Transcriptomics analysis of lungs and peripheral blood of crystalline silica-exposed rats

Abstract

Minimally invasive approaches to detect/predict target organ toxicity have significant practical applications in occupational toxicology. The potential application of peripheral blood transcriptomics as a practical approach to study the mechanisms of silica-induced pulmonary toxicity was investigated. Rats were exposed by inhalation to crystalline silica (15 mg/m(3), 6 h/day, 5 days) and pulmonary toxicity and global gene expression profiles of lungs and peripheral blood were determined at 32 weeks following termination of exposure. A significant elevation in bronchoalveolar lavage fluid lactate dehydrogenase activity and moderate histological changes in the lungs, including type II pneumocyte hyperplasia and fibrosis, indicated pulmonary toxicity in the rats. Similarly, significant infiltration of neutrophils and elevated monocyte chemotactic protein-1 levels in the lungs showed pulmonary inflammation in the rats. Microarray analysis of global gene expression profiles identified significant differential expression [>1.5-fold change and false discovery rate (FDR) p < 0.01] of 520 and 537 genes, respectively, in the lungs and blood of the exposed rats. Bioinformatics analysis of the differentially expressed genes demonstrated significant similarity in the biological processes, molecular networks, and canonical pathways enriched by silica exposure in the lungs and blood of the rats. Several genes involved in functions relevant to silica-induced pulmonary toxicity such as inflammation, respiratory diseases, cancer, cellular movement, fibrosis, etc, were found significantly differentially expressed in the lungs and blood of the silica-exposed rats. The results of this study suggested the potential application of peripheral blood gene expression profiling as a toxicologically relevant and minimally invasive surrogate approach to study the mechanisms underlying silica-induced pulmonary toxicity.

Figure 1

Lactate dehydrogenase activity in rat lungs. Rats were exposed to crystalline silica or filtered air and lactate dehydrogenase activity was determined in bronchoalveolar lavage fluid (BALF) as described in the text. Values represent mean ± SE of eight rats per group. *Statistically significant (p < 0.05) compared to the control rats.

Figure 2

Photomicrographs of lungs from control and silica-exposed rats. Lung samples obtained from control and crystalline silica-exposed rats were sectioned and stained with H & E (top panel) or Masson’s trichrome stain (bottom panel) as described in the text. The arrow and arrowhead in the H & E stained sections show type II pneumocyte hyperplasia and alveolar space filled with macrophages and neutrophils, respectively, in the silica-exposed rat lungs. The arrow and arrowhead in the trichrome stained sections show thickened alveolar septae and type II pneumocyte hyperplasia lining the alveolar septae, respectively, in the silica-exposed rat lungs Magnification: H & E = ×20; trichrome = ×40.

Figure 3

Bronchoalveolar lavage fluid (BALF) and blood parameters of inflammation in the control and silica-exposed rats. Rats were exposed to crystalline silica or air and total number of alveolar macrophages (AMs) (A) and polymorphonuclear leukocytes (PMNs) (B) and concentration of MCP-1(C) were determined in the BALF as described in the text. Number of neutrophils (D) in the blood of the rats was also determined. Values represent mean ± SE of eight rats per group. *Statistically significant (p < 0.05) compared to the control rats.

Figure 4

Number of significantly differentially expressed genes (SDEGs) in the lungs (L) and blood (B) of silica-exposed rats. Rats were exposed to crystalline silica or air and gene expression profile was determined in the lungs and blood by microarray analysis. Genes with >1.5-fold change and a false discovery rate (FDR) p < 0.01 in the silica-exposed rat lungs and blood compared with the corresponding control samples were considered as significantly differentially expressed. SDEGs – Significantly differentially expressed genes in the lungs and blood of the silica-exposed rats compared with the control samples; Upregulated – genes which were significantly overexpressed in the lungs and blood of the silica-exposed rats compared with the control samples; and Downregulated – genes whose expressions were significantly downregulated in the lungs and blood of the silica-exposed rats compared with the control samples.

Figure 5

Validation of microarray results by QRT-PCR. A set of seven genes whose expressions were significantly different in the lungs and blood of the silica-exposed rats as indicated by the microarray findings were analyzed by QRT-PCR as described in the “Materials and methods” section. The microarray data presented is the mean fold change (n= 6) and the QRT-PCR data presented is mean fold change ± SE (n= 6) of silica-exposed rats compared with the control. A. Lung results, B. Blood results. *Statistically significant (p < 0.05) compared to the control rats.

Figure 6

Enrichment of IPA biological functions in the lungs and blood of silica-exposed rats. Bioinformatics analysis of the significantly differentially expressed genes identified in the silica-exposed rat lungs and blood was done using IPA software. The top 10 significantly enriched biological functions in the lungs of the silica-exposed rat lungs compared with the control rat lungs and the same biological functions in the blood samples are presented to demonstrate the similarity in gene expression profile between lungs and blood of the silica-exposed rats. Data represents the mean of six rats per group.

Figure 7

Enrichment of IPA canonical pathways in the lungs and blood of silica-exposed rats. Bioinformatics analysis of the significantly differentially expressed genes in the silica-exposed rat lungs and blood was done using IPA software. The top eight significantly enriched canonical pathways in the silica-exposed rat lungs compared with the control rat lungs and the same pathways in the blood samples of the rats are presented to demonstrate the similarity in the gene expression profile between lungs and blood of the silica-exposed rats. Data represents the mean of six rats per group.