks following initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), respectively, resulted in significant increase of mammary adenocarcinoma incidence. A significant increase of endometrial atypical hyperplasia multiplicity was also observed. Furthermore, PM at doses of 0.3%, and more pronouncedly, at 1% induced dilatation, hemorrhage and inflammation of the uterine wall. In conclusion, postpubertal long-term PM administration to Donryu rats exerts estrogenic effects in the mammary gland and uterus, and at a dose of 200 mg/kg b.w./day was found to promote mammary carcinogenesis initiated by DMBA.

Genotoxicity and subacute toxicity studies of a new astaxanthin-containing Phaffia rhodozyma extract.

Experimental and clinical studies demonstrate that astaxanthin (AXN), a xanthophyll carotenoid, has protective effects against oxidative damage. Because most of these studies assessed AXN derived from Haematococcus pluvialis that were cultivated at industrial scales, few studies have examined the toxicity of AXN derived from Phaffia rhodozyma. To evaluate the safety of astaxanthin-containing P. rhodozymaextract (AXN-PRE), genotoxicity was assessed in bacterial reverse mutation test and mouse bone marrow micronucleus test, and general toxicity was assessed in 4-week repeated oral toxicity study in rats. AXN-PRE did not induce reverse mutations in the Salmonella typhimurium strains TA98 or TA100 at concentrations of 5,000 µg/plate with or without S9 mix, and no chromosome damage was observed at a dose of 2,000 mg/kg in mouse micronucleus test. In the subacute toxicity study, male and female Sprague-Dawley rats were given AXN-PRE at doses of 0, 500, and 1,000 mg/kg by gavage for 4 weeks. Body weights, urinalysis, hematology, serum biochemistry, organ weights, or histopathological lesions indicated no distinct toxicity. In conclusion, AXN-PRE had no effect in bacterial reverse mutation test and mouse bone marrow micronucleus test. The no-observed-adverse-effect level for AXN-PRE in 4-week repeated oral toxicity study in rats was determined to be greater than 1,000 mg/kg (corresponding to dose of 50 mg/kg AXN) regardless of gender.

From cartoon to real time MRI: in vivo monitoring of phagocyte migration in mouse brain.

Recent studies have demonstrated that immune cells play an important role in the pathogenesis of many neurological conditions. Immune cells constantly survey the brain microvasculature for irregularities in levels of factors that signal homeostasis. Immune responses are initiated when necessary, resulting in mobilisation of the microglial cells resident in the central nervous system (CNS) and/or of infiltrating peripheral cells. However, little is known about the kinetics of immune cells in healthy and diseased CNS, because it is difficult to perform long-term visualisation of cell motility in live tissue with minimal invasion. Here, we describe highly sensitive in vivo MRI techniques for sequential monitoring of cell migration in the CNS at the single-cell level. We show that MRI combined with intravenous administration of super-paramagnetic particles of iron oxide (SPIO) can be used to monitor the transmigration of peripheral phagocytes into healthy or LPS-treated mouse brains. We also demonstrate dynamic cell migration in live animal brains with time-lapse MRI videos. Time-lapse MRI was used to visualise and track cells with low motility in a control mouse brain. High-sensitivity MRI cell tracking using SPIO offers new insights into immune cell kinetics in the brain and the mechanisms of CNS homeostasis.

Hormonally active doses of isoflavone aglycones promote mammary and endometrial carcinogenesis and alter the molecular tumor environment in Donryu rats.

Our research is focused on modifying effects of an isoflavone aglycones (IAs)-rich extract at a hormonally active dose of 150 mg/kg body weight/day on mammary and endometrial carcinogenesis in female Donryu rats. IA administered for 2 weeks in a phytoestrogen-low diet exerted estrogenic activity and induced cell proliferation in the uterus of ovariectomized rats. Furthermore, administration for 4 weeks resulted in elevation of cell proliferation in the mammary glands of 7,12-dimethylbenz[a]anthracene (DMBA)-treated animals. Forty weeks of postpubertal administration of IA to 5-week-old rats after initiation of mammary and endometrial carcinogenesis with DMBA and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) caused significant increase of incidence and multiplicity of mammary adenocarcinoma, multiplicities of endometrial atypical hyperplasia, adenomatous polyps, and an increased trend of uterine adenocarcinomas. Liquid chromatography with tandem mass spectrometry and immunohistochemical analyses revealed significant elevation of tumorigenesis-related proteins such as S100 calcium-binding protein A8, kininogen 1, and annexins 1 and 2 in mammary adenocarcinomas and cadherin EGF LAG seven-pass G-type receptor 2, DEAD box polypeptide 1, and cysteine- and glycine-rich protein 1 in uterine proliferative lesions of IA-treated animals. Those changes are likely to be related to modulation of estrogen receptor (ER), AP1, nuclear factor-kappa B, and actin signaling pathways. Our results indicate that the postpubertal exposure of Donryu rats to IA at an estrogenic dose results in promotion of mammary and uterine carcinogenesis induced by DMBA and ENNG, which might be related to the activation of ER-dependent signaling and alteration of the molecular tumor environment in the mammary gland and endometrium.

Evaluation of the Subchronic Toxicity of Dietary Administered Equisetum arvense in F344 Rats.

Equisetum arvense, commonly known as the field horsetail, has potential as a new functional food ingredient. However, little information is available on its side effects, and the general toxicity of Equisetum arvense has yet to be examined in detail. In the present study, we evaluated the influence of administration in diet at doses of 0, 0.3, 1 and 3% for 13 weeks in male and female F344 rats. No toxicity was detected with reference to clinical signs, body weight, urinalysis, hematology and serum biochemistry data and organ weights. Microscopic examination revealed no histopathological lesions associated with treatment. In conclusion, the no-observed-adverse-effect level (NOAEL) for Equisetum arvense was determined to be greater than 3% in both sexes of F344 rat (males and females: >1.79 g/kg BW/day and >1.85 g/kg BW/day, respectively) under the conditions of the present study.