UVM Theses and Dissertations
Format:
Print
Author:
Perkins, Timothy Nicholas
Dept./Program:
Pathology
Year:
2011
Degree:
MS
Abstract:
Exposure to respirable crystalline silica particles, as opposed to inert, amorphous silica, is associated with lung inflammation and pulmonary fibrosis (silicosis). In addition, the number of potentially harmful particulates in the environment and workplace is growing rapidiy, especially in the field of nano-technology. Current toxicological testing in the realm of particulates and fibers is in dire need of standardization. We have, proposed a model of in vitro toxicological screening of particulates assessing viability, robust gene expression profiling and cytokine and chemokine elaboration, in human bronchial epithelial cells to determine comparative pathogenic potential. These methods have the ability to assess the potential toxicity of respirable particulates more thoroughly and efficiently than current in vitro and in vivo testing.
Methods used include assessment of cell viability by Trypan Blue exclusion, cellular uptake of particles by scanning electron microscopy-electron dispersion spectroscopy (SEM-EDS), and Affymetrix/GeneSifter microarray analysis to determine gene expression profiles of both BEAS 2B cells and (primary) NHBE cells exposed to equal surface area doses of cristobalite (crystalline) silica and synthetic, amorphous silica particles. Gene ontology analyses, as well as quantitative polymerase chain reaction (qRT-PCR), were used to analyze gene expression induced by silica particles. In addition, we performed Bioplex analysis of secreted cellular mediators induced by exposure to particles in BEAS 2B cells. Results show that both crystalline and amorphous silica had equitoxic effects on cell viability and both were phagocytosed by cells as early as 2 hours; however, crystalline silica induces a much more potent effect on gene expression and mediator secretion compared to amorphous silica, inducing nearly 6-fold more changes in gene expression and only crystalline silica induced secretion of cytokines and chemokines. Responses invoked by crystalline silica were also dose-responsive.
These studies indicate that toxicological testing of particulates surveying viability and metabolic activity alone are insufficient. Gene expression profiling with concurrent survey of mediator secretion in vitro, may be more efficient in toxicological screening of respirable materials than expensive and labor-intensive in vivo model. Lung epithelial cells have recently been described to have a more active role in lung defense. Early responses of the lung epithelium, such as up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators can be indicative of a pathological response. Assessment of such responses will be essential in determining the pathogenicity of potential harmful particulates. In addition to toxicity screening, utilization of in-depth pathway analyses may reveal key pathways and signaling networks involved in particle-induced disease. Elucidation of these vital factors may lead to new understandings in the mechanisms involved and potential therapeutic targets.
Methods used include assessment of cell viability by Trypan Blue exclusion, cellular uptake of particles by scanning electron microscopy-electron dispersion spectroscopy (SEM-EDS), and Affymetrix/GeneSifter microarray analysis to determine gene expression profiles of both BEAS 2B cells and (primary) NHBE cells exposed to equal surface area doses of cristobalite (crystalline) silica and synthetic, amorphous silica particles. Gene ontology analyses, as well as quantitative polymerase chain reaction (qRT-PCR), were used to analyze gene expression induced by silica particles. In addition, we performed Bioplex analysis of secreted cellular mediators induced by exposure to particles in BEAS 2B cells. Results show that both crystalline and amorphous silica had equitoxic effects on cell viability and both were phagocytosed by cells as early as 2 hours; however, crystalline silica induces a much more potent effect on gene expression and mediator secretion compared to amorphous silica, inducing nearly 6-fold more changes in gene expression and only crystalline silica induced secretion of cytokines and chemokines. Responses invoked by crystalline silica were also dose-responsive.
These studies indicate that toxicological testing of particulates surveying viability and metabolic activity alone are insufficient. Gene expression profiling with concurrent survey of mediator secretion in vitro, may be more efficient in toxicological screening of respirable materials than expensive and labor-intensive in vivo model. Lung epithelial cells have recently been described to have a more active role in lung defense. Early responses of the lung epithelium, such as up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators can be indicative of a pathological response. Assessment of such responses will be essential in determining the pathogenicity of potential harmful particulates. In addition to toxicity screening, utilization of in-depth pathway analyses may reveal key pathways and signaling networks involved in particle-induced disease. Elucidation of these vital factors may lead to new understandings in the mechanisms involved and potential therapeutic targets.