2009 Progress Report: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle CompositionEPA 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: Oberdörster, Günter
Title: Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition
Investigators: Finkelstein, Jacob N. , Prather, Kimberly A. , Rahman, Arshad , Oakes, David , Phipps, Richard
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: October 1, 2008 through September 30,2009
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air
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.
Figure 1 Production of IL-6 by human endothelial cells following treatment with concentrated ambient ultrafine particles, TiO2 and carbon black. Cells were incubated with a fixed concentration of the various particles for 24 hrs. Culture media was harvested and assayed for IL-6 by ELISA. A second group of cells were similarly exposed after growing in 30mM supplemental glucose.
1. Responses to particles by endothelial cells
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 and nitrite (as a surrogate for NOS activity) have the best predictive value. As shown in Figure 1, primary cultures of human endothelial cells do not produce significant amounts of IL6 in response to PM either well-characterized laboratory particles or PM recovered from an ambient particle concentrator.
Figure 2 Production of nitrate by human endothelial cells following treatment with concentrated ambient ultrafine particles, TiO2 and carbon black. Cells were incubated with a fixed concentration of the various particles for 24 hrs. Culture media was harvested and assayed for IL-6 by ELISA. A second group of cells were similarly exposed after growing in 30mM supplemental glucose.
2. In vitro models for diabetes.
In contrast production of NO, as measured by nitrite production and release into culture media can be higly responsive to particles ( Figure 2). Both commercially available bulk PM (TiO2 and Printex carbon) were found to be highly stimulatory under these conditions. Interestingly, PM recovered from the particle concentrator was capable to a significant response using this outcome measure. From a mechanistic perspective, this suggests that induction of IL6 gene expression and production of NO in endothelial cells is regulated by independent mechanisms and simply invoking particle induced oxidative stress is insufficient to our understanding of the process. One could speculate that since our previous work showed high levels of the antioxidant enzymes SOD and catalase in endothelial cells that this protection modulates the cellular response to PM.
As mentioned previously, a component of the current research of this PM center is to investigate the response of diabetics to the effects of PM and thus, our in vitro studies are designed to model this and offer some mechanistic basis. We have shown that culture of vascular endothelial cells in high glucose alters both the basal and particle induced cytokine responses. Using this model, we have begun to address the response of pulmonary cells to particles collected by a high volume sampler in Rochester. These would be similar to PM used in animal and human clinical studies carried out using the Harvard ultrafine particle concentrator.
Figure 3 Production of NO by pulmonary endothelial cells following treatment with PM collected with the Rochester high volume samples at different times. Effect of glucose and modulation by antioxidants. Cells were incubated with a fixed concentration of the various particles for 24 hrs. Cells were alternatively culture in 30 mM glucose with or without the antioxidants N-acetyl cysteine or BHA. Culture media was harvested and assayed for nitrite by the Griess assay.
Using production of NO as our benchmark may reflect a possiblealteration of vascular reactivity and altered regulation of vascular tone following particle inhalation and subsequent translocation to the vasculature.
One of the other questions raised in our proposed studies is the role of PM induced oxidative stress in the generation of cytokine or NO (Nitric Oxide) responses. The human clinical studies have been measuring vascular reactivity as a measure of response to inhaled PM. Included in that battery of outcomes was IL-6 and plasma Nitric Oxide (NO).To examine this response in a mechanistic manner, we began to measure changes in both of these outcomes in cells that have had their antioxidant status altered by culturing with exogenous antioxidants. For these initial studies, we used both a soluble sulfhydryl agent, N-acetyl cysteine (NAC) and a lipophilic antioxidant butylate hydroxyanisole (BHA). Culture with NAC is known to increase intracellular –SH groups, including glutathione while BHA as a lipophilic agent sequesters in cell membranes to trap lipid radicals. As shown in Figure 3, endothelialcells exposed to collected ultrafine particles respond through increased production of NO. This increase in NO is reduced in cells under hyperglycemic conditions. Consistent with our previous suggestion regarding the role of oxidative stress in the production of NO, none of the antioxidants had a significant effect on NO. The effect of the hypergycemia is much more dominant.
Lastly, we continue to mine results from particles collected as a result of the multi-center collaboration. We had previously examined production of cytokines and other inflammatory mediators, and we have returened to these samples to examine boththe production of NO and to continue to validate our hyperglycemia model. As shown in Figure 4, particles collected at the different sites show a modest but significant difference in their ability to induce NO production. In this circumstance, greater response appears to be generated from the no-urban samples. This suggests differences in PM composition ( or source) could lead to different outcome. We are working with our analysis 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.
Figure 4 Production of NO by pulmonary endothelial cells; effect of glucose pre-treatment and comparison of ultrafine PM collected from multiple sites.
Co-culture systems consisting of lung epithelial cells and lung endothelial cells are being established so that both PM-cell and cell-cell interactions can be evaluated. Cell type specific responses of cytokine induction (TNF-a; 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 first will 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 by Core 4.
Journal Articles:No journal articles submitted with this report: View all 13 publications for this subproject
Supplemental Keywords:RFA, Scientific Discipline, Health, 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
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