Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells

Abstract

BackgroundExposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression.ResultsMicroarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 106μm2/cm2) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 106μm2/cm2) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (FOS, ATF3, IL6 and IL8) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells.ConclusionsCristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses in vitro are suggested as predictive in determining the pathogenicity of potentially harmful particulates.

Induction of altered mRNA expression profiles caused by fibrous and granular dust

“…Our study and recent reports in the literature demonstrate that gene expression profiling in human lung epithelial cells can be an important tool for analyzing the pathogenicity of potentially harmful fibres and particles (42)(43)(44). Gene expression profiling, for example in response to asbestos, is valuable to define early molecular effects as demonstrated in diverse human cells, such as normal human bronchial epithelial cells (NHEC) (45), human lung adenocarcinoma cells (A549) (46,47), SV40-transformed human bronchial epithelial cells (BEAS-2B) and SV40-immortalized pleural mesothelial cells (MET5A) (47).…”

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: Grasping complex toxicity with a great depth of field

“…These results are consistent with those found in the literature for silica and other NPs [4,35,36]. Perkins et al previously compared crystalline and amorphous silica NPs, but found that only cristobalite upregulated IL6 and IL8 in lung BEAS-2B cells whereas amorphous silica NPs only modulated CCL5 (RANTES) expression [4].…”

“…This discrepancy between our data and previously published data illustrates the possible heterogeneity of the results in the NP literature, probably directly linked to the different nature of the NPs. In particular, fumed AERO-SIL® 200 NPs differ from the amorphous silica NPs of the previous study [4], with a surface area 100 times greater. The "TREM1 signaling pathway" was triggered, leading to proinflammatory immune responses.…”

“…Of the amorphous silicas examined, AERO-SIL® 200 induced the most severe changes in the lungs of rats, which only partly recovered. Previous in vitro studies also showed that amorphous silica nanoparticles were toxic to lung cells [4,6,7].…”

Global DNA methylation and PTEN hypermethylation alterations in lung tissues from human silicosis

“…Silica dust can also increase the mortality rate from respiratory diseases and cardiovascular diseases (3). Exposure to silica can cause abnormal gene expression (4). Regulation of gene expression not only depends on genetic mechanism, also with epigenetics mechanisms without genetic coding change, including DNA methylation and histone acetylation and so on (5).…”