2000 Progress Report: Lung Injury from Inhaled Ultrafine Particles in Compromised Rats of Old Age: Influence of Priming and AdaptationEPA Grant Number: R826784
Title: Lung Injury from Inhaled Ultrafine Particles in Compromised Rats of Old Age: Influence of Priming and Adaptation
Investigators: Oberdörster, Günter , Finkelstein, Jacob N.
Current 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 Period Covered by this Report: September 1, 1999 through September 20, 2000
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 goal of this research is to evaluate oxidative stress responses in the compromised aged organism induced by inhaled ultrafine carbonaceous particles in combination with transition metals. Specific objectives are 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.
Based on our previous results in a genetic emphysema mouse model (TSK mouse), which showed a slight inflammatory response after inhalation of ultrafine carbonaceous particles in combination with ozone, we started to characterize an emphysema model in rats to continue our studies on the effects of ultrafine particles in the emphysematous lung. We induced emphysema by two subsequent intratracheal instillations of elastase or using our intratracheal inhalation model for more uniform distribution of the emphysematous lesions. Although it turned out that intratracheal inhalation of elastase resulted in rather uniform mild emphysema with no significant increase in lavaged inflammatory cells, it appears that this model induced a state of adaptation/tolerance to subsequent stimuli with ultrafine particles, as determined by ultrafine TiO2 administration. We decided, therefore, not to use this model for future studies with ultrafine particles.
In Fischer-344 rats, we also evaluated a sensitization model using ovalbumin, although Fischer-344 rats in contrast to Brown Norway rats are less prone to become sensitized. We found that 3 weekly intraperitoneal injections of ovalbumin, followed 2 weeks later by an inhalation challenge, resulted in only a slight increase in neutrophils and eosinophils in lung lavage, but both cell types were increased in the pleural lavage of these animals. Using this model, we determined in young (8 weeks) and old (20 months) rats the effect of a 6-hour exposure to ultrafine carbon particles, which contained 20 percent iron. The bioavailability of iron was determined prior to the study in an in vitro citrate assay, which showed that these particles exhibited very high biological activities, 370 nmol of bioavailable iron per mg of particles. In young rats, ultrafine particle exposure following ovalbumin challenge of the sensitized rats showed that there was no significant change in the cellular and biochemical lung and pleural lavage parameters. In old rats, ultrafine particle exposure resulted in a slight inflammatory cell increase in the lung lavage and a decrease of neutrophils in the blood. The pleural lavage showed a slight increase in lymphocytes, compared to rats not exposed to the ultrafine particles.
Most of our studies used endotoxin (LPS) sensitization as a model of inflammation for evaluating particle effects. Such sensitization or priming can be done by either administration of LPS to the respiratory tract or systemically. We used inhalation of LPS aerosols to prime the respiratory tract, immediately followed by inhalation of ultrafine particles. We tested the LPS inhalation model first in mice with ultrafine particles of known toxicity, i.e., polytetrafluoroethylene (PTFE) fumes inhaled by C57 mice. These mice are more resistant than previously tested Fischer-344 rats, which showed high lethality after a 15-minute exposure to PTFE fumes at a concentration of ~50 µg/m3. In 8-week old C57 mice such exposure did not induce a significant pulmonary inflammatory response, however, prior priming with inhaled LPS resulted in a significant inflammatory response that was greater than that induced by LPS alone. We also evaluated this response in a genetic mouse model of emphysema, TSK mice, at 15 months of age. These mice showed a mild inflammatory response to PTFE fumes, which was significantly increased when primed with inhaled LPS.
We used the LPS priming model in 22-month old rats to determine effects of ultrafine carbon particles (~100 µg/m3, 6-hour inhalation) given either before or after LPS inhalation. LPS alone resulted in a significant inflammatory response, characterized by 30 percent neutrophils in lung lavage, and the ultrafine carbon particle exposure, either before or after LPS, did not show a significant additional effect. Our conclusion from these studies was that the LPS dose needs to be lowered; otherwise, a small effect of the inhaled ultrafine carbon particles may be masked by the high LPS response.
In a subsequent study in old rats, a 6-fold lower LPS dose was administered to prime their respiratory tract for evaluating the effects of inhaled ultrafine and fine carbon particles. The fine particles were generated by aging for several minutes of a high concentration of ultrafine particles resulting in their aggregation. This gave a count median diameter (CMD) of ~250 nm vs. ~30 nm for the fine vs. ultrafine particles. Aged particles were inhaled at a 2.7-fold higher concentration to adjust for predicted differences in pulmonary deposition between the two particle sizes. Endpoints evaluated included cellular and biochemical lung lavage parameters, and generation of reactive oxygen species (ROS) ex vivo by resting and stimulated lung lavage cells. The results showed that there were no significant differences between the fine and ultrafine carbon particle effects as measured by these endpoints. However, surface area measurements of the particles performed subsequently showed that the fine particles had the same specific surface area as the ultrafines. Apparently, surface area was not changed by the agglomeration during the aging process, which is an important dosemetric when comparing effects of particles. There is a need to find another source of fine carbon particles for such comparative studies.
One goal of our studies is to examine the role of adaptation and age on the response to particles. Adaptation needs to be considered when evaluating effects of repeat exposures to particles when they act via inducing an oxidative stress response. Such response may be different in the aged organism, which could make the elderly more susceptible to repeat exposures. As a preliminary step for these studies, we examined first the influence of adaptation/tolerance by LPS. The LPS inhalation model was used to adapt old and young rats and mice by short-term low-dose inhalation of LPS on 3 consecutive days followed on day 4 by a high dose of inhaled LPS. Three-day sham-exposed animals, followed by high-dose inhalation of LPS on day 4, served as the non-adapted group. Results showed an adaptative response in both rats and mice of young age evidenced by a significantly lower inflammatory response after high-dose LPS in adapted compared to non-adapted animals. There also was a lower release of ROS from lavaged cells ex vivo in the adapted animals. Such adaptive response is consistent with results from other studies of oxidant stress inducing stimuli. Old mice showed this adaptive response as well, whereas in old rats there was a greater variability, although an adaptive trend was apparent.
in vitro studies focused on the effect of age on the comparative response of macrophages and epithelial cells following exposure of cells to particles. Due to some technical difficulties, we have been unable to utilize our in vitro cell exposure system to deliver singlet particles to cells growing on an air liquid interface. We have, however, carried out some studies with cells grown in Transwell membranes but exposed to particles by addition to culture media.
Using this approach, we were able to establish vectorial secretion of the inflammatory chemokine MIP-2 by cultured pulmonary epithelial cells. When particles (silica or ultrafine TiO2) were added to the apical surface of confluent epithelial monolayers (direct particle cell contact), we were able to detect an increase in MIP-2 secretion into the apical compartment. Little MIP-2 was released through the basal face unless particle concentrations that led to increased cytotoxicity were used. A similar result was obtained for TNF. In contrast, experiments with cultured macrophages showed no directionality of cytokine or chemokine release.
In our studies focusing on age effects, we compared macrophage production of cytokines following LPS and particles from 22-27?month old rats to cells from 10-12?week old rats. When macrophages from young rats were treated with LPS, a clear dose response, with MIP-2 as the endpoint, was obtained. A similar dose response relationship was observed with carbon particles 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 TNF) was elevated 30-50 percent in these cells. In addition, response to LPS 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 AM 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 lead to synergistic effects at the lowest dose of particles in contrast to rats. Our initial studies with mouse macrophages have shown a somewhat blunted response in these cells in comparison to rats. We plan to continue these studies and characterize this response until aged rats become available.
We also have begun to develop 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. Preliminary experiments confirm particle-induced increase in reporter gene activity with a peak at 6 hours post treatment. We will continue to use this human cell line to investigate response to multiple exposures as well as antioxidants
We will continue our LPS priming model in rats and mice and evaluate the effects of mixed ultrafine (carbon + metals) exposures. The effect of age on the response to ultrafine particles as well as adaptive phenomena will be explored as well.
In the coming year, we plan to continue to characterize in vitro the difference in response to stimuli, alone and in combination, as a function of age. We also expect to extend these studies from macrophages to parenchymal cells, fibroblasts and epithelial cells, as well. We also will investigate other markers of response. Measurement of prostaglandin production and COX-2 activation may prove to be a useful marker. Studies have shown COX-2 to be important in the induction of the inflammatory response and systemic responses.
Also, in support of the in vivo projects, we will evaluate in vitro effects of particles of differing composition. We have begun to examine the cytokine response to particles containing carbon and iron. In our standard exposure model with mouse cell lines, enhanced cell death was seen but cytokine production was similar to pure carbon.