A Pathological Study of Acute Pulmonary Toxicity Induced by Inhaled Kanto Loam Powder

Abstract

The frequency and volume of Asian sand dust (ASD) (Kosa) are increasing in Japan, and it has been reported that ASD may cause adverse respiratory effects. The pulmonary toxicity of ASD has been previously analyzed in mice exposed to ASD particles by intratracheal instillation. To study the pulmonary toxicity induced by inhalation of ASD, ICR mice were exposed by inhalation to 50 or 200 mg/m³ Kanto loam powder, which resembles ASD in elemental composition and particle size, for 6 h a day over 1, 3, 6, 9, or 15 consecutive days. Histological examination revealed that Kanto loam powder induced acute inflammation in the whole lung at all the time points examined. The lesions were characterized by infiltration of neutrophils and macrophages. The intensity of the inflammatory changes in the lung and number of neutrophils in both histological lesions and bronchoalveolar lavage fluid (BALF) appeared to increase over time. Immunohistochemical staining showed interleukin (IL)-6- and tumor necrosis factor (TNF)-α-positive macrophages and a decrease in laminin positivity in the inflammatory lesions of the lung tissues. Electron microscopy revealed vacuolar degeneration in the alveolar epithelial cells close to the Kanto loam particles. The nitric oxide level in the BALF increased over time. These results suggest that inhaled Kanto loam powder may induce diffuse and acute pulmonary inflammation, which is associated with increased expression of inflammatory cytokines and oxidative stress.

Keywords: Asian sand dust; Kanto loam powder; inhalation; pulmonary toxicity.

Figure 1

Histological findings of the control lungs, showing no changes. HE. Bar = 50 µm.

Figure 2

Histological examination of the lung taken from mice treated with Kanto loam powder showed acute inflammatory changes. Representative lung sections taken from mice treated with 50 mg/m³ of the powder at one day (A); three days (C); six days (E); nine days (G); and 15 days (I) after exposure, and from mice treated with 200 mg/m³ Kanto loam powder at one day (B); three days (D); six days (F); nine days (H); and 15 days (J) after exposure. Diffuse deposition of the particles (white arrowheads) at the bronchial mucosa and alveolar wall, and infiltration of inflammatory cells (neutrophils and macrophages) (black arrowheads) in the alveolar wall and alveolar space are observed; these changes become prominent with both time and concentration of the particles. HE. Bars = 50 µm.

Figure 3

Higher power magnification of the histological findings of the control lungs (A); of mice treated with 50 mg/m³ of the powder for one day (B); treated for 15 days (C); of mice treated with 200 mg/m³ of the powder for 15 days (D). Infiltration of neutrophils into alveoli and increased number of macrophages (black arrowheads), and exudation of serum protein in the alveolar space (black arrow) (B). Macrophages with increased cytoplasm containing the particles are shown (black arrow heads) (C,D). HE. Bars = 50 µm.

Figure 4

Histological changes of the lymph nodes taken from mice treated with 50 mg/m³ of the powder for one day. Increased number of neutrophils (black arrowheads) and a macrophage containing the particles in the sinus (a white arrowhead). HE. Bar = 50 µm.

Figure 5

Alpha-SMA immunohistochemistory in the lungs of mice treated with 50 mg/m³ of the powder for one day (A) and treated for 15 days (B). Spindle-shaped to elongated cells in the inflammatory lesions show positive immunolabelings for α-smooth muscle actin ((B), white arrows). Bars = 50 µm.

Figure 6

Laminin immunohistochemistry in the lungs of control mice (A) and of mice treated with 50 mg/m³ of the powder for one day (B). Positive, continuous, distinct thin lines are shown in the basement membrane of the alveoli (A). Weakly positive discontinuous staining is shown in the basement membrane of the alveoli in the inflammatory lesions (B). Bars = 30 µm.

Figure 7

(a) TNF-α (A), IL-6 (B), and iNOS (C) immunohistochemistry of the lungs of mice treated with 50 mg/m³ of the powder for one day. Macrophages containing the brown particles show positive immunoreactivity for TNF-α, IL-6, and iNOS (black arrowheads). Control (lungs of mice with no treatment, TNF-α) (D). Bars = 10 µm. (b) A semi-quantative analysis of the immunohistochemical reactions (A: TNF-α, B: IL-6, C: iNOS) in the lungs of mice treated with 50 mg/m³ (open bars) and 200 mg/m³ (filled bars) of the powder for one day and for 15 days (** p < 0.05).

Figure 8

Transmission electron microscopic images of the inflammatory lesions in the lung from mice treated with 50 mg/m³ of the powder for three days. Destruction of the alveolar walls (black arrowhead) with epithelial cells showing vacuolar degeneration was observed in the inflammatory lesions. Note that the macrophage containing the particles is closely located to the alveolar wall (white arrowheads). AS: alveolar space, R: red blood cell, M: macrophage. Bar = 1 µm.

Figure 9

Cellular and biochemical parameters of the bronchoalveolar lavage fluid. Total cell numbers (A); The percentage of neutrophils (B); The percentage of lymphocytes (C); Nitric oxide (D). d: day, * significantly different from the control group, p < 0.05.

Figure 9

Cellular and biochemical parameters of the bronchoalveolar lavage fluid. Total cell numbers (A); The percentage of neutrophils (B); The percentage of lymphocytes (C); Nitric oxide (D). d: day, * significantly different from the control group, p < 0.05.

Figure 10

Diagram of the nose inhalation system.