Misaki Naota

Translocation Pathway of the Intratracheally Instilled C60 Fullerene from the Lung into the Blood Circulation in the Mouse: Possible Association of Diffusion and Caveolae-mediated Pinocytosis

Ultrafine particles are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular diseases. Recently, it has been demonstrated that ultrafine particles (UFPs) are translocated from the lung into the systemic circulation. The exact pathway, however, for the translocation in the lung remains unclear. In this study, we examined the translocation pathway of intratracheally instilled C60 fullerene particles from the lung into the blood circulation in the mouse. Using light microscopy, aggregated particles of fullerene were observed in the capillary lumen in the lung and the pulmonary lymph nodes immediately after instillation. Electron microscopic analysis demonstrated an increased number of pinocytotic vesicles (caveolae) of various sizes in the type 1 alveolar epithelial cells (AEC) and endothelial cells; occasional caveolae containing some particulate substances were observed. In addition, particles of various sizes were observed throughout the structure of the air-blood barrier (ABB). These findings suggest that fullerene particles may pass the ABB by both diffusion and caveolae-mediated pinocytosis, resulting in immediate translocation into the systemic circulation.

Vanadium Toxicity in Mice Possible Impairment of Lipid Metabolism and Mucosal Epithelial Cell Necrosis in the Small Intestine

Because precise information as to the toxicity of vanadium is required for practical use of vanadium compounds as antidiabetic drugs, we examined vanadium toxicity in mice fed normal diet or high-fat diet (C57BL/6N, male, 7 weeks) by oral administration of ammonium metavanadate (AMV) with a maximum dose of 20 mgV/kg/day. Marked lipid accumulation in hepatocytes, renal epithelial cells, and mucosal epithelial cells of the small and large intestines and severe degeneration, necrosis, and loss of mucosal epithelial cells in the small intestine were observed. These pathological changes were more severe in mice fed high-fat diet than mice fed normal diet, and the intensity of the changes increased with increase in the administered dose of AMV. By electron microscopy, the number and size of lipid droplets in hepatocytes were increased. In the small intestine, a TUNEL assay showed a decreased number of positive cells, and positive cells for acrolein immunohistochemistry were observed specifically in the mucosal epithelial cells indicating degeneration and necrosis in the AMV-treated group, suggesting that a possible factor responsible for cell necrosis in the small intestine could be oxidative stress. In conclusion, AMV may impair cellular lipid metabolism, resulting in lipid accumulation, and induce mucosal epithelial cell necrosis in the small intestine.

Pathological Study of Acute Pulmonary Toxicity Induced by Intratracheally Instilled Asian Sand Dust (Kosa)

The objective of this study was to investigate acute lung toxicity caused by Asian sand dust. Simulated Asian sand dust collected from the Tennger desert in China (CJ-2 particles) and Asian sand dust collected from the atmosphere in Japan (Tottori particles) were used. Saline suspensions of 50, 200, 800, and 3,000 µg Asian sand dust were intratracheally instilled to ICR mice. Localized accumulation of the dust particles was observed in the bronchioles and the alveoli of the lung tissues; acute inflammatory changes characterized by infiltration of macrophages and neutrophils were observed around the particles. Degenerated alveolar walls and bronchial epithelial cells, as well as a weakened positive immunolabeling for laminin, were observed to be associated with particle attachment. Positive immunolabelings for interleukin-6, tumor necrosis factor-α inducible nitric oxide synthase, and dimeric copper- and zinc-containing superoxide dismutase were observed mainly in the inflammatory cells in the lesions; these findings were not observed in the controls or in areas lacking lesions. These results suggest that Asian sand dust particles caused damage to the lung tissue through a direct physical effect. In addition, secondary released cytokines and oxidative stress generated in the lesion may be involved in the development of the acute lung toxicity.

Ultrastructural changes of the air–blood barrier in mice after intratracheal instillation of lipopolysaccharide and ultrafine carbon black particles

Epidemiological studies have indicated associations between exposure to increased concentrations of ambient ultrafine particles and adverse health effects especially in susceptible individuals. To ellucidate the mechanisms underlying the findings from epidemiological studies, mice pretreated with lipopolysaccharide (LPS) (acute lung injury model) were intratracheally instilled with ultrafine carbon black particles (UFCB), and the air-blood barrier was observed to examine the translocation pathway of UFCB from the lung into the systemic circulation. In addition, lung toxicity induced by the intratracheal instillation of LPS and UFCB was studied with the use of electron microscope. LPS treatment induced acute inflammatory changes with increased number of activated macrophages and neutrophils in the degenerated alveolar walls. UFCB were demonstrated on or in the denuded basement membrane in the air-blood barrier; these findings were associated with edematous changes and fragmentation of the cytoplasms of alveolar epithelial cell type 1, and the damages of alveolar epithelial cell type 1 were frequently observed in the close vicinity of the clumps of UFCB. These findings suggest that translocation of the exposed ultrafine particles may be enhanced in the lung tissues with acute inflammatory changes.

Caveolae-mediated Endocytosis of Intratracheally Instilled Gold Colloid Nanoparticles at the Air-Blood Barrier in Mice

Endocytosis is the primary mechanism by which nanoparticles are translocated over the alveolar epithelium. The purpose of this study was to elucidate the association between endocytosis and the translocation of nanoparticles at the air–blood barrier (ABB). Gold colloid particles (diameter, 20 nm) were intratracheally instilled into male ICR mice. Fifteen minutes after instillation, localized accumulation of agglomerated gold particles was observed in the cytoplasm of macrophages, on the surface of alveolar epithelial cells (AECs), and in alveoli. Electron microscopy revealed particles in the vesicles of macrophages, on the surface of AECs, and in caveolae-like vesicles in type 1 AECs. Immunohistochemistry demonstrated positive immunolabeling for caveolin-1 in the ABB of untreated lungs as well as lungs treated with gold particles. Double immunofluorescence and immunoelectron microscopy revealed the presence of caveolin-1 in AECs in the untreated lungs. These results suggest that instilled gold colloid particles are internalized into the alveolar epithelium at the ABB by caveolae-mediated endocytosis, which is regarded as a physiological function of AECs.

Pathological study of chronic pulmonary toxicity induced by intratracheally instilled Asian sand dust (Kosa): possible association of fibrosis with the development of granulomatous lesions

INTRODUCTION Exposure to Asian sand dust (ASD) is associated with enhanced pulmonary morbidity and mortality, and the reporting of such cases has rapidly increased in East Asia since 2000. The purpose of the study was to assess chronic lung toxicity induced by ASD. MATERIAL AND METHODS A total of 174 ICR mice were randomly divided into 5 control and 17 exposure groups. Suspensions of low dose (0.2, 0.4 mg) and high dose (3.0 mg) of ASD particles in saline were intratracheally instilled into ICR mice, followed by sacrifice at 24 hours, 1 week, and 1, 2, 3 and 4 months after instillation. Paraffin sections of lung tissues were stained with hematoxylin and eosin and by immunohistochemistry to detect α-smooth muscle actin, collagen III, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), CD3, CD20, immunoglobulin G, interleukin-1β and inducible nitric oxide synthase. RESULTS A lung histological examination revealed similar patterns in the lesions of the groups treated with high (3.0 mg) or low dose (0.4 mg) of ASD. Acute inflammation was observed 24 h after treatment and subsided after 1 week; persistent granulomatous changes were observed at 2 months, focal lymphocytic infiltration at 3 months, and granuloma formation at 4 months. An increase in the size of granulomatous lesions was observed over time and was accompanied by collagen deposition in the lesions. The cytoplasm of macrophages in inflammatory lesions showed positive immunolabeling for MMP-9 at 24 h, 1 and 2 months after instillation of 3.0 mg of ASD. Positive immunolabeling for TIMP-1 was demonstrated in the cytoplasm of macrophages at 2 and 4 months after instillation of 3.0 mg of ASD. These findings suggest association between the expression of MMP-9 and TIMP-1 with the development of lung granulomatous lesions. CONCLUSIONS These findings suggest that collagen deposition resulting from the altered regulation of extracellular matrix is associated with granuloma formation in the lungs of mice treated with ASD.

Specific pathologist responses for Standard for Exchange of Nonclinical Data (SEND)

The Standard for Exchange of Nonclinical Data (SEND), introduced by the US Food and Drug Administration (FDA), is a scheme for the computerization, electronic application, and screening of preclinical data. Since its establishment, related organizations have been working together to implement SEND. However, it is difficult for individual pharmaceutical companies that often outsource to achieve complete compliance with SEND; hence, the cooperation of contract research organizations (CROs) and SEND Registered Solution Providers (RSPs) is indispensable. In SEND, most data, including those on pathology findings, are converted into controlled terminology (CT), but it is not a simple process to convert findings or levels of severity in the field of pathology, which is a descriptive science. The authors have successfully completed an FDA trial submission for a toxicology test conducted at a CRO and in doing so acquired important knowledge. This article presents a clear picture of such important knowledge from a pathologist’s viewpoint.