2010 Progress Report: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition

EPA Grant Number: R832415C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R832415
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Rochester PM Center
Center Director: Oberd√∂rster, G√ľnter
Title: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition
Investigators: Finkelstein, Jacob N. , Oakes, David , Phipps, Richard , Prather, Kimberly A. , Rahman, Arshad
Institution: University of Rochester , University of California - San Diego
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: June 30, 2009 through July 1,2010
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The experiments proposed within this project are designed to address specific mechanistic hypotheses regarding the interactions between inhaled ultrafine particles and specific pulmonary cell populations. In vitro experiments are best utilized to provide a mechanistic link and biological plausibility for the whole animal and controlled clinical (human) exposures, described in the other programs of this Particle Center. In some circumstances they can be used to screen materials for acute toxicity or provide an initial ranking of response. In our original proposal we had suggested a focus on identifying specific responses and mechanisms that may be involved in the enhanced susceptibility of diabetic. We continue to explore this in the context of the oxidant stress hypothesis of PM induced effects.

Progress Summary:

Responses to particles by epithelial/endothelial co-cultures 

As more of the physiological effects noted in our in vivo studies have pointed towards cardiovascular endpoints our emphasis on cellular models has shifted to vascular cell populations. The main focus of our in vitro studies continues to be the endothelium but while continuing to test the validity of the cellular mechanisms of response in different cell types. As a means of better correlating the potential of various particles or other stimuli to induce effects in vivo and establish appropriate in vitro models we continue to examine multiple cellular endpoints, in particular those that are relevant to measurement made in other PM center projects. Our results have established that measurement of IL 6 has the best predictive value. Once we have established the appropriate end points our plans have been to create more relevant models. Co-culture systems consisting of lung epithelial cells and lung endothelial cells have established so that both PM-cell and cell-cell interactions can be evaluated. Cell type specific responses of cytokine induction (TNF-á; IL-6; IL-8; or homologue) and NO-production (endothelial cells) will be measured as well as uptake and PM size-specific translocation. Dose-response relationships using PM samples collected at different sites from different sources will first be established in epithelial and endothelial cell monocultures. These data will be used to select the most active fractions of the PM samples for the co-culture studies and for the in vivo instillation studies.

To address this question we initially grew endothelial cells in transwell cultures with the cell either growing on top of the transwell, similar to a classical monolayer or grew them on the underside of the membrane to be able to access the basal surface of the cell through the pores of the insert. The system was stimulated by adding particles or a soluble agent like LPS to either the As shown in Figure 1 when particles were added either to the insert or to the well below Il-6 production was stimulated.
 
 
 
Figure 1 Production of IL-6 by pulmonary endothelial cells; grown in transwell inserts to allow access to apical and basal surfaces of cells. Human endothelial cells were grown on gelatin coated transwell inserts either within the transwell or on the underside. After 7 days cells were incubated with particles at the concentrations shown for 24 hours and IL-6 production assayed
 
To create cocultures A549 were culture on the top surface of the transwell insert and endothelial cell grown in the lower surface after coating with gelatin. Cells were permitted to grow for 7 days to reach confluence and then stimulated by addition of LPS or particles to either the upper or lower chamber of the culture.
 
Adding a stimulus to the upper chamber would directly stimulate the epithelial cells while adding agents to the lower chamber would stimulate the apical face of the endothelium. By sampling media from both chambers we have the potential of determining the response of each cell and the possibility that stimulation of one of the cello types would lead to a response in the second. Our data with LPS and a silver nanoparticles suggests some specificity in the response for the particle as an increase in luciferase activity (indicating epithelial oxidative stress) was only observed when particles were added directly to the apical surface of the cells. Particle added to the lower chamber had no effect.
 
 
Figure 2 Characterization of Transwell co-culture model
 
A594 cells and vascular endothelial cells were cultured in transwell cells were stimulated with either LPS or particles added to the upper chamber or lower. Culture media was harvested from upper and lower chamber and assayed for luciferase as a response of the epithelial cells and IL-6 measured to indicate response of the endothelium. LDH was uses as a measure of cytotoxicity.
 
The data in Figure 2 examines a similar set of experiments with the cels grown individually as monolayers as is more typically used to evaluate particle responses. It seem clear that the monolayerssis core to attempt to model the different responses we have noted for the different particle samples. What was consistent across all samples was the ability of added glucose to blunt the response. We are currently investigating the mechanism of this suppression through effects on intracellular signaling pathways.
 
 
 
 

Future Activities:

We plan to continue to use the trans well and co-culture systems to measure cell type specific responses of cytokine induction (TNF-α; IL-6; IL-8; or homologue) and NO-production (endothelial cells) as well as uptake and PM size-specific translocation. Dose-response relationships using PM samples collected at different sites from different sources will first be established in epithelial and endothelial cell monocultures. These data will be used to select the most active fractions of the PM samples.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 13 publications 11 publications in selected types All 10 journal articles
Other center views: All 190 publications 156 publications in selected types All 143 journal articles
Type Citation Sub Project Document Sources
Journal Article Rushton EK, Jiang J, Leonard SS, Eberly S, Castranova V, Biswas P, Elder A, Han X, Gelein R, Finkelstein J, Oberdorster G. Concept of assessing nanoparticle hazards considering nanoparticle dosemetric and chemical/biological response metrics. Journal of Toxicology and Environmental Health, Part A 2010;73(5-6):445-461. R832415 (2010)
R832415 (2011)
R832415 (Final)
R832415C004 (2010)
R832415C004 (2011)
R832415C005 (2010)
R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor & Francis-Abstract
    Exit
  • Supplemental Keywords:

    RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Air, particulate matter, Genetics, Health Risk Assessment, Risk Assessments, Physical Processes, Biology, altered gene expression, atmospheric particulate matter, atmospheric particles, long term exposure, airway disease, exposure, human exposure, ambient particle health effects, atmospheric aerosol particles, PM, aersol particles

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2011 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R832415    Rochester PM Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832415C001 Characterization and Source Apportionment
    R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
    R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
    R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
    R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition