S. Takenaka

Distribution Pattern of Inhaled Ultrafine Gold Particles in the Rat Lung

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 μg/m3 (4 × 106/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma–mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5–8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma–mass spectrometry analysis of the lung, 1945 ± 57 ng (mean ± SD) and 1512 ± 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 ± 67 ng and 96 ± 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Ultrafine Particles Exert Prothrombotic but Not Inflammatory Effects on the Hepatic Microcirculation in Healthy Mice In Vivo

Background—Air pollution episodes are strongly associated with increased cardiovascular morbidity and mortality. The effect of ultrafine particles (UFPs), when translocated after inhalation, on the microcirculation of extrapulmonary organs remains unclear. Methods and Results—In C57BL/6 mice, either carbon black UFPs (1×107 and 5×107) or vehicle was infused intra-arterially. Two hours after infusion, platelet- and leukocyte-endothelial cell interactions, sinusoidal perfusion, endothelial fibrin(ogen) deposition, and phagocytic activity of Kupffer cells were analyzed by intravital video fluorescence microscopy in the liver microvasculature. Expression of fibrin(ogen), von Willebrand factor (vWF), and P-selectin on hepatic endothelium was determined by immunostaining. Apoptotic cells were quantified in TUNEL-stained tissue sections. Application of UFPs caused significantly enhanced platelet accumulation on endothelium of postsinusoidal venules and sinusoids in healthy mice. UFP-induced platelet adhesion was not preceded by platelet rolling but was strongly associated with fibrin deposition and an increase in vWF expression on the endothelial surface. In contrast, inflammatory parameters such as the number of rolling/adherent leukocytes, P-selectin expression/translocation, and the number of apoptotic cells were not elevated 2 hours after UFP exposure. In addition, UFPs did not affect sinusoidal perfusion and Kupffer cell function. Conclusions—UFPs induce platelet accumulation in the hepatic microvasculature of healthy mice that is associated with prothrombotic changes on the endothelial surface of hepatic microvessels. Accumulation of particles in the liver exerts a strong procoagulatory impact but does not trigger an inflammatory reaction and does not induce microvascular/hepatocellular tissue injury.

Fate and toxic effects of inhaled ultrafine cadmium oxide particles in the rat lung.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 μg Cd/m3 (1× 106/cm3) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)–mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53± 0.12 μg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 μg Cd/m3 for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 μg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.

Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 μg m−3), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.

A Morphologic Study on the Fate of Ultrafine Silver Particles: Distribution Pattern of Phagocytized Metallic Silver in Vitro and in Vivo

The distribution pattern of inhaled particles is an important factor for the evaluation of health effects. In this study, we morphologically investigated the fate of agglomerated ultrafine particles in macrophages in vitro and in vivo. Metallic silver (Ag) was chosen as a test particle, since it can be easily produced and detected by elemental and morphologic analyses. Ultrafine Ag particles generated by an electric spark generator in an argon atmosphere were collected on PTFE filters. The particles were suspended in distilled water and adjusted to different concentrations (10 μg/ml to 1 mg/ml) with phosphate-buffered saline (PBS). For the in vitro study, 1774 macrophage cell suspensions (200,000 cells in 400 μl medium) were plated in small chambers. Six hours later, 100 μl of the silver-PBS suspension was added to each chamber. For the next 9 days, the chamber slides were examined daily with an inverted microscope in order to detect agglomerated particles in the cell. The medium was changed every day, and Ag in the medium was checked by inductively coupled plasma mass spectrometry (ICP-MS). On days 1, 3, 5, 7, and 9, cells in the chambers were fixed with 2.5% buffered glutaraldehyde and examined ultrastructurally. For the in vivo study using F344 rats, 50 μg Ag particles were instilled intratracheally. On days 1, 4, and 7 following instillation, rats were sacrificed and the lungs were examined morphologically. The Ag content in the lung, liver, and lung-associated lymph nodes was analysed by ICP-MS. In the in vitro study, the dose-dependent presence of agglomerated particles was observed in 1774 cells. The size and form of particles remained unchanged throughout the observation period. Electron microscopy with x-ray microanalysis showed that both single and agglomerated Ag particles were observed in the dilated phagolysosome of 1774 cells. In the in vivo study, focal accumulation of Ag-particle-laden alveolar macrophages was found. Ag particles were also observed in the alveolar wall. Ag content in the lung was constant between day 1 and day 7, indicating that no rapid particle translocation from the lung to other organs had taken place in this time period. In vitro and in vivo studies suggested that agglomerated Ag particles remained in targets for a given period of time—at least up to 7 days.

Early Response of the Canine Respiratory Tract Following Long-Term Exposure to a Sulfur(IV) Aerosol at Low Concentration. V. Morphology and Morphometry

AbstractThe respiratory system of eight male beagle dogs was examined morphologically and morphometrically after continuous exposure for 290 days to a respirable sulfur(IV) aerosol at a concentration equivalent to a sulfur dioxide (SO2)concentration of 0.6 mg m−3. Three dogs served as unexposed controls.Seven of eight exposed dogs showed hyperplastic changes in the respiratory mucosa of the posterior nasal cavity. The changes were characterized by a thickened epithelial layer resulting from epithelial proliferation, by a loss of secretory material, and by moderate mononuclear cell infiltration. Four of eight exposed dogs had an increased number of nonciliated cells and slight mononuclear cell infiltration in the larynx. Tracheal changes were limited to an increased number of nonciliated cells in the membranous portion. Transmission electron microscopy TEM) showed the changes in the trachea to be caused by disorder in the development of ciliated cells.Morphometric examination of the pulmonary alveolar regi...

Effects of Inhaled CdO Particles on the Sphingolipid Synthesis of Rat Lungs

Surfactant lipids of the alveolar space protect the lung from various environmental stimuli. We investigated the influence of ultrafine (UF) CdO particles inhalation on two key enzymes involved in lung sphingolipid metabolism, serine palmitoyltransferase (SPT), and sphingomyelinase (SMase). Rats inhaled either 0.63 mg UF-CdO/m 3 for 6 h (group 1), or 1.08 mg UF-CdO/m 3 12 h/day for 10 days (group 2). Two corresponding control groups inhaled filtered clean air. Additional rats intratracheally instilled with lipopolysaccharide (LPS) were used as positive controls. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) of lung tissue showed a significant increase in the level of SPT mRNA (LCB2 subunit) expression in group 2 compared to the corresponding controls (p < .01). Group 1 and LPS were not statistically different from control. No alteration in the mRNA level of SMase was detected in any exposure group. The immunohistochemical analysis showed that SPT (LCB2 subunit) localization was stronger in the alveolar type II cells of group 2 lungs compared to the corresponding controls. These results were correlated with alterations in BALF cellular and biochemical parameters and lung morphology. Since SPT is the key enzyme for de novo sphingolipid synthesis in lung surfactant and SMase is responsible for sphingomyelin catabolism, we can postulate that high-dose UF-CdO exposure for 10 days induces an increase in sphingolipid synthesis in the type II cells of rat lungs that would not be promptly followed by its degradation.

HEALTH EFFECTS OF SULFUR-RELATED ENVIRONMENTAL AIR POLLUTION. V. Lung Structure

The lungs of 8 male beagle dogs were examined morphologically and morphometrically after exposure for 13 mo to a respirable sulfur(IV) aerosol at a mass concentration of 1.53 mg m(-3) (16.5 h/day), and to an acidic sulfate aerosol carrying 15.2 micromol m(-3) hydrogen ions into the lungs (6 h/day). An additional eight dogs served as unexposed controls. Standard morphometric analyses of both the surface epithelia of the conducting airways and the alveolar region were performed. These analyses showed no difference between the exposure group and control group. However, there was a tendency to an increase in the volume density of bronchial glands in the exposure group. Five of eight exposed animals showed thickened ridges (knob-like structures) at the entrance to alveoli in the alveolar duct and alveolar sac. Transmission electron microscopy revealed that the thickening was mainly due to type II cell proliferation. As the previous experiment using sulfite aerosol only showed no alterations in the proximal alveolar regions, the changes observed may be considered as effects of acidic sulfate aerosol alone or in combination with sulfite. These findings suggest that sulfur aerosols have the potential to induce epithelial alterations in the proximal alveolar region, which is a primary target for air pollutants.

Morphometric evaluation of bronchial glands of beagle dogs

Using lung tissues of beagle dogs, we developed a method for objective estimation of bronchial glands. Defined generations (5th, 10th, 15th and 20th) of bronchi were collected according to the microdissection method by Plopper et al. (1983) [1]. Transverse sections were prepared for both light microscopic and electron microscopic studies. The point counting using prints made by a video copy processor was performed for estimation of the volume density of bronchial glands. Bronchial glands were observed throughout the canine bronchial tree. Moreover, a distinct difference was found in the volume density of bronchial glands depending on the lobe examined and the generation number. Recently, we performed a long-term inhalation study using beagle dogs in order to elucidate toxic effects of sulfur(IV) and acidic sulfate aerosols (Heyder et al., 1994) [2]. In addition to the quantitative analysis of surface epithelia, the volume density of submucosal glands in the bronchial wall was measured according to the method described above. As preliminary result, there was a tendency to an increased volume density of glands in the exposure group. Detailed quantitative evaluation of bronchial glands may enable detection of finite changes induced by low level concentrations of air pollutants.

Investigations on the solubility of corrosion products on depleted uranium projectiles by simulated body fluids and the consequences on dose assessment

Ingestion and inhalation of corrosion products covering weathered penetrators made of depleted uranium (DU) represent potential radiological exposure pathways. In order to study the bioavailability of these corrosion products, their solubility was determined using simulated gastric and pulmonary juices. About 75 and 36% of the uranium in the corrosion products were found to be soluble in simulated gastric and pulmonary juices, respectively. The effective dose coefficient for adults after ingestion was calculated to be 0.61xa0μSvxa0mg−1 DU. This compares to an effective dose coefficient for an adult of 0.71xa0μSvxa0mg−1 for DU materials given by the World Health Organization (WHO). The effective dose coefficient for inhalation was calculated to be 3.7xa0×xa010−6xa0Svxa0Bq−1 for workers and 5.3xa0×xa010−6xa0Svxa0Bq−1 for members of the public, respectively, which is between those of particles of Types M and S as defined by the International Commission on Radiological Protection (ICRP). The speciation of the corrosion products was investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The mean oxidation state of uranium was found to be 4.6, which suggests that the uranium in the corrosion products consists of a mixture of U(IV) and U(VI) species.