2003 Progress Report: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events

EPA Grant Number: R827354C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R827354
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Airborne PM - Rochester PM Center
Center Director: Oberdörster, Günter
Title: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
Investigators: Oberdörster, Günter
Current Investigators: Oberdörster, Günter , Elder, Alison C.P.
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2003 through May 31, 2004
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air

Objective:

The objective of this research project is to use animal studies to focus on factors including particle size, dosimetric aspects (lung deposition and disposition), and host susceptibility (advanced age; cardiovascular disorders). Exposures were to laboratory-generated model particles and to particles on highways.

This is one of the projects of the Rochester PM Center. The progress for the other projects is reported separately (see reports for R827354 C001 through R827352C003 and R827352C005).

Progress Summary:

Translocation of Inhaled Manganese Oxide Nanoparticles to the Olfactory Brain Following Inhalation Exposure with One Naris Occluded

Clearance pathways for inhaled solid ultrafine particles (UFP < 100 nm) depositing in the respiratory tract include their translocation to extrapulmonary sites. Our previous results in rats with inhaled ultrafine carbon particles suggested that nasally-deposited solid UFP may be taken up by olfactory rods in the olfactory nasal mucosa and translocated along the olfactory dendrites and axons. Olfactory nerve translocation has been well demonstrated for inhaled soluble metal compounds. To test this hypothesis for solid UFP and determine the significance for causing health effects, we exposed groups of rats to ultrafine MnO2 particles (CMD = 31 nm) in whole-body exposure chambers for up to 11 days. Other groups were exposed with either both nostrils patent or the right nostril occluded. Atomic absorption spectrophotometry analysis of Mn in lung, liver, kidney, olfactory bulb, and several other brain regions as well as Atlas gene array and antibody protein array analyses of these brain regions were performed.

After 11 days of exposure, Mn concentrations in the olfactory bulb were 3.6-fold higher whereas lung Mn concentrations only doubled, and there were also small but significant increases of Mn in striatum and frontal cortex and cerebellum. TNF-α mRNA and protein were significantly increased about 30-fold in the olfactory bulb and showed 2-3 fold increases in the other brain regions associated with increased Mn levels. MIP-2 and NCAM mRNA also increased significantly. With the right nostril occluded, Mn accumulated only in the olfactory bulb of the side with the patent nostril. This confirmed the olfactory neuron translocation pathway for inhaled nano-sized particles to the CNS. We conclude that the olfactory neuronal pathway is very efficient for translocating inhaled solid UFP to the CNS and that translocated UFP, depending on their chemistry, can cause serious adverse effects. We suggest further that despite differences between the human and rodent olfactory systems this pathway is likely to be operative in humans as well.

These results together with our earlier data on CNS translocation of inhaled ultrafine carbon are of significance for ambient UFP because of the potential of their translocation to the CNS. Even if such translocation is not as efficient in humans as it is in rats, continued exposure and accumulation in sensitive CNS structures may affect brain function. Future studies are aimed at investigating this potential.

Effects of Ultrafine Particle Exposure on the Formation of Thrombi in Rat Ear Veins

The method is based on activating a photoreactive agent, Rose Bengal (RB), in the blood vessels of rats which results in local generation of superoxide and subsequent activation of endothelial cells leading to local thrombus formation. Initial experiments were aimed at optimizing RB plasma levels and keeping them stable for an extended period of time. Animals received either intraperitoneal (IP, 200 mg/kg), intravenous (IV, 10 mg/kg) or IVinfused (24 mg/kg/hr) RB in separate experiments. Blood was collected at different times to determine the kinetics of RB concentration up to 90 minutes. Our data indicate that the IVbolus resulted in a plasma RB concentration that decreased rapidly 5 minutes after injection, whereas the IP dose caused a continuous increase in plasma even 60 minutes after RB administration. This change in blood concentration produced by these routes of administration resulted in RB-induced endothelial damage that was not reproducible. In contrast, a steady state RB level of 60 μg/mL plasma was reached after 30 minutes of continued infusion and a consistent reaction was observed after 30 seconds illumination of the ear vein. These data suggest that continuous IVinfusion leads to more consistent plasma RB levels.

Intravenous administration of ultrafine aminated-polystyrene particles (positively charged; 0.02, 0.5, and 50 mg/kg) 30 minutes after the start of the RB infusion significantly shortened the time of thrombus formation in our RB ear model. However, carboxylated polystyrene particles (negatively charged) of the same size failed to affect thrombus formation at any dose. These data suggest that UFP can affect coagulation directly, but that the effect will depend on particle charge. These results were presented at the recent meeting of the Society of Toxicology (Silva, et al., 2004).

Other experiments studied the effect of positively charged UFP on coagulation in the same system without RB. Intravenous administration of particles to this system showed a significant reduction in the time of thrombus formation. Likewise intratracheal instillation of the 60 nm -polystyrene particles shortened the time to about 42 percent of the baseline level. This data indicates that particles can induce changes in thrombus formation without RB and that they might translocate from the lung into the blood stream to produce an effect in our ear model.

More environmentally relevant particles were used in our ear vein model to assess their effect on thrombus formation. Laboratory-generated ultrafine (30 nm) elemental carbon particles (4, 20, 100, and 500 μg/kg) were administered intravenously to rats and their effects on coagulation studied. It was shown that doses as low as 4 μg/kg (1 mg per rat) were able to significantly shorten the time of thrombus formation. Although different doses are not significantly different from each other, there seems to be a trend indicating that the lower the dose of UFP in the system the greater the effect. Similar results were obtained when particles were intratracheally instilled. In this case, even a lower dose (0.8 µg/kg or 0.2 µg per rat) was able to produce a significant effect. Inhalation studies are in progress; some of our preliminary data indicates that inhaled UFP particles produce increased thrombus formation independent of the time of exposure.

Effects of Exposure to Freshly-Generated On-road Fine/Ultrafine Particles (Truck Study II)

In the last update, we reported on results from on-road exposures using freshly-generated vehicle exhaust aerosols that were entrained into a mobile emissions laboratory (MEL; David Kittelson, University of Minnesota). Results from these studies have been published (Elder, et al., 2004; Kittelson, et al., 2004). One limitation of these studies was the variability in the aerosol number concentration caused by both the high dilution factor and long periods of low particle number concentrations. To achieve more continuous aerosol sampling, we performed another series of studies with MEL, this time orienting two telescoping inlets on the top in the back of the trailer so that they would sample the engine exhaust plume from MEL itself and—to a lesser degree—those particles and gases from surrounding vehicles.

Old rats (18-22 month F-344) were exposed directly on highways to either the gas phase/particle mixture, gas phase only, or filtered air. Some were pretreated with a low dose of inhaled endotoxin (immediately prior to on-road exposures) or with instilled human influenza virus (2 days prior to on-road exposures) to induce lung inflammation. Other groups of rats were exposed to on-road exhaust aerosols first and then to instilled virus. Virus-exposed animals were euthanized three days after exposure. The on-road exposures consisted of 6-hour driving periods on I-90 between Rochester and Utica, NY, once or 3 days in a row. Endpoints related to lung inflammation, inflammatory cell activation, and acute phase responses were assessed. In addition, two experiments were conducted in telemetered spontaneously hypertensive rats. For both experiments, inhaled LPS was used to prime respiratory tract cells. One experiment consisted of a single 6-hour on-road exposure and is similar to the one done in last year’s truck study; the second consisted of three consecutive days of exposures to the emission aerosols. Telemetry data was collected continuously for 5 days after exposure; data was also collected between each day’s exposure for those rats exposed for three days in a row (i.e. ~12 hours recorded). The telemetry data is currently being analyzed. The daily average number concentration in the filtered air chamber was less than 4.5 x 103 particles/cm3. The in-coming sampled air had a number concentration of 1.6-4.3 x 106 particles/cm3, but some losses were experienced when that air was directed into the chambers. Chamber number concentrations were 4.4-7.6 x 105/cm3, 4.3 to 5.7-fold lower than in-coming air; this same phenomenon was observed in the previous year’s studies. Despite these losses, the number concentrations in the exposure cages were consistent and about twice as high as in the previous study and the in-coming particle number concentration was an order of magnitude higher.

For the parameters that have been analyzed thus far, no differences in response were observed in rats exposed to gas-phase components alone versus the gas-phase/particle mixture. This was also observed in the previous year’s study. We observed the expected responses to the priming agents, for example,increased percentages of neutrophils (PMNs) in bronchoalveolar lavage (BAL) fluid and increased reactive oxygen species release from BAL inflammatory cells. The exhaust emission aerosols were found to have small but significant effects on several of the endpoints examined thus far. In one experiment, a single 6-hour exposure to on-road aerosols was found to increase the total number of cells in BAL fluid 3 days after exposure in comparison to filtered air controls. In a separate experiment, the aerosols were found to induce a decrease in the percentage of circulating PMNs relative to filtered air controls after a single 6-hour exposure regardless of pretreatment (LPS or saline aerosol given immediately prior to on-road exposures). In addition, on-road aerosols were found to decrease the concentration of plasma fibrinogen in rats exposed for 3 consecutive days and euthanized 3 days after the last exposure. However, when virus pre-treated rats were given a single 6-hour exposure, fibrinogen was significantly increased relative to filtered air controls. Although not all of the results have been analyzed, the changes described above are similar to what was reported for the previous study. That more significant effects of on-road emission aerosols were not found may reflect that there was only a two-fold difference in particle number concentration in the exposure chambers from the current study compared to the previous one. It is intriguing, however, that some of the effects of the particle/gas phase mixture persisted up to several days after the end of the on-road exposures (e.g., increased total BAL cell number, decreased plasma fibrinogen). This may suggest a lag in response or that more profound effects would have been observed if measurements were made sooner following exposure.

On-road Exposure Studies: Gene Expression Changes in Extrapulmonary Organs, Heart and CNS

We harvested tissues to examine gene expression changes in the lungs, heart, and selected brain regions (olfactory bulb, frontal cortex, cerebellum, striatum) after exposure as analyzed by microarray analyses (J. Carter, Proctor and Gamble Co.). Samples have been analyzed from those rats exposed to filtered air or exhaust aerosols for 1 day in MEL. Priming exposures consisted of either low-dose LPS aerosol inhalation or human influenza virus instillation; controls were exposed to sterile saline. The rats were euthanized approximately 18 hours after the end of the exposures in MEL (24 hours after LPS or 3 days after virus priming exposures).

The expression of two genes, in particular, was found to be increased consistently in lung, heart, and olfactory bulb, although to different degrees in each tissue. TNF-α and TNF-α receptor I were both increased in response to the exhaust particles or to LPS or influenza virus alone; when the exhaust aerosol and priming agent were combined, expression was the highest. The expression of TNF-α and its receptor was increased by approximately 3-fold over control (saline plus filtered air) in lung tissue after combined exposure. In heart tissue, increases in the two genes were not evident unless inhaled LPS or exhaust aerosols were combined; no changes were found in either of the virus-exposed groups. Gene expression changes in olfactory tissue were similar to that in the lung. A notable difference was that the exhaust aerosol alone did not alter the TNF-α or TNF-α receptor I gene. Both priming agents alone induced increases in expression, but the combination of the priming agents with exhaust aerosol produced the highest response, approximately 2.5-fold over control in the olfactory bulb of the brain. No changes in TNF-α or its receptor were detected in the striatum, cerebellum, or frontal cortex. These results will be confirmed using slot-blot or Northern analyses.

Heart Rate Variability Studies

The heart rate variability (HRV) analysis program that was developed and tested by the Cardiology Core (Couderc) has been applied to data from telemetered spontaneously hypertensive rats exposed to on-road aerosols for 6 hours after priming with IP-injected endotoxin or saline. The results suggest that inhaled on-road aerosols activate irritant receptors in the respiratory tract, leading to increased vagal activity. These responses were of greater magnitude and were potentiated in rats pre-treated with endotoxin. Although we did not evaluate arrhythmias, it could be expected that a prolonged and unchecked imbalance favoring parasympathetic drive would lead to bradycardia. The results showed that heart rate, SDNN, and vagosympathetic balance decreased and the high- and low-frequency components of the power spectrum increased in response to on-road aerosols, suggesting a dysregulation of autonomic input. The results suggest that activation of CNS receptors occurs either directly or indirectly by inhaled ultrafine on-road particles and their gaseous co-pollutants. They further suggest, given evidence of parasympathetic activation and the fact that pulmonary irritant receptors are under vagal control, that the observed effects are mediated at the level of the lung as opposed to a systemically-derived response. Further studies with more continuous exposures are needed to confirm these findings. Results from these studies are being submitted for publication. This custom analysis program has recently been distributed to other investigators who study the effects of various exposures on heart rate and HRV in rats.


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other subproject views: All 33 publications 31 publications in selected types All 27 journal articles
Other center views: All 104 publications 98 publications in selected types All 90 journal articles
Type Citation Sub Project Document Sources
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. R827354 (Final)
R827354C003 (Final)
R827354C004 (2003)
R827354C004 (Final)
R827354C005 (Final)
R826784 (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 Elder ACP, Gelein R, Oberdorster G, Finkelstein J, Notter R, Wang Z. Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate. Experimental Lung Research 2004;30(2):105-120. R827354 (Final)
    R827354C003 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R827354C005 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Journal Article Elder A, Gelein R, Finkelstein J, Phipps R, Frampton M, Utell M, Kittelson DB, Watts WF, Hopke P, Jeong C-H, Kim E, Liu W, Zhao W, Zhuo L, Vincent R, Kumarathasan P, Oberdorster G. On-road exposure to highway aerosols. 2. Exposures of aged, compromised rats. Inhalation Toxicology 2004;16(Suppl 1):41-53. R827354 (Final)
    R827354C003 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R827354C005 (Final)
    R828046 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
    R832415C005 (2011)
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  • Abstract: Taylor and Francis-Abstract
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  • Journal Article Kittelson DB, Watts WF, Johnson JP, Remerowki ML, Ische EE, Oberdorster G, Gelein RM, Elder A, Hopke PK, Kim E, Zhao W, Zhou L, Jeong C-H. On-road exposure to highway aerosols. 1. Aerosol and gas measurements. Inhalation Toxicology 2004;16(Suppl 1):31-39. R827354 (Final)
    R827354C001 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
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  • Journal Article Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W, Cox C. Translocation of inhaled ultrafine particles to the brain. Inhalation Toxicology 2004;16(6-7):437-445. R827354 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
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  • Abstract: Taylor and Francis-Abstract
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  • Journal Article Silva VM, Corson N, Elder A, Oberdorster G. The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP). Toxicological Sciences 2005;85(2):983-989. R827354 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
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  • Supplemental Keywords:

    pollution prevention, urban air pollution, atmosphere, metals, air, health, waste, atmospheric sciences, biochemistry, children's health, environmental chemistry, epidemiology, genetics, virology, molecular biology, health risk assessment, risk assessments, incineration, combustion, combustion engines, air toxics, tropospheric ozone, PM2.5, particulates, ultrafine particles, particulate matter, particle exposure, particle size, aerosol, aerosols, ambient air, ambient air monitoring, ambient air quality, animal model, atmospheric, cardiopulmonary, cardiopulmonary responses, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, cytokine production, fine particles, human exposure, human health, human health effects, environmental health effects, inhalation toxicology, lead, lung, lung inflammation, metals, morbidity, mortality, pathophysiological mechanisms, pulmonary, pulmonary disease, stratospheric ozone, sensitive populations, susceptible populations,, RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Atmospheric Sciences, Molecular Biology/Genetics, ambient air quality, cytokine production, particle size, particulates, sensitive populations, biostatistics, health effects, risk assessment, cardiopulmonary responses, fine particles, human health effects, morbidity, ambient air monitoring, lung, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient air, environmental health effects, particle exposure, ambient monitoring, particulate exposure, lung inflamation, pulmonary, coronary artery disease, inhalation toxicology, tropospheric ozone, urban air pollution, mortality, urban environment, aerosol, cardiopulmonary, human health, aerosols, cardiovascular disease, ultrafine particles, pathophysiological mechanisms

    Relevant Websites:

    Relevant Web Sites: http://www2.envmed.rochester.edu/envmed/PMC/ Exit

    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2000 Progress Report
  • 2001 Progress Report
  • 2002 Progress Report
  • 2004 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R827354    Airborne PM - Rochester PM Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827354C001 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles
    R827354C002 Inflammatory Responses and Cardiovascular Risk Factors in Susceptible Populations
    R827354C003 Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
    R827354C004 Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
    R827354C005 Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
    R827354C006 Development of an Electrodynamic Quadrupole Aerosol Concentrator
    R827354C007 Kinetics of Clearance and Relocation of Insoluble Ultrafine Iridium Particles From the Rat Lung Epithelium to Extrapulmonary Organs and Tissues (Pilot Project)
    R827354C008 Ultrafine Oil Aerosol Generation for Inhalation Studies