Akinori Shimada

Demonstration of the Clathrin- and Caveolin-Mediated Endocytosis at the Maternal–Fetal Barrier in Mouse Placenta after Intravenous Administration of Gold Nanoparticles

ABSTRACT Exposure to nanoparticles during pregnancy is a public concern, because nanoparticles may pass from the mother to the fetus across the placenta. The purpose of this study was to determine the possible translocation pathway of gold nanoparticles across the maternal–fetal barrier as well as the toxicity of intravenously administered gold nanoparticles to the placenta and fetus. Pregnant ICR mice were intravenously injected with 0.01% of 20- and 50-nm gold nanoparticle solutions on the 16th and 17th days of gestation. There was no sign of toxic damage to the placentas as well as maternal and fetal organs of the mice treated with 20- and 50-nm gold nanoparticles. ICP-MS analysis demonstrated significant amounts of gold deposited in the maternal livers and placentas, but no detectable level of gold in the fetal organs. However, electron microscopy demonstrated an increase of endocytic vesicles in the cytoplasm of syncytiotrophoblasts and fetal endothelial cells in the maternal–fetal barrier of mice treated with gold nanoparticles. Clathrin immunohistochemistry and immunoblotting showed increased immunoreactivity of clathrin protein in the placental tissues of mice treated with 20- and 50-nm gold nanoparticles; clathrin immunopositivity was observed in syncytiotrophoblasts and fetal endothelial cells. In contrast, caveolin-1 immunopositivity was observed exclusively in the fetal endothelium. These findings suggested that intravenous administration of gold nanoparticles may upregulate clathrin- and caveolin-mediated endocytosis at the maternal–fetal barrier in mouse placenta.

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.

Excitatory amino acid transporter 2 downregulation correlates with thalamic neuronal death following kainic acid-induced status epilepticus in rat.

Recurrent seizures without interictal resumption (status epilepticus) have been reported to induce neuronal death in the midline thalamic region that has functional roles in memory and decision‐making; however, the pathogenesis underlying status epilepticus‐induced thalamic neuronal death is yet to be determined. We performed histological and immunohistochemical studies as well as cerebral blood flow measurement using 4.7 tesla magnetic resonance imaging spectrometer on midline thalamic region in Sprague–Dawley rats (n = 75, male, 7 weeks after birth, body weight 250–300 g) treated with intraperitoneal injection of kainic acid (10 mg/kg) to induce status epilepticus (n = 55) or normal saline solution (n = 20). Histological study using paraffin‐embedded specimens revealed neuronal death showing ischemic‐like changes and Fluoro‐Jade C positivity with calcium deposition in the midline thalamic region of epileptic rats. The distribution of neuronal death was associated with focal loss of immunoreactivity for excitatory amino acid transporter 2 (EAAT2), stronger immunoreaction for glutamate and increase in number of Iba‐1‐positive microglial cells showing swollen cytoplasm and long processes. Double immunofluorescence study demonstrated co‐expression of interleukin‐1 beta (IL‐1β) and inducible nitric oxide synthase (iNOS) within microglial cells, and loss of EAAT2 immunoreactivity in reactive astrocytes. These microglial alterations and astrocytic EAAT2 downregulation were also observed in tissue without obvious neuronal death in kainic acid‐treated rats. These results suggest the possible role of glutamate excitotoxicity in neuronal death in the midline thalamic region following kainic acid‐induced status epilepticus due to astrocytic EAAT2 downregulation following microglial activation showing upregulation of IL‐1β and iNOS.

Comparative Study on the Pathogenesis of the Generated 9a5b Newcastle Disease Virus Mutant Isolate Between Chickens and Waterfowl

Lentogenic Newcastle disease viruses (NDVs), circulating among waterfowl, have the potential to become highly pathogenic by replication in chickens. The pathological studies that compare NDV infections in chickens and waterfowl are rare. The virulent 9a5b mutant NDV isolate was generated by passaging the lentogenic Goose/Alaska/415/91 NDV isolate in chickens. The pathogenesis of the virulent 9a5b mutant isolate is unknown in both chickens and waterfowl. In this study, the virulent 9a5b mutant NDV isolate was inoculated intranasally in 32-day-old specific pathogen-free white Leghorn chickens and Japanese commercial ducks. Unlike ducks, which remained clinically normal throughout the study, chickens had depression, gasping, oral discharges, and greenish-white soft feces. Gross and histologic lesion patterns as well as viral replication supported the differing clinical outcome. Ducks had slight inflammation mainly in respiratory and digestive tracts, whereas slight nonpurulent encephalitis, necrotizing pancreatitis, tubulointerstitial nephritis, and mild inflammation in respiratory and digestive tracts were detected in chickens. In agreement, interferon-beta (IFN-β)–immunopositive signals were more intense in lung tissue of ducks than that of chickens, and NDV replications were detected intensively in chicken tissues. These results suggest that the 9a5b mutant NDV isolate is more virulent in chickens, and slight histological lesions were induced in ducks even with virulent NDVs.

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.

Different Regulation of p53 Expression by Cadmium Exposure in Kidney, Liver, Intestine, Vasculature, and Brain Astrocytes

Chronic exposure to cadmium (Cd) is known to adversely affect renal function. Our previous studies indicated that Cd induces p53-dependent apoptosis by inhibiting gene expression of the ubiquitin-conjugating enzyme (Ube) 2d family in both human and rat proximal tubular cells. In this study, the effects of Cd on protein expression of p53 and apoptotic signals in the kidney and liver of mice exposed to Cd for 12 months were examined, as well as the effects of Cd on p53 protein levels and gene expression of the Ube2d family in various cell lines. Results showed that in the kidney of mice exposed to 300 ppm Cd for 12 months, there was overaccumulation of p53 proteins in addition to the induction of apoptosis, which was triggered specifically in the proximal tubules. Interestingly, the site of apoptosis was the same as that of p53 accumulation in the proximal tubules. In the liver of mice chronically exposed to Cd, gene expression of the Ube2d family tended to be slightly decreased, together with slight apoptosis without the accumulation of p53 protein. In rat small intestine epithelial (IEC-6) cells, Cd decreased not only the p53 protein level but also gene expression of Ube2d1, Ube2d2 and Ube2d4. In human brain microvascular endothelial cells (HBMECs), Cd did not suppress gene expression of the Ube2d family, but increased the p53 protein level. In human brain astrocytes (HBASTs), Cd only increased gene expression of UBE2D3. These results suggest that Cd-induced apoptosis through p53 protein is associated with renal toxicity but not hepatic toxicity, and the modification of p53 protein by Cd may vary depending on cell type.

Accumulation of p53 via down-regulation of UBE2D family genes is a critical pathway for cadmium-induced renal toxicity.

Chronic cadmium (Cd) exposure can induce renal toxicity. In Cd renal toxicity, p53 is thought to be involved. Our previous studies showed that Cd down-regulated gene expression of the UBE2D (ubiquitin-conjugating enzyme E2D) family members. Here, we aimed to define the association between UBE2D family members and p53-dependent apoptosis in human proximal tubular cells (HK-2 cells) treated with Cd. Cd increased intracellular p53 protein levels and decreased UBE2D2 and UBE2D4 gene expression via inhibition of YY1 and FOXF1 transcription factor activities. Double knockdown of UBE2D2 and UBE2D4 caused an increase in p53 protein levels, and knockdown of p53 attenuated not only Cd-induced apoptosis, but also Cd-induced apoptosis-related gene expression (BAX and PUMA). Additionally, the mice exposed to Cd for 6 months resulted in increased levels of p53 and induction of apoptosis in proximal tubular cells. These findings suggest that down-regulation of UBE2D family genes followed by accumulation of p53 in proximal tubular cells is an important mechanism for Cd-induced renal toxicity.

Cerebellar Ataxia Suspected to Be Caused by Oxytropis glabra Poisoning in Western Mongolian Goats

ABSTRACT In the last five years in western Mongolia, a neurological disorder and resultant economic loss have developed in goats, sheep, cattle and horses: association of the disease with ingestion of Oxytropis glabra, a toxic plant, was suggested. Affected goats showed neurological signs, including ataxia, incoordination, hind limb paresis, fine head tremor and nystagmus. Three goats, one with moderate clinical signs and the other two with severe clinical signs, were necropsied and examined to describe and characterize the histologic, immunohistochemical and ultrastructural lesions. Although no gross pathological changes were observed in a variety of organs including the central nervous system of these goats, microscopic examination of the cerebellum demonstrated degenerative changes in all these goats, such as vacuolar changes and loss of Purkinje cells, torpedo formation in the granular layer, increased number of spheroids in the cerebellar medulla, and loss of axons and myelin sheaths of Purkinje cells. The chemical analysis of the dried plant detected 0.02–0.05% (dry weight basis) of swainsonine. This is the first report describing the clinical and pathological findings in Mongolian goats suspected to be affected by O. glabra poisoning.

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.