Grantee Research Project Results

2010 Progress Report: Project 4 -- Transport and Fate Particles

EPA Grant Number: R832414C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Project 4 -- Transport and Fate Particles
Investigators: Wilson, Dennis , Barakat, Abdul , Louie, Angelique
Current Investigators: Wilson, Dennis , Louie, Angelique
Institution: University of California - Davis
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
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

Objective:

To characterize the time course, tissue distribution, and mechanisms of particulate matter (PM) accumulation in the systemic circulation and target organs.
To evaluate the effects of size and surface-fixed charge on this process.
To determine how altered lung structure affects systemic particle distribution.
To characterize how fluid flow modulates particle interactions with vascular endothelial cells.

Progress Summary:

Specific Aim 1: To characterize the time course and distribution of circulating particulates in vivo. In the last year, we have focused on determining the effects of a co-mixture containing labeled (64-Cu) polystyrene particles and PM2.5 (Kent Pinkerton) on lung retention and transport in a normal mouse model. Ambient particles have been shown to cause a physiological response in the lung (increased inflammation, permeability, oxidative stress). We hypothesized that delivering ambient particles could induce these changes in the lung and we could observe differences in transport/retention with our current radiolabeled model of PM (polystyrene). We have focused on two PM2.5 concentrations: 50 µg, and 500 µg doses. The region of interest analysis of the PET images of the 50 µg co-mixture dose is shown below in Figure 1.

Figure 1. Biodistribution of 50 µg co-mixture dose over 48 hours in the normal mouse. Lung retention at 48 hours was 82.8%DD +/- 10 and significant (p<0.05) changes in secondary organ accumulation were shown in the bladder, liver and spleen (*). DD = deposited dose.

For the 500µg dose similar retention and secondary organ accumulation was observed (Figure 2).

Figure 2. Biodistribution of 500µg dose over 48 hours. Lung retention at 48 hours was 79.7%DD +/- 12 and significant changes (p<0.05) were observed in the bladder, liver and spleen (*).

Comparing the lung retention with only labeled particles and both co-mixture doses we observed a decreasing trend over the 48 hour study period (Figure 3).

Figure 3. Lung retention over 48 hours for PS only and co-mixture doses (50µg and 500µg).

In addition to changes in secondary organ accumulation, we also observed potential lymphatic signal for both co-mixture doses (Figure 4). This potential signal is currently under further investigation.

Figure 4. Potential lymphatic signal after co-mixture delivery. Sites of accumulation are located near the mesenteric and inguinal lymph nodes.

To verify that the PM2.5 used for these studies is inducing the expected inflammation reported in the literature we looked at histological sections of the lungs for neutrophil content. (Work done by Laurel Plummer-Kent Pinkerton Lab) A dose-response relationship was observed for neutrophil and cellular influx in the lungs. Other work for this year included quantitative analysis of asthma PET data from the previous year and radio-labeling optimization for new models of PM. Future work includes utilizing new model of PM (20nm polystyrene) for both co-mixture and PS only experiments with either PET or SPECT. Also, repeating co-mixture experiment for ex vivo analysis of lymphatic vessels.

Specific aim 3: To evaluate potential mechanisms of PM transport across epithelial and endothelial barriers. Mechanisms of Endothelial and Epithelial transport using synthetic fluorescent and electron dense ultrafine particles. In previously reported work, we characterized the transport of 30 nm synthetic iron oxide particles across endothelial cell monolayers and demonstrated vesicular transport though caveolar like structures by 4 hours. We further characterized this as vesiculocaveolar transport using fluorescence tagged silica particles with confocal microscopy. We now extended this work to ask whether similar rates of transport occur in cultured human airway epithelial cells. We found, in contrast to studies with endothelial cells, airway epithelium did not allow transport during a 4 hour incubation period.

Specific aim 4: To characterize how fluid flow modulates particle interactions with vascular endothelial cells. (Barakat) In vivo, vascular endothelial cells are constantly exposed to flow. Research over the past two decades has demonstrated that fluid flow regulates endothelial cell phenotype. Furthermore, the details of this regulation depend on the type of flow to which the cells are subjected. Endothelial cells exposed to either steady (time-independent) or non-reversing pulsatile flow develop an anti-inflammatory phenotype that is largely protected from atherosclerosis. On the other hand, endothelial cells subjected to oscillatory flow exhibit a pro-inflammatory phenotype and are prone to the development of vascular pathology. Oscillatory flow occurs at arterial branches and bifurcations, and these regions correlate with the development of early atherosclerotic lesions.

We have been investigating how different types of flow modulate PM-induced inflammation in cultures of human aortic endothelial cells (HAECs). The experiments involve pre-shearing HAECs with either steady or oscillatory flow for a period of 24 hrs in order to establish either an anti- or pro-inflammatory cellular phenotype. At the end of the flow period, HAECs are exposed to PM for 4 hrs under static conditions, and the mRNA and protein levels of the three inflammatory markers ICAM-1, IL-8, and MCP-1 are subsequently assessed using real-time PCR, Western blotting, or ELISA. The results are compared to control cells that are not exposed to flow. Our initial experiments focused on laboratory nanoparticles, most notably zinc oxide (ZnO). These particles were selected because we had previously shown that they induce HAEC inflammation under static (no flow) conditions (Gojova et al., Environmental Health Perspectives, 2007). As illustrated in Figure 4.1, HAECs pre-conditioned with oscillatory flow exhibit a higher level of ZnO-induced inflammation, as measured by the mRNA expression levels of the inflammatory markers IL-8, ICAM-1, and MCP-1, than cells not exposed to flow or cells pre-conditioned with steady flow. The same behavior is also observed at the protein level (Figure 4.2). These results, if conformed in vivo, suggest that particle-induced inflammation would be exacerbated considerably in arterial regions susceptible to atherosclerosis (where oscillatory flow occurs) than in atherosclerosis-resistant regions.

Figure 4.1: Relative mRNA levels of three inflammatory proteins (A) IL-8, (B) ICAM-1 and (C) MCP-1 in HAECs as determined by real time PCR after ZnO treatment of HAECs grown under static condition or pre-sheared with steady laminar or oscillatory flow for 24h. Values are expressed as mean ± SEM. * denotes statistically significant increase (p<0.05) relative to static control (CT) group; # denotes statistically significant increase (p<0.05) relative to static ZnO group.

Figure 4.2: Inflammatory protein levels in HAECs after ZnO treatment. Cells were maintained under static conditions or pre-sheared with either steady or oscillatory flow for 24h. (A) IL-8; (B) MCP-1; (C) ICAM-1. IL-8 and MCP-1 secretion levels were determined by ELISA. ICAM-1 levels were established by Western blotting and were normalized relative to GAPDH which served as an internal control. Values are expressed as mean ± SEM. * and ** respectively denote statistically significant difference at p<0.05 or p<0.01 relative to static control (CT) group; # and ## respectively denote statistically significant difference at p<0.05 or p<0.01 relative to static ZnO group.

Why HAEC pre-conditioning with oscillatory flow leads to amplification of PM-induced inflammation remains unknown and is a question of critical importance. We have performed some transmission electron microscopy (TEM) experiments that may shed some light on this issue. As illustrated in Figure 4.3, ZnO nanoparticles induce considerable vacuole formation in HAECs that have not been pre-exposed to flow. This vacuolization appears even more pronounced in cells pre-conditioned with oscillatory flow but is largely absent in cells pre-conditioned with steady flow. In previous studies, we had shown that such vacuoles are involved in particle internalization into and transport within the cells. Therefore, it is possible that the results shown in Figure 4.3 reflect enhanced particle internalization in cells pre-conditioned with oscillatory flow and reduced particle uptake in cells pre-conditioned with steady flow.

Figure 4.3: Transmission electron micrographs demonstrating ZnO nanoparticle-induced vacuole formation in HAECs. Note the prominent vacuoles in cells that were either not pre-sheared or pre-sheared with oscillatory flow. These vacuoles are absent in cells pre-sheared with steady flow.

To follow up on the laboratory particle work described above, we have initiated studies on fine ambient particles derived either from the urban (Fresno) or rural (Westside) SAHERC testing sites. In these experiments, HAECs were exposed to particles for 4, 8, 12 or 24 hrs under static conditions after which cell viability and inflammation were assessed. Cell viability was measured using two techniques: a fluorescent assay (Live/Dead) which determines viable vs. dead cells and the WST-1 assay which measures mitochondrial metabolic activity. As depicted in Figure 4.4, exposure of HAECs to Fresno and Westside particles did not lead to significant cell death, even at the highest concentrations tested.

Figure 4.4: Effect of Fresno (Winter 2009 or Summer 2008 campaigns) and Westside (Winter 2008 campaign) ultrafine particles on HAEC viability as assessed by the fluorescence Live/Dead assay. Green denotes live cells while red denotes dead cells. The particles did not lead to significant cell death.

Although the particles did not lead to cell death, they did affect cell viability in more subtle ways as assessed by the mitochondrial activity-sensitive assay WST-1. As illustrated in Figure 4.5, ambient particles applied for a period of 24 hrs reduced HAEC viability in a dose-dependent fashion. Interestingly, particles from the Fresno Summer 2008 campaign had the most pronounced effect. At the highest concentration tested (6.6 µg/cm2, equivalent to 50 µg/ml), particles from this campaign reduced HAEC viability by ~50%.

Figure 4.5: Effect of Fresno (Winter 2009 or Summer 2008 campaigns) and Westside (Winter 2008 campaign) ultrafine particles on HAEC viability as assessed by the WST-1 assay which measures mitochondrial metabolic activity. The cells were exposed to the particles for 24 hrs.

We also assessed the effect of the ambient particles on HAEC inflammation. As shown in Figure 4.6, particles elicited cellular inflammation as assessed by mRNA levels of the three inflammatory markers ICAM-1, IL-8, and MCP-1. The inflammatory response was both time- and dose-dependent. Once again, the Fresno Summer 2008 particles had the most pronounced effect.

Figure 4.6: Effect of Fresno (Winter 2009 or Summer 2008 campaigns) and Westside (Winter 2008 campaign) ultrafine particles on HAEC inflammation as assessed by the mRNA levels of ICAM-1, IL-8, and MCP-1.

Particle interactions with cells are affected by the state of the particles as they make contact with the cell surface. Of particular importance in this regard is particle aggregation. In order to understand particle-cell interactions, it is important to establish the extent and dynamics of particle aggregation as well as how aggregation is affected by various parameters including the cell culture media and any proteins present in the media. Furthermore, because we are interested in flow, it is important to establish if flow has an effect on particle aggregation. We have used dynamic light scattering (DLS) to determine the aggregation dynamics of laboratory nanoparticles, most notably iron oxide (Fe2O3) and ZnO particles. Figure 4.7 depicts the DLS results of Fe2O3 and ZnO nanoparticle aggregation in deionized water. Previous studies had established that the primary particle size in both cases is of the order 60-100 nm. Several measures of particle aggregation are shown including the “effective” hydrodynamic diameter (based on the mobility of the aggregates within the measurement volume, a value heavily driven by larger aggregates), the “peak” diameter, and the “number average” diameter. These different measures represent different moments of the measured distributions. The results demonstrate that Fe2O3 particles in water (the “no sonication” bars in Figure 4.7) aggregate considerably. These aggregates are very effectively broken down after sonication for 1 min. Following sonication, the aggregates are slow to re-form. ZnO particles behave in a largely similar way to Fe2O3 particles, but the initial aggregates are significantly smaller.

Figure 4.7: DLS measurements to assess aggregation for A: Fe2O3 and B: ZnO nanoparticles in deionized water. Both types of particles aggregate significantly upon storage in water. Sonication effectively breaks down the aggregates, which are slow to re-form.

We have performed DLS measurements to assess the effects of media composition as well as the presence of serum in the media on Fe2O3 and ZnO particle aggregation dynamics following probe sonication. The results, depicted in Figure 4.8, demonstrate that particles aggregate to a greater degree in either PBS or cell culture medium (EBM) than in water. Furthermore, the presence of serum generally reduces aggregation. This effect is especially pronounced in water, where virtually no aggregation occurs. We hypothesize that the increased aggregation in PBS and cell culture media relative to water is attributable to differences in ionic strength. We also suspect that the reduction of aggregation by serum incubation is due to the fact that serum coating renders particle surfaces negatively charged, and the electrostatic repulsion limits the extent of aggregation.

Figure 4.8: Dynamic light scattering (DLS) measurements of aggregation dynamics following probe sonication for A: Fe2O3 and B: ZnO nanoparticles in different suspension media. Each bar corresponds to a time step of 1 hr. Measured values from three replicates were averaged at each time step. Data are mean ± SEM. PBS: phosphate-buffered saline. EBM: endothelial basal media. Complete: EBM plus all additives used in routine HAEC culture including serum.

We studied the aggregation of Fe2O3 particles in experiments that were intended to simulate our system for exposing cells to physiologic levels of shear stress. We conjectured that the combination of shear stress (potentially breaking up soft aggregates) and continuous mixing (altering the dynamics of particle collisions) would affect the kinetics of aggregation. Figure 4.9 shows the effective diameter measured by DLS for the static control in consecutive 30-minute periods and for the suspension circulated in the flow loop. The difference is readily apparent. The static samples reached an average effective diameter of 830 nm during the first 30 min, increasing to 1550 nm over four hrs. Fluid shear completely negated the increase in diameter over this period, yielding an effective diameter of 620 nm at the end of the four-hr treatment. We attribute this effect to shear-induced breakup of aggregates. A detailed microscopic study by Tolpekin et al. (2004) showed that dispersions of weakly aggregating particles under shear flow reach a steady-state maximum size. The size limit is determined by the balance between aggregation and breakup events, and is highly sensitive to both particle concentration and shear rate.

Figure 4.9: Effect of flow on aggregation of iron oxide nanoparticles in complete medium. Evolution of particle diameter over 4 hrs under static conditions. Also shown is particle diameter under flow conditions at the end of the 4-hr period. Data are mean ± SEM with n=4.

The previous experiment does not indicate the time scale for shearing of aggregates. Therefore, we performed a complementary experiment in which effective diameter was measured over the duration of flow (Figure 4.10). The results are approximated well by an exponential decay with a time constant of 1.1 hrs (dashed curve). Based on these results, we believe that the particles used in our cell culture system are probably minimally aggregated for most of the duration of a 6-24 hr flow experiment.

Figure 4.10: Effect of flow on aggregation of iron oxide nanoparticles in complete medium. Evolution of particle diameter over 4 hrs under flow conditions. Also shown are particle diameters under flow conditions at the beginning and end of the 4-hr period. Data are mean ± SEM with n=2.

Mechanisms of ultrafine PM transport across vascular barriers

The endothelial response to nanoparticulate silica exposure is dependent on particle size

Our previous work evaluating mechanisms of trans-endothelial transport of synthetic ultrafine particles demonstrated that PM in the nanoparticle range (20-50 nM) is transported across endothelial cell barriers relatively quickly through veiscuol-caveolar transport while larger particles (500 nM) are not. We have expanded these studies to evaluate the effect of size on cellular function using theoretically inert laboratory generated monosized silica. We have evaluated effects on barrier function by electrical conductance measurements (ECIS system Applied Biophysics). Results of these studies, described below, suggested that 20 nM silica particles were toxic in cultured endothelium. This suggests that internalization of PM is important in generating toxicity. We have further investigated the mechanism of nanofine silica toxicity by comparing intracellular ROS generation and assays for apoptosis.

ECIS

20nm and 500nm silica suspended in complete medium was applied to confluent monolayers of Human Pulmonary Microvascular Endothelial cells (HPMVEC) in ECIS chamber plates. The ECIS apparatus measured monolayer resistance (tests the integrity of intracellular junctions) and monolayer impedance (tests the integrity of connections between the basal side of the cell and the growth substrate). As shown in Figure 1, an image generated by the ECIS proprietary software, the ability of the monolayer to maintain resistance was obliterated by the application of 20nm silica at 50µg/ml within 12 hours of treatment application (p < 0.05). The figure also shows that 20nm silica at 10µg/ml has an effect on monolayer resistance but it is not as profound as the 50µg/ml treatment, though it differs significantly from control by 40 hours post treatment (p=0.012). Figure 2 shows the mean for each treatment when the experiment was repeated three times. There was no difference from control with the 500nm particles at either treatment dose.

Figure1:

Figure 2:

The monolayer impedance showed much the same trends. As shown in figure 3, the 20nm silica treatment at 50µg/ml showed the same precipitous decrease in monolayer impedance by 12 hours post-treatment (p<0.05) and the 10µg/ml treatment was significantly different from control at 40 hours post treatment. Figure 4 shows the mean data from three separate experiments. There was no difference from control after treatment with the 500nm silica particles at either dose.

Figure 3:

Figure 4:

Overall, the ECIS experiments show that there is a significant difference in the endothelial response to silica treatment that is based on particle size and both the intracellular junctions and cell-substrate junctions are affected.

Reactive Oxygen Species Generation Human Pulmonary Artery Endothelial Cells (HPAEC) exposed to 20nm and 500nm silica show an increase in ROS generation as measured with 5-(and-6)-carboxy-2',7'-dichlorofluorescein dictate (carboxy-DCFDA) dye (Molecular Probes) which passively diffuses into cells and is then cleaved by esterases active in the production of ROS. Figure 5 shows the number of pixels positive for intracellular ROS generation 3 and 24 hours after silica treatment. 0.25µM PMA was used as a positive control. 20nm silica at 50µg/ml shows a significant difference from PMA control at 3 hours (p=0.004) and 24 hours (p=0.023). At 24 hours, there is significant difference between 20nm silica 10µg/ml dose and the PMA control (p=0.0056). By 24 hours, there is very little ROS detection in the 20nm 50µg/ml treatment, leading to the conclusion that many of the cells have died at by this time point. At 24 hours, the 500nm silica at both 10µg/ml and 50µg/ml doses are not different from the PMA control, showing that ROS are generated after 500nm silica exposure, but more slowly than with the 20nm silica. These experiments have been repeated in HPMVEC, but have been unsuccessful due to the cell sensitivity to the dye loading protocol.

Figure 5:

Silica Cytotoxicity Cytotoxicity due to silica exposure was accessed by evaluating nuclear morphology using the fluorescent nuclear dye DAPI. Confluent monolayers of HMVECs were treated with 20 or 500 nM silica and evaluated by fluorescent microscopy with objective evaluation by image analysis using ImageJ software evaluation of digital images. We found nuclei of treated cells varied in size with 3 distinct groups: normal/large (nuclei with a pixel count of at least 2000 pixels), small/contracted (pixel count less than 2000 pixels), and fragmented. Since these findings suggested induction of cellular apoptosis, 0.1ng/ml TNFα was used as a positive control. Figure 6 shows that the total number of nuclei present on each coverslip is consistent between treatments, though there are fewer nuclei present 4 hours after treatment. Figure 7 shows that greater than 60% of the nuclei in the 20nm treatment groups were greater than 2000 pixels. Figure 7 also shows that there were fewer nuclei greater than 200 pixels in the 500nm treatment groups. Figure 8 shows that there are far more contracted nuclei (less than 2000 pixels) present after treatment with the 500nm particles, which is very similar to the positive control. There are also fewer contracted nuclei in the 20nm treatment groups. Figure 9 shows that the number of fragmented nuclei across the treatment groups is consistent, except for a small increase in the 500nm 50µg/ml treatment groups, but treatment groups had fewer fragmented nuclei than the positive control. Overall, these data show that there is a difference at the nuclear morphological level in how the 20nm and 500nm silica particles are exerting their effects on the HMVECs. 20nm particles seem to have little effect on the nuclear morphology, while 500nm particles cause a contraction to occur making the nuclei appear pyknotic. These results suggested differing mechanisms of cytotoxicity for 20 vs 500 nM silica. We further addressed these differences using an assay for apoptosis.

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Caspase-3/Annexin V Assay

To address the idea that the difference between 20nm and 500nm responses is due to a mechanistic difference in toxicity, HMVEC were grown to confluence on 12mm diameter coverslips coated with fibronectin then treated with 20nm or 500nm silica particles at either 10µg/ml or 50µg/ml dose. The cells were fixed after 1 hour or 24 hours of treatment and then assayed for Caspase 3 nuclear translocation and Annexin V activity of phosphotidyl serine flipping in the phospholipid bilayer. After 1 hour, there was no Caspase 3 or Annexin V activity detected. 1µg/ml tunicamycin was used as a positive control. After 24 hours of treatment, there is no detectable Caspase 3 in the nuclei of any treatment group, though it does appear that there is some Annexin V activity in both the 20nm and 500nm 50µg/ml silica treatments. Since there is no Caspase 3 activation, it is likely that the cells treated with both sizes of silica are undergoing ROS-driven oncosis rather than directed apoptosis.

Future Activities:

Journal Articles:

No journal articles submitted with this report: View all 3 publications for this subproject

Supplemental Keywords:

ambient air, ozone, exposure, health effects, human health, metabolism, sensitive populations, infants, children, PAH, metals, oxidants, agriculture, transportation, Air, Health, RFA, Risk Assessments, particulate matter, human health risk, toxicology, epidemiological studies, lung disease, long term exposure
, RFA, Health, Air, Risk Assessments, particulate matter, lung disease, long term exposure, epidemiological studies, PM, toxicology

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R832414 UC Davis Center for Children's Environmental Health and Disease Prevention

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832414C001 Project 1 -- Pulmonary Metabolic Response
R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
R832414C004 Project 4 -- Transport and Fate Particles
R832414C005 Project 5 -- Architecture Development and Particle Deposition

Top of Page

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.