2001 Progress Report: Pulmonary and Systemic Effects of Inhaled Ultrafine Particles in Senescent Rats with Cardiovascular Disease

EPA 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. , Oberd√∂rster, G√ľnter , 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, 2001 through March 23, 2002
Project Amount: $408,859
RFA: Airborne Particulate Matter Health Effects (1999) RFA Text |  Recipients Lists
Research Category: Particulate Matter , Air , Health Effects


The objectives of this research project are to: (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 toxins 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 (HRV), arrhythmia, gene expression) after systemic bacterial toxin delivery. 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. 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 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:

Analyses of HRV. Our hypothesis is that inhaled ultrafine particles can have effects on the cardiovascular system, either alone or in combination with systemic endotoxin. Such effects may be observed by measuring exposure-related changes in heart rate, blood pressure, and HRV. To accomplish this, we used radiotelemeters from DataSciences International (DSI) that have been described in previous reports. Our collaborators (Drs. Couderc and Zareba) have developed custom programs that extract heart rate and blood pressure data from the radiotransmitters, and then perform time and frequency domain-based analyses of HRV. In our preliminary analyses of data from rats, we confirmed that electrocardiogram (ECG) recordings obtained using the DSI system could provide reliable information about HRV in unrestrained rats (Couderc, et al., 2002). Software subsequently was designed (C++ platform; compatible with any window-based personal computer) to analyze HRV in short- and long-term rat ECG recordings. The program includes three different user interfaces allowing for: (1) a check of the recording quality, (2) a visual validation of the quasi-random signal (QRS) detection, and (3) the computation of time and frequency quantifiers of HRV. The software automatically reads files from the DSI System and creates its own data format for the analysis. Accurate detection of the R peak was validated by comparing it to two commercial systems. The detection visually can be checked by the user of the program. The frequency-domain parameters are computed based on the power spectral density of the tachogram; the time-domain parameters are automatically computed. Several frequency-domain parameters were included according to reported methodology:

(1) The energy of the high frequency (HF) band (0.8-3Hz in ms2) to estimate vagal control (parasympathetic tone), as well as the HF peak value (ms2/Hz) and its location (Hz).

(2) The low-frequency band (LF) to estimate sympathetic tone (0.1-0.7 Hz in ms2), as well as the peak value (ms2/Hz) and its location (Hz).

(3) The time-domain parameters are SDNN and RMSSD, both expressed in ms: SDNN is the standard deviation of the normal-to-normal RR intervals over a specific recording period; RMSSD is the root mean square of successive differences between normal-to-normal RR intervals.

In a preliminary study, the validity of the algorithm was confirmed for short-term ECGs (< 10 minutes). Because optimal length of short-term ECG recordings has not been determined for the analysis of HRV in rats, the stability of various parameters according to the length of the ECG signal was studied. This analysis led to the conclusion that at least 1,500 beats are needed to obtain reliable and reproducible estimations of HRV parameters. It also was found that the stability of the HRV parameters is dependent on the average heart rate: a maximum of 5 percent variation between averaged heart rate from continuous ECG segments is allowable to insure reliable HRV measurements.

Effects of Systemic Endotoxin and Inhaled Ultrafine Particles on Heart Rate Variability in Old Spontaneously Hypertensive Rats. Two separate experiments have been completed, in which radiotelemetered SH rats were exposed to inhaled ultrafine particles after systemic sensitization of the respiratory tract with endotoxin. The first study was conducted in aged SH rats (13-16 months) using a cross-over study design, where rats were exposed to combinations of systemic endotoxin and inhaled ultrafine iron-containing (25 percent) carbon particles. Preliminary analyses showed that the iron in these laboratory-generated ultra-fine particles (UFPs) is highly bioavailable. There were two rats per treatment group per period of the study, and each rat was exposed to all of the treatment combinations in a random manner over time.

Although the analysis of the ECG and blood pressure data obtained from this experiment is ongoing, preliminary results suggest changes in HRV associated with exposure. The contribution of the exposure components to these changes will be analyzed carefully with input from personnel in the Departments of Biostatistics and Cardiology.

The second study with telemetered SH rats was conducted using a newly designed on-road exposure system.

Effects of Systemic Endotoxin on the Responses to Inhaled Ultrafine Particles in Old Spontaneously Hypertensive 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 aged animals. 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. Lastly, we wished to investigate the impact of a compromised cardiovascular system on these responses.

In the last annual report, details were given of studies using aged healthy (F-344) rats. In subsequent studies, we obtained retired breeder SH rats and aged them inhouse for 11-14 months. Immediately before being exposed to ultrafine carbon particles (0 or 130 µg/m3, 6 hours; CMD=37 nm), the 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 assess endpoints of inflammation, oxidant stress, coagulability, and the acute phase response.

Neither systemic LPS nor inhaled ultrafine carbon particles, alone or combined, had an effect on the percentage of neutrophils (PMNs) in BAL fluid. Likewise, no significant exposure-related effects were observed in the phorbol myristate acetate (PMA)-stimulated release of oxidants from BAL cells (chemiluminescence). These results are similar to what was observed in old F-344 rats. However, unlike old F-344 rats, neither component nor their combination had an effect on the intracellular oxidation reactions in lavage macrophages.

Systemically, endotoxin was found to increase the number of circulating PMNs, the intracellular oxidation of 2,7-dichlorofluorescein acetate (DCFD) in blood PMNs, the concentration of plasma fibrinogen, the viscosity of whole blood and plasma, and the unstimulated release of TNF-alpha by whole blood cells in culture. It decreased the concentration of plasma thrombin-anti-thrombin (TAT) complexes and the stimulated release of TNF-alpha by cultured whole blood cells. Interestingly, inhaled UFPs independently and significantly increased the concentration of plasma TAT complexes and in vitro TNF-alpha release of resting whole blood cells. Plasma levels of fibrinogen and IL-6, as well as the stimulated release of TNF-alpha by blood cells in culture, significantly were decreased by UFPs. Many of the independent effects of systemic endotoxin and inhaled UFPs were in opposite directions. UFPs and endotoxin also interacted to increase, relative to control, the oxidation of DCFD in blood PMNs and the resting blood cell release of TNF-alpha, and decrease the stimulated blood cell release of TNF-alpha. In the case of in vitro TNF release, these interactions resulted in remarkable amplification of the effects of individual components.

Effects of Ultrafine Highway Aerosols in Aged, Compromised Rats. The Rochester PM Center recently undertook a series of studies in collaboration with David Kittelson (University of Minnesota) using his newly designed system for exposures to on-road highway aerosols. This system allows the collection of aerosols and the enrichment of the ultrafine mode without chemical or physical modification of the particles. It was possible to collaborate with the Center such that some limited studies were supported by this U.S. Environmental Protection Agency Science To Achieve Results (STAR) grant. A portion of the overall study involved the systemic administration of endotoxin or saline in telemetered SH rats that were then exposed to on-road aerosols or filtered air (exposures separated by 4-5 weeks). ECG, blood pressure, and HRV parameters were measured in these animals. In addition, the study was designed such that numerous extrapulmonary parameters were measured in exposed aged F-344 rats, and these results can be compared to ones obtained in studies supported by this investigation. Preliminary analyses show the effects of exposure on various endpoints, but the contribution of specific exposure components is the subject of ongoing analyses. The ECG and blood pressure data also are undergoing analysis at this time.

Future Activities:

Our future activities during this investigation are to: (1) continue with the analysis of telemetry data from the cross-over study, in which SH rats were exposed to combinations of systemic endotoxin and inhaled UFPs; and (2) analyze the ECG and blood pressure data from the SH rats exposed to systemic endotoxin and on-road aerosols.

Journal Articles:

No journal articles submitted with this report: View all 11 publications for this project

Supplemental Keywords:

urban air quality, coagulation, age, cardiotoxicity, health effects, sensitive populations, elderly, stressor., RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Health Risk Assessment, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, genetic susceptability, Atmospheric Sciences, sensitive populations, urban air, fine particles, human health effects, morbidity, respiration, exposure and effects, cardiovascular vulnerability, exposure, pulmonary disease, animal model, air pollution, carbon particles, cardiopulmonary response, chronic health effects, lung inflammation, particulate exposure, bacterial toxin induced stress, Acute health effects, elderly, PM, cardiotoxicity, inhaled particles, mortality, senescent rats, aerosols, air quality, human health risk, ultrafine particles, ultrafine carbon, toxics, animal inhalation study

Progress and Final Reports:

Original Abstract
  • 2000 Progress Report
  • 2002
  • Final Report