Age specific responses to acute inhalation of diffusion flame soot particles: cellular injury and the airway antioxidant response

Abstract

Current studies of particulate matter (PM) are confounded by the fact that PM is a complex mixture of primary (crustal material, soot, metals) and secondary (nitrates, sulfates, and organics formed in the atmosphere) compounds with considerable variance in composition by sources and location. We have developed a laboratory-based PM that is replicable, does not contain dust or metals and that can be used to study specific health effects of PM composition in animal models. We exposed both neonatal (7 days of age) and adult rats to a single 6-h exposure of laboratory generated fine diffusion flame particles (DFP; 170 µg/m(3)), or filtered air. Pulmonary gene and protein expression as well as indicators of cytotoxicity were evaluated 24 h after exposure. Although DFP exposure did not alter airway epithelial cell composition in either neonates or adults, increased lactate dehydrogenase activity was found in the bronchoalveolar lavage fluid of neonates indicating an age-specific increase in susceptibility. In adults, 16 genes were differentially expressed as a result of DFP exposure whereas only 6 genes were altered in the airways of neonates. Glutamate cysteine ligase protein was increased in abundance in both DFP exposed neonates and adults indicating an initiation of antioxidant responses involving the synthesis of glutathione. DFP significantly decreased catalase gene expression in adult airways, although catalase protein expression was increased by DFP in both neonates and adults. We conclude that key airway antioxidant enzymes undergo changes in expression in response to a moderate PM exposure that does not cause frank epithelial injury and that neonates have a different response pattern than adults.

Figure 1

Particle characterization. Mobility size distribution of the particles in the exposure chamber indicates a geometric mean particle size of 192 nm (A) Values are expressed as mean ± SD. Electron micrographs (B, C) of the particle morphology indicate that the particles varied in shape and that they consisted of primary particles 20–40 nm in diameter and formed larger fractal aggregates.

Figure 2

Morphologic changes in 7 day postnatal and adult rats 24 hours following DFP exposure. Resin sections were analyzed at 60× magnification for histologic changes. (A) In 7 day old postnatal rats reared in filtered air, a simple cuboidal epithelium of mostly ciliated and nonciliated cells was observed in the proximal airways. (B) After DFP exposure, more vacoulated cells were present (indicated by arrows). The epithelium of the large conducting airways was morphologically similar for filtered air (C) and DFP exposed (D) adult rats. Scale bar is 50μm. (E) Cell morphologies of five types: basal, mucous, nonciliated, ciliated, and vacuolated were quantified using point and intercept counts in large intrapulmonray airways. Calculated cell masses are shown (Vs). Although vacoulated cell mass increased and ciliated cell mass decreased following DFP exposure compared to filtered air controls, interactions between exposures were insignificant in both ages. Basal, mucous, and nonciliated cell mass in addition to epithelial thickness remained relatively consistent between exposure groups. Data are mean ± SEM (n=6 rats/group). (F) Lactate dehydrogenase (LDH), a marker for cytotoxicity, was quantified in bronchioalveolar lavage fluid. Significantly more LDH was detected in DFP exposed 7 day old rats, but was not changed in adult animals. Data are plotted as means ± SEM (n=6 rats/group). * = P <0.05, as compared to filtered air controls of the same age.

Figure 3

Heatmaps of all genes that were differentially expressed in the airways of rats as a combination of age or exposure. RNA was isolated from microdissected intrapulmonary airways obtained from RNAlater stabilized lung tissue and quantified using the RT² qPCR Array platform (SABiosciences, Frederick, MD). All filtered air adult genes were compared against 7 day old filtered air controls using HPRT as the reference gene. Each age exposed to DFP was also compared to its respective age matched FA control tissue. A heat map of all genes that were differentially expressed at a P value less than 0.05 is shown. The relative magnitude of expression is indicated on a spectrum ranging from minimum (green) to the maximum detected (red). Expression patterns in 65 genes differed significantly as a combination of age and/or exposure effects (n=3–5 rats/group).

Figure 4

Histochemical staining of glutamate-cysteine ligase (GCL). Paraffin sections from filtered air (A and C) or DFP exposed (B and D) neonatal (A and B) or adult rats (C and D) were immunostained for GCL. GCL protein abundance was low in filtered air animals regardless of age (compare 7 day postnatal FA in A with adult FA in C). Twenty-four hours after the cessation of DFP exposure, GCL protein expression was markedly increased in both ages (B and D), but the staining patterns differed between the two ages. In DFP exposed 7 day old neonates (B), GCL protein was abundant in subepithelial cells (white arrowheads). In contrast, GCL protein was immunolocalized primarily within the airway epithelium in exposed adults (black arrowheads, D). Scale bar for A, B, C, and D (shown in B) is 50μm.

Figure 5

Gene expression (A) and immunohistochemical staining (B–E) of glutathione S-transferase mu isoform (GSTμ) in the airways of filtered air (B and D) or DFP exposed (C and E) neonatal (B and C) or adult rats (D and E). Basally, FA adults had significantly greater GSTμ gene expression in microdissected airways than 7 day old FA neonates (A). Following DFP exposure, GSTμ gene expression remained unchanged in neonates and trended downwards in DFP exposed adults, but was not statistically significant (P=0.07). Gene expression of GSTμ was calculated using the comparative Ct method and displayed as a mean fold difference ± SEM (n=3–5 rats/group) compared against FA 7 day postnatal animals using HPRT as the reference gene. † = P <0.05, as compared to FA 7 day postnatal controls. Immunohistochemical staining for GSTμ protein indicates a similar pattern compared with gene expression data. GSTμ protein was diffusely localized to the terminal bronchiolar airway epithelium in both FA (B) and DFP exposed (C) 7 day old neonates with a few cells containing more abundant staining (arrows). In contrast, cells with increased GSTμ protein immunolocalization were more abundant in FA adult rats (D) and the number of cells with abundant GST μ protein was decreased in adult animals after DFP exposure (E). Scale bar in E for B, C, D and E is 50μm.

Figure 6

Gene expression (A) and immunohistochemical staining (B–E) of catalase in the airways of filtered air (B and D) or DFP exposed (C and E) neonatal (B and C) or adult rats (D and E). Gene expression of catalase (A) in microdissected airways was calculated using the comparative Ct method and displayed as a mean fold difference ± SEM (n=3–5 rats/group) compared against FA 7 day postnatal animals using HPRT as the reference gene. After DFP exposure, catalase gene expression remained unchanged in neonates, while a significant decrease in gene expression was found in adults. * = P <0.05, as compared to filtered air controls of the same age. Immunohistochemical staining for catalase protein in FA 7 day neonates was localized to the airway epithelium (B). After DFP exposure (C), catalase staining remained intense within the airway epithelium but increased in other lung compartments. Compared to FA neonates, catalase protein in adult FA exposed rats (D) was increased in abundance. After DFP exposure (E), intense catalase positive staining could be observed within the terminal bronchiole, as denoted by asterisks, in the airway epithelium. Areas with asterisks are pictured in the high magnification insets in D and E respectively. Scale bars are 50μm.

Figure 7

Gene expression (A) and immunohistochemical staining (B–E) of glutathione peroxidase 1 in the airways of filtered air (B and D) or DFP exposed (C and E) neonatal (B and C) or adult rats (D and E). Gene expression of glutathione peroxidase 1 (A) in microdissected airways was calculated using the comparative Ct method and displayed as a mean fold difference ± SEM (n=3–5 rats/group) compared against FA 7 day postnatal animals using HPRT as the reference gene. After DFP exposure, glutathione peroxidase 1 gene expression remained unchanged in neonates, while a significant decrease in gene expression was found in adults. * = P <0.05, as compared to filtered air controls of the same age. Protein expression in FA 7 day neonates was faint (B) but increased markedly with DFP exposure (C). Compared to FA neonates, protein expression in adult FA exposed rats (D) was higher. After DFP exposure (E), intense glutathione peroxidase 1 positive staining was observed in the airway epithelium but was diminished in the most distal portion of the terminal bronchiole. Scale bars are 50μm.