2007 Progress Report: Assessing Toxicity of Local and Transported Particles Using Animal Models Exposed to CAPs

EPA Grant Number: R832416C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R832416
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Harvard Particle Center
Center Director: Koutrakis, Petros
Title: Assessing Toxicity of Local and Transported Particles Using Animal Models Exposed to CAPs
Investigators: Godleski, John J. , Koutrakis, Petros
Institution: Harvard University
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, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The objective of Project 3 is to differentiate the toxicological effects of locally emitted and transported particles. To do so, short-term 5 hr animal exposures to concentrated ambient fine particles (CAPs) are conducted during the time periods of 5-10 am and 10:30 am-3:30 pm. Starting inhalation exposures at 5 am, before significant vertical mixing takes place, permits us to capture particles predominantly from local sources. We expect that exposures starting about 10:30 am are relatively more enriched in transported particles. Specific biologic outcomes will include: breathing patterns, indicators of pulmonary and systemic inflammation, blood pressure, endothelin-1, endothelial nitric oxide synthase, atrial naturetic peptide, in vivo oxidant responses in the heart and lung, and quantitative morphology of lung and cardiac vessels. To control for circadian variations all outcomes are assessed during both time periods, in relation to those of filtered air (sham) exposures as well as those of positive controls using particles of known toxicity. Animal exposures are characterized using continuous measurements of particle mass, size, number, and black carbon, as well as integrated measurements of particle mass, sulfate, elements, and organics. Strains of rats used include Sprague Dawley (SD), which we have used extensively in previous CAPs studies, Spontaneously Hypertensive Rats (SHR), a sensitive model in many studies, and Wistar Kyoto (WKY) the strain control for SHR rats.

Progress Summary:

Table 1 shows the target number of animals for each outcome in the exposure plan for these studies. Because limited numbers of animals can be used in each outcome at a time, 21 repetitions of these experiments were carried out.

Table 1. Target numbers of outcomes per group of rats with any 5 hr-exposure

Treatment
Group
5-10 AM

Total #
Exposed

Blood

Buxco

In Vivo
Oxidant

RNA, BAL
or
Morphology

CAPs

5

3

5

2

3

SHAM

5

3

5

2

3

When
Assessed

 

24hrs
After
Exposure

During
Exposure

< 30 min
after
Exposure

24 hrs
After
Exposure

Table 2 lists the number of animals studied for breathing pattern, BAL, and chemiluminescence outcomes, respectively in each treatment group for each strain. The numbers of animals studied provided sufficient power to find differences in each of these groups based on our previous studies.

Table 2. Numbers of animals of each Strain assessed for breathing pattern data (Buxco), Broncholaveolar Lavage (BAL), and In Vivo Chemiluminescence (IVC) either in the early morning (AM) or mid-day and afternoon (PM)

Outcome
Assessed

Group/
Exposure

All AM

All PM

SD AM

SD PM

WKY AM

WKY PM

SHR AM

SHR PM

 

 

 

 

 

 

 

 

 

 

Buxco

CAPs

105

105

50

50

28

27

27

28

Buxco

Sham

105

105

50

50

28

27

27

28

 

 

 

 

 

 

 

 

 

 

BAL

CAPs

63

63

30

30

15

18

18

15

BAL

Sham

63

63

30

30

17

16

16

17

 

 

 

 

 

 

 

 

 

 

IVC

CAPs

21

22

12

11

4

5

5

6

IVC

Sham

21

22

11

11

5

5

5

6

Exposure data from all of these experiments are shown in Table 3. For these experiments the CAPs mass concentrations (Early - 505.8 ± 75.8 and Late 407.2 ± 45.7) were slightly higher than previous published studies from our laboratory, but not significantly different from each other using a paired two-tailed t-test. However, it is clear that there are significant differences in black carbon and elemental carbon between the early and late exposure, supporting the premise that the early exposure would be more influenced by local (primarily traffic sources) whereas the exposures later in the same day were more likely to contain transported particles. Since these experiments were not done in the summer, there is no significant difference in sulfur between the morning and afternoon. In these studies, in addition to greater black and elemental carbon in the morning, iron, nickel, and copper levels were also significantly higher in the morning.

When these data were analyzed using the ratio of a specific component to the total fine mass (or fraction of the component) essentially the same findings were observed with more robust p-values. Thus, elemental carbon, copper, and nickel were significantly higher in the morning. In addition, several components were found to be significantly higher in the afternoon. These include sodium, potassium, magnesium, manganese, and silicon. The sulfur fraction was higher in the afternoon than the morning, but this difference was not significant. Therefore, while statistically significant differences were found between AM and PM exposures, the differences were modest.

Table 3. CAPs mass and component concentrations during the early and late exposure periods (concentrations are reported in micrograms per m3)

Measures

Early mean ± SE

Late mean ± SE

p =

CAPs Mass

505.8 ± 75.8

407.2 ± 45.7

0.083

*Black Carbon Mass

10.5 ± 0.9

7.3 ± 1.1

0.002

*Elemental Carbon

22.5 ± 2.6

16.5 ± 2.8

0.032

Organic Carbon

72.2 ± 6.9

67.3 ± 6.9

0.505

Total Carbon

94.9 ± 9.0

83.8 ± 9.5

0.261

Sodium

8.9 ± 2.6

10.4 ± 2.9

0.181

Chlorine

9.7 ± 3.6

13.8 ± 6.2

0.174

Silicon

9.5 ± 1.6

8.8 ± 1.1

0.448

Aluminum

3.4 ± 0.6

3.1 ± 0.4

0.437

Sulfur

37.0 ± 5.9

35.7 ± 5.3

0.832

Calcium

6.3 ± 0.9

6.1 ± 0.8

0.761

Titanium

0.33 ± 0.05

0.26 ± 0.04

0.108

Potassium

2.8 ± 0.3

2.7 ± 0.3

0.465

*Iron

13.3 ± 1.9

9.7 ± 1.0

0.035

Zinc

1.0 ± 0.1

1.1 ± 0.1

0.857

*Nickel

0.07 ± 0.015

0.04 ± .007

0.033

Vanadium

0.03 ± 0.01

0.01 ± .009

0.144

Magnesium

1.2 ± 0.3

1.4 ± 0.3

0.293

*Copper

0.4 ± 0.06

0.2 ± 0.03

0.019

Manganese

0.2 ± 0.03

0.3 ± 0.04

0.349

*EC Percent of Mass

5.3 ± 0.5

3.9 ± 0.4

0.007

OC Percent of Mass

17.8 ± 1.7

18.7 ± 1.5

0.586

TC Percent of Mass

23.1 ± 2.1

22.5 ± 1.8

0.753

*Sodium Percent of Mass

2.3 ± 0.7

2.7 ± 0.7

0.026

Chlorine Percent of Mass

2.3 ± 0.9

3.1 ± 1.3

0.180

*Silicon Percent of Mass

2.5 ± 0.4

2.9 ± 0.5

0.040

Aluminum Percent of Mass

0.89 ± 0.17

1.03 ± 0.19

0.107

Sulfur Percent of Mass

7.5 ± 0.7

8.6 ± 0.8

0.123

Calcium Percent of Mass

1.6 ± 0.3

2.0 ± 0.4

0.056

Titanium Percent of Mass

0.09 ± 0.01

0.09 ± 0.02

0.814

*Potassium Percent of Mass

0.68 ± 0.07

0.77 ± 0.08

0.017

Iron Percent of Mass

3.3 ± 0.4

2.9 ± 0.4

0.226

Zinc Percent of Mass

0.25 ± 0.03

0.29 ± 0.04

0.429

*Nickel Percent of Mass

0.019 ± 0.004

0.009 ± 0.001

0.025

Vanadium Percent of Mass

0.007 ± 0.002

0.003 ± 0.002

0.074

*Magnesium Percent of Mass

0.29 ± 0.07

0.39 ± 0.07

0.048

*Copper Percent of Mass

0.09 ± 0.01

0.07 ± 0.01

0.043

*Manganese Percent of Mass

0.06 ± 0.01

0.08 ± 0.01

0.040

For all rat strains, breathing pattern and in vivo chemiluminescence studies show significant differences between CAPs and Sham exposures, as we have previously reported. With breathing pattern there is an increase in respiratory frequency with concomitant shortening of the time of inspiration and expiration. Statistical modeling was used to assess the size and strength of association between CAPs or Sham exposure and each respiratory outcome. Additive mixed models were applied to 10-minute averaged data collected from all CAPs and Sham animals during AM or PM exposure. A form of the repeated measures model for longitudinal data, additive mixed models represent an extension of linear regression models that (1) estimate potentially non-linear effects of independent variables, and (2) include random effects as independent variables in order to account for clustering of observations that result from repeated measurements being taken on the same animal during the same exposure period. For each outcome, additive mixed models were fit using as independent variables (1) a general nonlinear mean trend for sham animals over the exposure period, (2) an exposure indicator, which implies a constant shift in the mean trend due to the exposure, and (3) random animal effects reflecting animal-to-animal heterogeneity that results in correlation among 10-minute averages taken on the same animal over time. All models were fit using the gamm function in R software (R Development Core Team 2004). Finally, a more general model that relaxed the assumption of a constant shift due to exposure was also fit to the data. This model specified distinct mean trends over the exposure period for the sham and exposed animals, again including random animal effects to account for the repeated measurements taken on each animal. The difference between these estimated trends represents the time-varying effect over the exposure period.

All three strains show an increase in respiratory frequency with CAPs exposure, with slight but significant strain differences. The differences between the AM CAPs and Sham exposures and the PM CAPS and Sham exposures are similar. These data are shown in Figure 2 in which the mean respiratory response for the number of animals per group listed in Table 2 is plotted over the entire exposure period. To simplify the data presentation, only the means are shown for frequency and inspiratory time.

Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time

Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time
 
Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time
Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time
 
Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time
Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time
 
Figure 2. Respiratory breathing patterns illustrating frequency and inspiratory time

Analyses of BAL data did not show significant differences between morning and afternoon exposures for any BAL parameter.

Data from the in vivo chemiluminescence studies are presented in Table 4. These studies show that in these exposures, the lung had significant effects of CAPs exposures whereas chemiluminescence changes in the heart did not reach significance. There were significant changes with both early and late exposures and analyses showed that these were not significantly different.

Table 4. In vivo chemiluminescence of the heart and lungs with early and late exposures

Sites

Effect

CAPs – Sham
Difference
Mean ± SE

 

P value

Heart

All Exposures

5.47 ± 4.2

 

0.197

 

Testing whether
CAPs effects are different
in the 3 species

 

 

0.644

 

Testing whether early and late
CAPs effects are different

 

 

0.366

 

 

 

 

 

Lung

All Exposures

10.28 ± 3.07

 

0.001

 

Testing whether
CAPs effects are different
in the 3 species

 

 

0.640

 

Early Exposure

12.28 ± 4.41

 

0.007

 

Late Exposure

8.38 ± 4.31

 

0.055

 

Testing whether early and late
CAPs effects are different

 

 

0.529

Because the CAPs effects from the different rat strains were not significantly different from one another, and the CAPs effects during early and late exposures were also not significantly different, component analyses with the heart and lung data estimated overall concentration slopes not segregated by strain or early/late exposures. In these analyses, no cardiac effects were found using univariate or multivariate analyses. Table 5 illustrates many components with significant univariate relationships to lung chemiluminescence, but none of these were significant in multivariate analyses.

Table 5. Effects of specific exposure components on in vivo lung chemiluminescence.
using univariate analyses

Element/
Component

Estimate ± SE

P value

CAPs Mass

0.012 ± 0.005

0.027

Organic Carbon

0.106 ± 0.038

0.0064

Elemental Carbon

0.298 ± 0.121

0.016

Al

1.573 ± 0.739

0.036

Si

0.577 ± 0.267

0.034

S

0.132 ± 0.061

0.033

Fe

0.509 ± 0.218

0.022

Overall, the analyses thus far largely confirm our earlier studies and findings with CAPs exposures in Boston. It is of particular interest that, apparently, despite statistically significant differences in the composition of early and late exposures on given days, there is no significant difference in toxicity. It seems that our studies have adequate statistical power, since we were able to detect significant diurnal differences with respiratory patterns, significant strain differences, as well as CAPs vs Sham differences. Since both early and late exposures show significant toxicity, with no significant difference in the biological outcomes between the two exposure periods, these results do not suggest any difference in the toxic potential of local and transported sources.

Future Activities:

We are doing exposures of WKY and SH rats, monitoring blood pressure, electrocardiogram, and blood parameters. We expect to complete these exposure sets by December 2007, and have much of the exposure data analyzed. In the coming year we expect to complete analyses of all the outcomes. This project is scheduled to be finished within the first two and a half years of the grant.

Journal Articles:

No journal articles submitted with this report: View all 14 publications for this subproject

Supplemental Keywords:

concentrated air particles, acute cardiovascular effects, coarse particles, fine particles, vascular dysfunction,, RFA, Health, Scientific Discipline, Air, particulate matter, Environmental Chemistry, Health Risk Assessment, Risk Assessments, ambient air quality, atmospheric particulate matter, human health effects, chemical characteristics, automobile exhaust, airborne particulate matter, cardiovascular vulnerability, chemical composition, biological mechanism , biological mechanisms, ambient particle health effects, mobile sources, human exposure, autonomic dysfunction, oxidative stress

Relevant Websites:

http://www.hsph.harvard.edu/epacenter Exit

Progress and Final Reports:

Original Abstract
  • 2006 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010
  • Final Report

  • Main Center Abstract and Reports:

    R832416    Harvard Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832416C001 Cardiovascular Responses in the Normative Aging Study: Exploring the Pathways of Particle Toxicity
    R832416C002 Cardiovascular Toxicity of Concentrated Ambient Fine, Ultrafine and Coarse Particles in Controlled Human Exposures
    R832416C003 Assessing Toxicity of Local and Transported Particles Using Animal Models Exposed to CAPs
    R832416C004 Cardiovascular Effects of Mobile Source Exposures: Effects of Particles and Gaseous Co-pollutants
    R832416C005 Toxicological Evaluation of Realistic Emission Source Aerosol (TERESA): Investigation of Vehicular Emissions