2000 Progress Report: Pulmonary and Systemic Effects of Inhaled Ultrafine Particles in Senescent Rats with Cardiovascular DiseaseEPA Grant Number: R828046
Title: Pulmonary and Systemic Effects of Inhaled Ultrafine Particles in Senescent Rats with Cardiovascular Disease
Investigators: Elder, Alison C.P. , Zareba, Wojciech , Frampton, Mark W. , Oberdörster, Günter , Couderc, Jean-Philippe
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: March 24, 2000 through March 23, 2003 (Extended to September 23, 2003)
Project Period Covered by this Report: March 24, 2000 through March 23, 2001
Project Amount: $408,859
RFA: Airborne Particulate Matter Health Effects (1999) RFA Text | Recipients Lists
Research Category: Particulate Matter , Air , Health Effects
Objective:Epidemiological studies associate cardiovascular- and pulmonary-related morbidity and mortality in elderly individuals with cardiopulmonary disease with exposure to particulate air pollution. This consistent statistical link led to the hypothesis that inhaled particles can exert effects, either directly or indirectly, on the cardiovascular system. The three objectives of this research project test that hypothesis: (1) determine the effects of systemic bacterial toxin-induced stress on the pulmonary responses to inhaled ultrafine carbon particles; (2) measure the interactions between inhaled ultrafine carbon particles and systemically delivered bacterial toxin that affect peripheral blood cell activation, coagulability, and the acute phase response; and (3) determine the effects of inhaled ultrafine particles on cardiac function (heart rate, blood pressure, heart rate variability, arrhythmia, gene expression) after systemic bacterial toxin delivery. All studies are being carried out in healthy and compromised (hypertensive and hypertensive with heart failure) old rats. Endotoxin, a component of gram-negative bacteria, is given intraperitoneally in these studies to simulate a mild systemic infection, thus modeling a group with sensitivity to the effects of inhaled ambient air pollution. Ultrafine carbon particles generated by electric spark discharge in an argon atmosphere are used to simulate the carbonaceous fraction of the nucleation mode of ambient air particulate matter.
Progress Summary:Preliminary Findings on the Effects of Systemic Endotoxin on Extrapulmonary Tissues. Part of this project is focused on the interactions between inhaled ultrafine carbonaceous particles and systemically delivered endotoxin, as described above. Several pilot-type projects were performed to determine baseline levels of various coagulability parameters and acute phase reactants in various strains and age groups of rats. The time course and dose-related changes in plasma fibrinogen were used to determine an optimal intraperitoneal dose for endotoxin to be used in these studies. We found that the early response (4 hours) to injected endotoxin in rats is a decrease in the plasma fibrinogen concentration; the later response (24 hours) is an increase. The dose that was chosen (2 mg/kg) produced a 50 percent decrease in fibrinogen at 4 hours and a doubling in its concentration at 24 hours. This dose also did not change the body weight of exposed rats or appear to produce inactivity or labored breathing. Higher doses (e.g., 5 mg/kg) had these latter effects and, in some cases, caused mortality. In addition, preliminary studies showed that intraperitoneal endotoxin did not induce PMN accumulation in rat lungs or induce any changes in lactate dehydrogenase or b-glucuronidase activities or the concentration of total protein in lavage fluid.
Analyses of HRV. Part of the hypothesis that we are testing in these studies is that inhaled ultrafine particles can have effects on the heart alone or when combined with systemic endotoxin. Such effects may be observed by measuring exposure-related changes in heart rate, blood pressure, and heart rate variability. To accomplish this, we use radiotelemeters from DataSciences International (DSI) that transmit ECG waveforms, blood pressure waveforms, body temperature, and activity from conscious, untethered rats. In collaboration with Drs. Couderc and Zareba, custom programs have been developed that read the DSI-formatted data and perform time and frequency domain-based analyses of heart rate variability (HRV) on both the original ECG and blood pressure data. Validation of this methodology was achieved by using autonomic blockers (atropine or propranolol) in telemetered rats and by studying their effect on heart rate and HRV. A manuscript detailing the technical aspects of these analyses currently is being submitted for publication.
Other aspects of HRV analyses in unanesthetized rats were discovered by doing these preliminary experiments. We explored many commercially available analysis software packages and found our custom program to be superior for our needs. The main reason for this was that other programs failed to recognize many of the R-peaks in the tachogram, giving erroneous estimations of variability. Another lesson we learned from these analyses was that both the physical activity of the animals and the duration of the recording have significant effects on the stability of the HRV estimation. We defined a set of recommendations for HRV analyses based on these preliminary studies. These recommendations include that the recording duration must be greater than 5 minutes long and that the averaged heart rate between segments in an analysis must vary by less than 10 percent to obtain stable values.
With these analysis tools, we have done some pilot-like studies on the effects of systemic (i.p.) endotoxin by itself. It has been shown in humans that intravenous endotoxin causes a decrease in HRV, and we wished to see if similar changes could be measured in rats. Preliminary data analyses suggest dramatic decreases in HRV, and the results will be used in future experiments to target the appropriate post-exposure times for analyses and to gauge the magnitude of expected changes in HRV.
Effects of Systemic Endotoxin on the Responses to Inhaled Ultrafine Particles in Old Rats. One goal of this project is to characterize the response of lung cells to inhaled ultrafine carbonaceous particles after priming them with systemic endotoxin in old, healthy F-344 rats as well as in old spontaneously hypertensive (SH) rats and SH rats with heart failure (SHHF). A second goal is to learn how inhaled ultrafine particles affect extrapulmonary tissues by monitoring changes in blood cell oxidant production, coagulability, and the acute phase response with and without systemic endotoxin priming.
To begin answering these questions, we obtained healthy old F-344 rats and aged them to 23 months. Immediately before being exposed to ultrafine carbon particles (0 or 175 mg/m3, 6 hours; CMD=35 nm), rats were injected with endotoxin (0 or 2 mg/kg, i.p.). Bronchoalveolar lavage (BAL) fluid, BAL cells, and blood were obtained 24 hours after exposure to examine endpoints of inflammation, oxidant stress, coagulability, and the acute phase response.
Neither systemic LPS alone, inhaled ultrafine carbon particles alone, nor the combination of the two had an effect on the percentage of neutrophils (PMNs) in BAL fluid. Our preliminary studies predicted this outcome for endotoxin by itself. Likewise, no significant exposure-related effects were observed in the PMA-stimulated release of oxidants from BAL cells (chemiluminescence). However, ultrafine carbon particles were found to independently and significantly decrease the intracellular oxidation of a fluorescent probe (2',7'-dichlorodihydro-fluorescin diacetate [DCFD]) in BAL cells. Endotoxin had the opposite effect, namely that DCFD oxidation was increased significantly. The net effect of the combination of the two was to decrease intracellular oxidation. These results are interesting in that an effect on BAL cell oxidant production is found in response to systemic endotoxin and that it can interact with inhaled ultrafine particles. This cell-specific flow cytometric analysis was made possible by combining light scattering characteristics of the cells with their staining intensity for CD45 (pan-leukocyte surface antigen).
Systemically, endotoxin was found to increase the number of circulating PMNs, increase the concentrations of fibrinogen, thrombin-anti-thrombin (TAT) complexes, and interleukin (IL)-6, and to decrease the intracellular oxidation of DCFD in blood PMNs. Interestingly, inhaled ultrafine particles were found to have independent systemic effects, including a significant increase in blood PMN DCFD oxidation and a decrease in the number of circulating PMNs. Both of these effects were in the opposite direction from the effects of endotoxin. Inhaled ultrafine particles and systemic endotoxin also interacted in the extrapulmonary compartments (number of circulating PMNs; concentrations of fibrinogen and IL-6) and, in all cases, the resultant response was increased relative to control.
Future Activities:The experiments that are planned for the immediate future include the following:
- Perform a similar study as to what was described above for aged F-344 rats, but use SH rats instead to model cardiovascular disease. The same endpoints and post-exposure time will be examined.
- Perform a crossover study with telemetered SH rats that will be exposed to combinations of systemic endotoxin and inhaled ultrafine carbonaceous particles (carbon/20% Fe) to measure the effects on heart rate, blood pressure, body temperature, and HRV. The iron in such particles has been shown to be highly bioavailable. If these latter studies show dramatic effects in the first few hours after exposure, this may justify experiments to examine BAL and blood cells changes at a time point earlier than 24 hours after exposure.