Final Report: Lung Injury from Inhaled Ultrafine Particles in Compromised Rats of Old Age: Influence of Priming and Adaptation

EPA Grant Number: R826784
Title: Lung Injury from Inhaled Ultrafine Particles in Compromised Rats of Old Age: Influence of Priming and Adaptation
Investigators: Oberdörster, Günter , Elder, Alison C.P. , Finkelstein, Jacob N.
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: September 1, 1998 through September 20, 2001 (Extended to September 20, 2002)
Project Amount: $606,545
RFA: Health Effects of Particulate Matter and Associated Air Pollutants (1998) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Air , Health Effects , Particulate Matter

Objective:

The overall objective of this research project was to evaluate oxidative stress responses in the compromised aged organism induced by inhaled ultrafine carbonaceous particles in combination with transition metals. Specific objectives were to investigate the characterization of models of emphysema and allergic sensitization as well as of respiratory infection, and the in vitro evaluation of age-related differences in mechanisms of particle effects.

Epidemiological studies found that ambient particles at low concentrations are associated with mortality and serious morbidity in susceptible parts of the population (e.g., the elderly with cardiorespiratory conditions). Based on results of our previous studies, we hypothesize that the ultrafine particles (UFPs) (particles < 100 nm)-as part of the ambient fine mode particles-caused oxidative stress in the compromised aged organism, which escalates into a significant pulmonary inflammation in the primed organism. Priming of humans can occur following exposures to a variety of agents, including endogenous (intestinal) as well as exogenous (airborne) exposure to endotoxin, or exposure to the influenza virus. Some experimental studies have shown that depending on the timing of a second stimulus after priming, either a state of sensitization with heightened response or a state of tolerance with lowered response can occur. We focused on UFPs (< 100 nm) because: (1) at low mass concentrations, they have a high number concentration and large surface area, which is of toxicological significance; (2) some of them (20 nm) have a higher deposition efficiency in the alveolar region than any other particle size; (3) they may penetrate rapidly across the pulmonary epithelium and reach interstitial sites; and (4) they generally are more biologically reactive than larger particles. We also hypothesized that UFPs translocate to extrapulmonary organs to a significant degree via access to the pulmonary interstitium and across endothelial epithelium into the blood circulation.

Summary/Accomplishments (Outputs/Outcomes):

We conducted studies comparing UFP effects in old and young rats with endotoxin priming and co-exposure to ozone. A significant inflammatory cell influx into the alveolar space was observed for both age groups, whereby the combination of inhaled ultrafine elemental carbon particles and ozone after endotoxin priming resulted in the greatest increase of neutrophils in the lung lavage fluid. When assessing oxidant release from lung lavage cells 24 hours after the exposure, it was found that the unstimulated and stimulated release of reactive oxygen species (ROS) from lavage inflammatory cells correlated well with the neutrophil response in the lavage. There were significant main effects of the ultrafine carbon particles, as well as a consistent interaction between ultrafine and ozone as determined by analysis of variance. However, this interaction was in the opposite direction in young rats compared to old rats: UFPs and ozone interacted such that ROS activity was depressed in young rats, whereas it was enhanced in old rats, indicating age-dependent functional differences in the elicited pulmonary inflammatory cells. These results demonstrate that ultrafine carbon particles inhaled for short periods of time can induce significant pulmonary inflammation and oxidative stress that are modified by age, copollutant, and a compromised respiratory tract. The results show that the aged organism is at greater risk of oxidative stress when exposed to UFPs.

Studies with our intratracheal human influenza virus model comparing young and old rodents also showed that old age significantly increases the effect of inhaled ultrafine carbon particles. Both the endotoxin and the influenza virus model appear to be relevant models to evaluate effects of inhaled particles. We also performed studies using a mixed carbon/iron UFP with the same multigroup study design. Although the bioavailability of iron in these particles was determined prior to the study, in an in vitro citrate assay-showing that these particles exhibited very high biological activity-we did not find that exposure to a mixed carbon/iron particle as opposed to carbon alone showed a significantly different response.

Adaptation to endotoxin is a well-known phenomenon, and because our priming model involved the inhalation of endotoxin, we determined potential differences in young versus the aged organism with respect to their ability to adapt to inhaled endotoxin. As described above, in our priming studies, young and old rats and mice were exposed to low-dose endotoxin by inhalation on 3 consecutive days, and on day 4, received a high dose of inhaled endotoxin. The "nonadapted" group of animals were sham exposed to saline aerosols, and received only the high endotoxin dose on day 4, and another group received only filtered air as unexposed controls. Twenty-four hours after the last exposure, lung inflammatory parameters showed that an adaptive response had occurred in both rats and mice. Adapted animals showed significantly fewer bronchoalveolar lavage neutrophils compared to nonadapted animals. Likewise, the spontaneous and stimulated oxidant released from lavaged cells was significantly lower in the adapted as compared to the nonadapted animals. It also was found that adaptation to inhaled endotoxin occurs in the older age group. However, whereas the adaptive response in older mice was clearly evident, there is much greater variability in this response in the aged rats. The importance of tolerance development after lipopolysaccharide (LPS) inhalation lies in the fact that there also may be a development of cross tolerance towards the inflammatory response of inhaled particles. Indeed, we found in a pilot type study that particles administered after inhalation of endotoxin, elicited a lower response when given 24 hours postendotoxin, whereas immediately after endotoxin, the inflammatory response was significantly increased. This is of importance for our LPS priming model, implying that particles have to be administered shortly after LPS exposure to exploit the priming event of activated cells, whereas an adaptive response in the late phase after the priming event would attenuate the particle response.

We have developed a method to generate 13C UFPs for use in rat dosimetry studies. After several methodological improvements, we performed a study to determine whether ultrafine elemental carbon particles translocate to the liver and other extrapulmonary organs following inhalation as singlet particles by rats. We generated 13C UFPs as an aerosol with a median diameter of 29 nm using electric spark discharge of 13C graphite electrodes in argon. Nine Fischer-344 rats were exposed to these particles for 6 hours in whole-body inhalation chambers at concentrations of 180 and 80 µg/m3; three animals were killed at 0.5, 18, and 24 hours postexposure. Six unexposed rats served as controls. Lung lobes, liver, as well as heart, brain, olfactory bulb, and kidney were excised, homogenized, and freeze dried for analysis of the added 13C by isotope ratio mass spectrometry. Organic 13C was not detected in the 13C particles. The 13C retained in the lung at 0.5 hours postexposure was about 70 percent less than predicted by rat deposition models for UFPs, possibly indicting some rapid initial elimination from the lung. Lung levels did not change significantly during the 24-hour postexposure period. Normalized to exposure concentration, the added 13C per gram of lung on average in the postexposure period was approximately 9 ng/g organ/µg/m3. Significant amounts of 13C had accumulated in the liver by 0.5-hours postinhalation only at the high exposure concentration, whereas by 18 and 24 hours postexposure, the 13C concentration of the livers of all exposed rats was more than one-third of the 13C concentration found in the lung. Considering the approximately 10-fold greater weight of the liver compared to the lung, the 13C amount in the liver was approximately 4-fold greater than in the lung by 18 and 24 hours after exposure. No significant increase in 13C was detected in the other organs that were examined. These results demonstrate effective translocation of ultrafine elemental carbon particles to the liver by 1 day after inhalation exposure. Potential translocation pathways include direct input into the blood compartment from carbon UFPs deposited throughout the respiratory tract as well as uptake into the blood circulation of UFP particles from the gastrointestinal tract after swallowing. These studies also indicated a slight nonsignificant increase of 13C in the olfactory bulb. In a subsequent pilot study with a prolonged 7-day postexposure period, 13C of lung and extrapulmonary organs was analyzed on day 1 and day 7 post-exposure to 13C UFP. Again, results showed significant amounts of added 13C in the liver on day 1, but no longer on day 7. However, on day 7, significant increases in added 13C in heart, brain, and olfactory bulb were found. This prompted us to design a follow-up study focusing on the uptake of inhaled solid UFP into the central nervous system to be conducted within the Rochester PM Center.

We have developed in vitro models that will be useful in understanding the mechanism of UFP-induced gene expression in various cell types. Additionally, these models should prove useful in studies of other size fractions, and we are using them to attempt to differentiate between particles of differing toxicity and activation potential. One objective of these experiments is to define the mechanisms of cellular activation, the effects of age or prior activation on cytokine gene activation, and the differential responses of epithelial cells and macrophages to particles of different sizes.

In our studies focusing on age effects, we compared macrophage production of cytokines following endotoxin and particle exposure to cells from rats 22-27 months old to cells from rats 10-12 weeks old. When macrophages from young rats were treated with endotoxin, a clear dose response, with the chemokine MIP-2 as the endpoint, was obtained. A similar dose-response relationship was observed with carbon UFPs alone. When the two stimuli were combined, no enhanced effect was observed except at the highest dose of particles. When a similar study was performed with macrophages from "old rats," a number of clear differences were observed. Interestingly, baseline (unstimulated) production of MIP-2 (and the cytokine TNF) was elevated 30-50 percent in these cells. In addition, response to endotoxin was enhanced at every dose. Response to particles alone was similar to that observed in young cells. Most significant, in the context of our investigation of age effects in mice alveolar macrophages, and the ability of particles to induce effects at low dose, was the fact that in the aged animals, co-administration of particles and endotoxin led to synergistic effects at the lowest dose of particles in contrast to rats.

Previously, we had proposed using isolated cells from aged animals as a model to study age-related, particle-induced gene activation. We have further characterized the aged mice and begun to evaluate the utility of isolated cells from these mice as a model of aging. Because one of the outcomes being measured was changes in cytokine gene expression, we first measured baseline cytokine levels in the plasma of aged mice. Both TNFalpha and IL-6 were significantly elevated in the aged mice.

An important advance was the development and use of laboratory-generated UFPs containing various metals. We compared macrophage production of cytokines following LPS and particles (with C/Fe) incubation with cells from mice 20-22 months old and mice 8-10 weeks old. Baseline MIP-2 and TNF was significantly elevated in cells from the old mice. After stimulation, the old mice also were found to be more responsive.

When particles and LPS were combined as a stimulus, an enhanced effect is observed only in the "old" cells from the older animals, except at the highest dose of particles. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose, was the fact that in the aged animals, co-administration of particles and LPS led to synergistic effects at the lowest dose of particles. This result is somewhat similar to results obtained in the in vivo studies, where enhanced response to combined insult was noted in aged rats.

We also have developed reagents and approaches that would allow extension of our in vitro studies to human cells, while also developing a test of our oxidant stress hypothesis. We have developed a human lung cell line, A549, that was stably transfected with a reporter gene that other studies have shown was responsive to oxidant stress. Experiments confirmed particle-induced increase in reporter gene activity with a peak at 6 hours posttreatment.

Our studies have confirmed and established several important facts about the effects and behavior of inhaled UFPs. We conclude that:

· Age: the aged organism is at greater risk to experience oxidative stress.

· Pollutant co-exposure: combined exposure of UFPs with the oxidant gas ozone results in significant interactions and greater effects.

· Priming of the respiratory tract: priming of rodents to mimic respiratory tract infections in humans increases effects of inhaled UFPs; this is consistent with epidemiological findings of PM effects.

Additional findings of these studies are that:

· The age effect is even observed in vitro: primary cells of aged rodents show significantly greater effects to the same dose in vitro than cells from young rodents;

· Inhaled UFPs can induce systemic effects; an acute phase response and effects on circulating neutrophils were found.

· Inhaled solid UFPs gain access to the blood circulation and translocate to extrapulmonary organs.

Collectively, our studies show that inhaled solid UFPs consisting of elemental carbon or carbon/iron can induce respiratory and systemic effects in rodents. A crucial issue, which needs further investigation, is whether specific UFPs in the ambient air will cause similar effects. Because our laboratory-generated particles consisted only of elemental carbon and-in the mixed-particle studies-carbon/Fe, it is likely that urban UFPs emitted from internal combustion engines, and consisting to a large degree of organic compounds will cause even greater effects. Therefore, our results contribute to the mounting evidence that UFPs can cause significant adverse effects, particularly in the compromised organism. This has to be considered when regulatory decisions are made aimed at reducing urban particulate air pollution by reducing particulate mass. UFPs contribute very little to urban air PM by mass; in fact, reducing PM mass (fine PM mass) based on regulatory standards can increase ultrafine number concentrations significantly. This is because UFPs cannot agglomerate as effectively onto fine-mode particles, which are a natural sink for UFP elimination. Because ambient UFPs, particularly those smaller than 20 nm, are composed, to a high degree, of organic carbon compounds, future studies should focus on organic carbon as well to determine its contribution to UFP effects. Such studies should make use of methods to either expose laboratory animals directly to relevant ambient UFPs or generate in the laboratory relevant surrogate particles containing organics that have been found on ambient UFPs.


Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other project views: All 24 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Elder ACP, Finkelstein J, Johnston C, Gelein R, Oberdorster G. Induction of adaptation to inhaled lipopolysaccharide in young and old rats and mice. Inhalation Toxicology 2000;12(3):225-243. R826784 (Final)
  • Abstract: InformaWorld Abstract
    Exit
  • Journal Article Elder ACP, Gelein R, Finkelstein JN, Cox C, Oberdorster G. Pulmonary inflammatory response to inhaled ultrafine particles is modified by age, ozone exposure, and bacterial toxin. Inhalation Toxicology 2000;12(Suppl 4):227-246. R826784 (Final)
  • Abstract from PubMed
  • Abstract: InformaWorld Abstract
    Exit
  • Journal Article Elder ACP, Gelein R, Azadniv M, Frampton M, Finkelstein J, Oberdorster G. Systemic interactions between inhaled ultrafine particles and endotoxin. Annals of Occupational Hygiene 2002;46(Suppl 1):231-234. R826784 (Final)
    R827354 (Final)
    R827354C003 (Final)
    R827354C004 (Final)
    R828046 (Final)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
  • Full-text: Oxford Journals-Full Text PDF
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  • Abstract: Oxford Journals-Abstract
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  • Journal Article Elder ACP, Gelein R, Azadniv M, Frampton M, Finkelstein J, Oberdorster G. Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains. Inhalation Toxicology 2004;16(6-7):461-471. R826784 (Final)
    R827354 (Final)
    R827354C003 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R827354C005 (Final)
    R828046 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor&Francis-Abstract
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  • Journal Article Oberdorster G. Toxicology of ultrafine particles: in vivo studies. Philosophical Transactions of The Royal Society of London A-Mathematical Physical and Engineering Sciences 2000;358(1775):2719-2739. R826784 (Final)
  • Abstract: Royal Society Publishing Abstract
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  • Other: Royal Society Publishing PDF
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  • Journal Article Oberdorster G. Pulmonary effects of inhaled ultrafine particles. International Archives of Occupational and Environmental Health 2001;74(1):1-8. R826784 (Final)
    R827354 (Final)
    R827354C004 (2000)
    R827354C004 (2001)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
  • Abstract from PubMed
  • Full-text: Precaution.org-Full Text PDF
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  • Abstract: SpringerLink-Abstract
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  • Journal Article Oberdorster G, Utell MJ. Ultrafine particles in the urban air:to the respiratory tract—and beyond? Environmental Health Perspectives 2002;110(8):A440-A441. R826784 (Final)
    R827354 (Final)
    R827354C003 (Final)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
  • Full-text from PubMed
  • Abstract from PubMed
  • Journal Article Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, Kreyling W, Cox C. Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. Journal of Toxicology and Environmental Health, Part A: Current Issues 2002;65(20):1531-1543. R826784 (Final)
    R827354 (Final)
    R827354C004 (2001)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
  • Abstract from PubMed
  • Full-text: UMN-Full Text PDF
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  • Abstract: Taylor & Francis-Abstract
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  • Supplemental Keywords:

    particulates, exposure, sensitive population, animal, extrapulmonary, translocation, air, ambient air, ultrafine particles, UFPs, fine particles, effects, health effects, human health, bioavailability, mammalian, organism, cellular, elderly, age, susceptibility, metals, heavy metals, pathology,, RFA, Health, Scientific Discipline, Air, Toxicology, particulate matter, Environmental Chemistry, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, genetic susceptability, Atmospheric Sciences, Environmental Engineering, health effects, particulates, sensitive populations, air toxics, human health effects, inhalability, morbidity, air pollutants, effects assessment, health risks, pulmonary disease, emphysema, airway disease, respiratory problems, tissue damage, laboratory animals, air pollution, chronic health effects, lung inflammation, lung dysfunction, Acute health effects, elderly, sensitive subgroups, tolerance, highrisk groups, human susceptibility, mortality, metals, oxidant stress, respiratory, ultrafine particles, exposure assessment, air contaminant exposure, air quality, environmental hazard exposures

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • 2001
  • 2002