Yoshiyuki Tago

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.

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.

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.

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

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.

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.

Role of deltaNp63posCD44vpos 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 deltaNp63posCD44vposCK5/6posCC10pos clara cells as key constituents of early precancerous atypical bronchiolar hyperplasia. In addition, deltaNp63posCD44vpos 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 deltaNp63posCD44vpos 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 deltaNp63posCD44vpos 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 deltaNp63posCD44vpos 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.

Abstract 3232: The proteome analysis of mice lung tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have previously shown that lung squamous cell carcinoma (SCC) is induced by 20 weeks treatment with N-nitroso-tris-chloroethylurea (NTCU) The aim of the present study was to investigate the expression patterns of proteins in lung adenocarcinoma (AdCa) and SCC Seven mice were treated with single i p injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2 mg/0 1 ml saline/mouse) on the first experimental day and sacrificed after 55 weeks (AdCa-induced model) Twenty six mice were treated topically with NTCU (0 01 M 75 μl/mouse) twice a week for eighteen weeks and sacrificed after 22 weeks (SCC-induced model) SCC and AdCa samples were collected from each group and proteome analysis was carried out by using QSTAR Elite LC-MS/MS 187 proteins were identified with 95% confidence or higher and quantified with ProteinPilot 2 0 Software Significant overexpression of 52 proteins (e g SP-C, TIM) were detected in lung AdCa compared to normal-appearing tissues and 46 proteins (e g cytokeratins 6A, 5, 19) were detected in SCC Expression patterns of proteins largely differed between AdCa and SCC Furthermore AdCa and SCC are suggested to be derived from type II pneumocytes and clara cell, respectively Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3232.