Induction of pulmonary fibrosis by cerium oxide nanoparticles

Abstract

Cerium compounds have been used as a diesel engine catalyst to lower the mass of diesel exhaust particles, but are emitted as cerium oxide (CeO(2)) nanoparticles in the diesel exhaust. In a previous study, we have demonstrated a wide range of CeO(2)-induced lung responses including sustained pulmonary inflammation and cellular signaling that could lead to pulmonary fibrosis. In this study, we investigated the fibrogenic responses induced by CeO(2) in a rat model at various time points up to 84 days post-exposure. Male Sprague Dawley rats were exposed to CeO(2) by a single intratracheal instillation. Alveolar macrophages (AM) were isolated by bronchial alveolar lavage (BAL). AM-mediated cellular responses, osteopontin (OPN) and transform growth factor (TGF)-β1 in the fibrotic process were investigated. The results showed that CeO(2) exposure significantly increased fibrotic cytokine TGF-β1 and OPN production by AM above controls. The collagen degradation enzymes, matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitor of MMP were markedly increased in the BAL fluid at 1 day- and subsequently declined at 28 days after exposure, but remained much higher than the controls. CeO(2) induced elevated phospholipids in BAL fluid and increased hydroxyproline content in lung tissue in a dose- and time-dependent manner. Immunohistochemical analysis showed MMP-2, MMP-9 and MMP-10 expressions in fibrotic regions. Morphological analysis noted increased collagen fibers in the lungs exposed to a single dose of 3.5mg/kg CeO(2) and euthanized at 28 days post-exposure. Collectively, our studies show that CeO(2) induced fibrotic lung injury in rats, suggesting it may cause potential health effects.

Fig. 1

Cerium oxide exposure induced TGF-β1 and OPN in AM culture medium. AM were isolated by bronchoalveolar lavage and cultured at 37 °C for 24 h. TGF-β1 (A) and OPN (B) levels were measured in the cultured supernate using ELISA. The values are expressed as means±SE, n=6. *Significantly different from saline controls, p<0.05. The dotted line indicates control.

Fig. 2

MMP-2, MMP-9 and TIMP-1 levels in the first BAL fluid monitored at 1- and 28-day post CeO₂ exposure. The first BAL fluid was isolated from saline and CeO₂-exposed rats, as described in the Materials and methods section. The MMPs and TIMP levels were determined using ELISA. The values are expressed as means±SE, n=6. *Significantly different from saline control group at p<0.05.

Fig. 3

Effect of CeO₂ exposure on phospholipids content in the BAL fluid and micrographs of TEM analysis of AM. (A) The phospholipid content in the first BAL fluids obtained from saline and various concentrations of CeO₂-exposed rats at 28 and 84 days post exposure. TEM of AM isolated by bronchoalveolar lavage from (B) control and (C) CeO₂ (3.5 mg/kg)-exposed rats at 10 days post-exposure. (Bar=2 μm). The values are expressed as means± SE, n=6. *Significantly different from saline control group at p<0.05. +Significantly different from 28-day exposure groups, at p<0.05.

Fig. 4

Cerium oxide particles in lung tissue and pulmonary fibroblasts isolated from CeO₂ (a single intratracheal dose of 7 mg/kg)-exposed rats, at 28 days post exposure. Control lung tissues exhibit no particles under high resolution, dark field illumination (A). Illuminated CeO₂ particles, using darkfield-based illumination, were clearly detected in macrophages (MAC), the interstitium (arrow), in acellular surfactant clumps (arrow head), in the airspace as free particles (B). Panel C gives representative intensity versus wavelength spectra of points (pixels) of CeO₂ particles in the cerium oxide-exposed tissue section (upper panel) and spectra of control tissue. Each different colored curve represents a different point. Small arrow: MAC; big arrow: interstitium; Arrow head: acellular mass of surfactant-cerium oxide in the air space.

Fig. 5

Immunohistochemical localization of MMP-2, -9, -10 and TIMP-1 in lung tissues. Exposure of rats to cerium oxide at 28 days after a single intratracheal instillation of 7 mg/kg of cerium oxide. Lungs were isolated and treated as described in the Materials and methods section. Representative immunolocalization for MMP-2, MMP-9, MMP-10 and TIMP-1 (brown-DAB precipitate) of saline control lung sections is shown in the upper panels (Panels A, C, E and G) and corresponding cerium oxide-treated lung sections in the lower panels (Panels B, D, F and H). MMP-2, MMP-9, and TIMP-1 (Panels B, D and H) were intensely expressed in cells of fibrotic areas in CeO₂-exposed lung, respectively. MMP-10 (Panel F) was intensely expressed in fibrotic areas of CeO₂-exposed lungs and was also expressed diffusely throughout the lungs. MMP-10 expression in saline controls was minimal (Panel E). Arrow head: acellular mass of surfactant-cerium oxide in the air space, containing PMN (small arrows).

Fig. 6

Hydroxyproline content and Sirius red staining for collagen in the lung tissue and quantitative morphometric analysis of alveolar wall thickness and collagen fiber volume in CeO₂ exposed rat lungs. (A) Lung tissues exposed to various levels of CeO₂ at 28 and/or 84 days after exposure. Hydroxyproline content in the lung tissues was monitored as described in the Materials and methods section. The values are expressed as means±SE, n=6. (B) Light micrograph of Sirius red staining for collagen formation in the lung tissues (arrow) at 28 days post exposure (cerium dose: 7 mg/kg). (C) Quantitative analysis of dose-dependent increase in the thickness of alveolar wall connective tissue. (D) Quantitative analysis of collagen volume expressed as a percentage of total tissue volume, based on the morphometric analysis of Sirius Red stained sections. *Significantly different from saline controls; p<0.05. +Significantly different from 28-day exposure groups, at p<0.05.