2008 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. , 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, 2007 through September 30,2008
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Human Health , 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. The in vitro experiments in this core are intended 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. Our ability to use defined populations of cells and well characterized particles allow us to test specific hypothesis that arise from the in vivo studies described elsewhere. A particular focus of the in vitro studies is to attempt to identify mechanisms that may be involved in the enhanced susceptibility of cells from diabetics. Also by working with our particle characterization and analytical cores we will attempt to correlate cellular effects with composition that is related to specific sources.

Approach:

Studies will use isolated and cultured cells that have been identified as the principle targets of inhaled PM and carry out hypothesis testing studies that support the in vivo studies (Cores 3 and 4). Cell specific endpoints of PM induced toxicity for inflammatory, epithelial and vascular endothelial cells will be determined in a dose-responsive manner. These cells will be obtained from both human and animal tissues. Our use of multiple endpoints ensures that we can determine the relationship between a cellular response and the critical variable of a specific particle. We plan studies with both "real world" and laboratory generated PM of known composition that may represent specific sources to establish source related effects.

Progress Summary:

1.      Differential 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 away from the typical pulmonary cell targets, namely alveolar macrophages and respiratory epithelium, to vascular cell populations. The main focus of our in vitro studies continues to be the endothelium but we continue to examine epithelial responses to PM for comparison and to test the validity of the cellular mechanisms of response in different cell types. An important direction of these experiments has been a more thorough examination of the role of oxidant stress in the response to PM and the effect of cellular and exogenous antioxidants as protectors against PM induced cellular injury.
 
 
In this regard we have compared the activity of SOD and catalase in 3 different pulmonary cell lines cultured under identical conditions. Both catalase and SOD activity was found to be highest in the microvascular endothelial line RLMVEC. Both the type I (R3/1) and type II alveolar (RLE.6TN) lines had significant activity but catalase was found to be substantively lower in the epithelial cells. On the basis of this result one might predict that the endothelial cells would be most resistant to oxidant induced damage.
 
2.      In vitro models for diabetes.
 
A major thrust of the current research of this PM Center is to investigate the proposed increased sensitivity of diabetics to the effects of PM. Our in vitro studies are designed to model this under
controlled conditions.
 
 
 
Figure 2.  Production of IL-6 by pulmonary epithelial cells following treatment with PM collected with the Rochester high volume samples at different times.  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 30 mM supplemental glucose.
 
 
As we reported previously, one of the hallmarks of the diabetic is the increased blood glucose and 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. Using production of IL-6 as our benchmark may reflect a possible acute phaase response following particle inhalation and subsequent translocation to the vasculature. As shown in Figure 2 human respiratory epthelial cells exposed to collected ultrafine particles respond through increased production of IL-6. In contrast to previous work that focussed only on the production of NO and endothelium epithelial cells maintained under conditions of hypergycemia actually produce increased amounts of IL-6. This is in contrast to results shown in Figure 3 where HUVEC were cultured under similar conditions. It is also evident that the ambient samples show a somewhat different response in these cells for this marker. In collaboration with our Analysis core we will attempt to determine if differences in compostion can help explain the differences in activity.
 
 
 
 
Figure 3.  Production of IL-6 by pulmonary endothelial cells HUVEC following treatment with PM collected with the Rochester high volume samples at different times.  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 30 mM supplemental glucose.
 
 
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 thes initial studies we used both a soluble sulfhydryl agent , N-acetyl cysteine (NAC)and a lipophilic antioxidant butylate hydroxyanisole (BHA).
 
 
Figure 4. IL-6 production by HUVEC in culture after treatment with LPS:  Effect of hyperglycemia and antioxidant pretreatment. RLMVEC cells grown with or without 30 mM supplemental glucose were culture for 24 hrs in the presence of various antioxidants and then treated with LPS.  Media was collected and IL-6 determined by ELISA.
 
 
Culture with NAC is know to increase intracellular –SH groups including glutathione while BHA as a lipophilic agent sequesters in cell membranes to trap lipid radicals. Our initial studies looked at cells stimulated by LPS which is know to stimulate the production of IL-6 by endothelial cells. Results in Figure 4 show that LPS stimulated IL-6 production was not altered by either antioxidant nor did glucose pretreatment interact with the antioxidants. Interestingly it did show that hyperglycemia inhibited LPS induced endothelial IL-6 production while stimulating PM induced IL-6 production by epithelail cells( see figure 2). This difference also emphasizes the need for examining multiple cell types when attempting to assess the effects of PM as there appears to be a degree of cellular specificity. As shown in Figure 5 pretreatment with antioxidant did have some effect on PM induced IL-6 but this was limited to the NAC treatment and generally appeared due to an overall reduction of IL-6 rather than a specific effect on induction by PM.
 
We are currently investigating the mechanism of this suppression through effects on
intracellular signaling pathways.
 
 
Figure 5.  Effect of antioxidant pretreatment on IL-6 production by vascular endothelial cells.

Expected Results:

The results of these in vitro studies will identify specific mechanisms that are triggered following particle cell contact in a cell specific context and identify markers of cellular response. We predict that many of the subsequent physiologic effects in vivo are the consequences of cellular oxidative stress, cell activation and apoptosis. The proposed in vitro experiments will provide a link between the whole animal and controlled clinical (human) exposures, described in the other programs of this PM Center, By evaluating "source specific" responses in vitro we can determine the relative potency of a given particle type and allow for studies in vivo to be more focused and mechanism based. These studies may also provide information regarding strategies to reduce PM effects. As correlations are established between in vitro responses and whole organism studies, the in vitro studies, which require less material and a shorter time frame, may be applied as a routine screen for potential PM risk and as support for setting regulatory standards.

Future Activities:

In the coming year we plan to continue to characterize the response of the microvascular endothelium to particles and begin to model for the effects of diabetes on these responses. Recent studies suggest that hyperglycemia can significantly alter the oxidative stress response in the epithelium and we plan to assess the effect of this treatment on cytokine production following particles. Also, in collaboration with Core 4 we plan to determine the effect of serum isolated from diabetic animals, both PM exposed and controls, on the ability of vascular endothelial cells to be restimulated by concentrated PM. We also expect to extend these studies to cells of neuronal origin and possibly cardiac muscle. Measurement of prostaglandin production, and COX-2 activation will be evaluated with respect to its usefulness as a marker.
 
Also, in support of the in vivo projects, we will evaluate in vitro effects of particles of differing composition. We will continue to examine the cytokine response to ultrafine particles containing elemental carbon and iron and organic carbon compounds and begin studies of concentrated real world particles.

Journal Articles:

No journal articles submitted with this report: View all 13 publications for this subproject

Supplemental Keywords:

Cytokines, antioxidants, diabetes,, 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
  • 2006 Progress Report
  • 2007 Progress Report
  • 2009 Progress Report
  • 2010 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