Pulmonary effects of inhaled limonene ozone reaction products in elderly rats

Abstract

d-Limonene is an unsaturated volatile organic chemical found in cleaning products, air fresheners and soaps. It is oxidized by ozone to secondary organic aerosols consisting of aldehydes, acids, oxidants and fine and ultra fine particles. The lung irritant effects of these limonene ozone reaction products (LOP) were investigated. Female F344 rats (2- and 18-month-old) were exposed for 3 h to air or LOP formed by reacting 6 ppm d-limonene and 0.8 ppm ozone. BAL fluid, lung tissue and cells were analyzed 0 h and 20 h later. Inhalation of LOP increased TNF-alpha, cyclooxygenase-2, and superoxide dismutase in alveolar macrophages (AM) and Type II cells. Responses of older animals were attenuated when compared to younger animals. LOP also decreased p38 MAP kinase in AM from both younger and older animals. In contrast, while LOP increased p44/42 MAP kinase in AM from younger rats, expression decreased in AM and Type II cells from older animals. NF-kappaB and C/EBP activity also increased in AM from younger animals following LOP exposure but decreased or was unaffected in Type II cells. Whereas in younger animals LOP caused endothelial cell hypertrophy, perivascular and pleural edema and thickening of alveolar septal walls, in lungs from older animals, patchy accumulation of fluid within septal walls in alveolar sacs and subtle pleural edema were noted. LOP are pulmonary irritants inducing distinct inflammatory responses in younger and older animals. This may contribute to the differential sensitivity of these populations to pulmonary irritants.

Figure 1

Flow diagram of aerosol generation and animal exposure system. Mixture of ozone and d-limonene generates a secondary organic aerosol at fixed relative humidity (RH) in the flow tube reactor. Particle size, mass, number, ozone concentration, temperature and RH were monitored in the exposure chamber.

Figure 2

Particle number size distribution in the exposure chamber. Particle number from 0.1- 2 μm in diameter by LASAIR optical particle counter; particle number from 0.01-0.1 μm in diameter determined by difference between the condensation particle counter (CPC) and LASAIR. Each bar is the mean ± SD (n=13).

Figure 3

Effects of inhalation of LOP on TNF-α expression. Lung sections (6 μm) were prepared from younger (2 mo) and older (18 mo) rats immediately (0 h) and 20 h after exposure to air (panels A and B) or LOP (panels C-F). Sections were stained with antibody to TNF-α (panels A-F) or IgG (panels G and H). One of three similar experiments is presented. Brown staining is indicative of TNF-α expression. Arrows, alveolar macrophages; Arrowheads, Type II cells.

Figure 4

Effects of inhalation of LOP on COX-2 expression. Lung sections (6 μm) were prepared from younger (2 mo) and older (18 mo) rats immediately (0 h) and 20 h after exposure to air (panels A and B) or LOP (panels C-F). Sections were stained with antibody to COX-2 (panels A-F) or IgG (panels G and H). One of three similar experiments is presented. Brown staining is indicative of COX-2 expression. Arrows, alveolar macrophages; Arrowheads, Type II cells.

Figure 5

Effects of inhalation of LOP on Cu/Zn SOD expression. Lung sections (6 μm) were prepared from younger (2 mo) and older (18 mo) rats immediately (0 h) and 20 h after exposure to air (panels A and B) or LOP (panels C-F). Sections were stained with antibody to Cu/Zn SOD (panels A-F) or IgG (panels G and H). One of three similar experiments is presented. Brown staining is indicative of Cu/Zn SOD expression. Arrows, alveolar macrophages; Arrowheads, Type II cells.

Figure 6

Effects of inhalation of LOP on MAP kinase expression in alveolar macrophages (AM) and Type II cells (TII). Cells were isolated from younger (2 mo) and older (18 mo) animals immediately (0 h) and 20 h after exposure to air or LOP. Cytoplasmic extracts were prepared and analyzed for p38, p44/42 and JNK MAP kinase expression by western blotting. One representative gel from three separate experiments is shown.

Figure 7

Effects of inhalation of LOP on NF-κB, C/EBP and CREB nuclear binding activity. Alveolar macrophages (AM) and Type II cells (TII) were isolated from younger (2 mo) and older (18 mo) rats immediately (0) and 20 h (20) after exposure to air or LOP. Nuclear extracts were prepared and analyzed for NF-κB, C/EBP and CREB binding activity by EMSA. For supershift analysis, extracts prepared immediately after exposure of older rats to LOP (18 mo/ 20 h) were pre-incubated with 50-fold excess of unlabeled specific competitor probes (C). One representative of three gels is shown.

Figure 8

Effects of LOP on BAL protein and cells. BAL fluid was collected immediately (0 h) or 20 h after exposure of younger (open bars) and older (closed bars) animals to air or LOP. Upper panel: Protein in BAL. Each bar is the average ± SEM of triplicate samples from three groups of animals (n=3/group). Lower panel: Number of viable alveolar macrophages (AM) and Type II alveolar cells (TII) recovered from the lung. Each bar is the average ± SE (n=3). *Significantly different (p<0.05), from younger animals (One way ANOVA).

Figure 9

Effect of LOP on lung histology. Lung sections (6 μm) prepared from younger (2 mo) and older (18 mo) rats immediately (0 h) and 20 h after exposure to air (panels A and B) or LOP (panels C-F) were stained with H & E, Magnification, 40×.