Pulmonary and Systemic Effects of Zinc-Containing Emission Particles in Three Rat Strains: Multiple Exposure Scenarios

Urmila P. Kodavanti*,1, Mette C. J. Schladweiler*, Allen D. Ledbetter*, Russ Hauser{dagger}, David C. Christiani{dagger}, James M. Samet{ddagger}, John McGee*, Judy H. Richards* and Daniel L. Costa*

* Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; {dagger} Harvard School of Public Health, Boston, Massachusetts 02115; and {ddagger} Human Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Chapel Hill, North Carolina 27599

Received May 1, 2002; accepted July 25, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a common component of ambient particulate matter (PM), zinc has been proposed to play a role in PM-induced adverse health effects. Although occupational exposures to high levels of zinc-fume have been associated with metal-fume fever accompanied by pulmonary inflammation and injury, the effects of PM-associated zinc are unclear. We hypothesized that an oil combustion emission PM (EPM) containing bioavailable zinc would induce pulmonary injury and systemic hematological changes attributable to the leachable zinc following acute as well as longer-term exposures in a rat strain-specific manner. In order to initially characterize the pulmonary response to EPM, male Sprague-Dawley (SD) rats were intratracheally (IT) instilled with 0.0, 0.8, 3.3, or 8.3 mg/kg EPM in saline. To further determine if the pulmonary injury was associated with the EPM leachable zinc, subsequent studies included IT instillation of SD rats with either saline, whole EPM suspension, the saline leachable fraction of EPM, the particulate fraction of EPM (all at 8.3 mg/kg, soluble Zn = 14.5 µg/mg EPM), or ZnSO4 (0.0, 33.0, or 66.0 µg/kg Zn). Finally, to ascertain the cumulative impact of inhaled EPM in the causation of acute pulmonary and systemic effects as well as long-term fibrotic responses, we exposed three rat strains of differential susceptibility to PM. Male SD, normotensive Wistar-Kyoto (WKY), and spontaneously hypertensive (SH) rats (90 days old) were exposed nose-only to either filtered air or EPM: 2, 5, or 10 mg/m3 (6 h/day x 4 days/week x 1 week); or 10 mg/m3 (6 h/day x 1 day/week for 1, 4, or 16 weeks) and assessed at 2 days postexposure. IT exposures to whole EPM suspensions were associated with a dose-dependent increase in protein/albumin permeability and neutrophilic inflammation. Pulmonary protein/albumin leakage and neutrophilic inflammation caused by the leachable fraction of EPM and ZnSO4 were comparable to the effect of whole suspension. However, protein/albumin leakage was not associated with the particulate fraction, although significant neutrophilic inflammation did occur following instillation. With EPM nose-only inhalation, acute exposures (10 mg/m3 only) for 4 days resulted in small increases in bronchoalveolar lavage fluid (BALF) protein and n-acetyl glucosaminidase activities (~50% above control). Surprisingly, unlike IT exposures, no neutrophilic influx was detectable in BALF from any of the inhalation groups. The only major effect of acute and long-term EPM inhalation was a dose- and time-dependent increase in alveolar macrophages (AM) regardless of the rat strain. Histological evidence also showed dose- and time-dependent accumulations of particle-loaded AM. Particles were also evident in interstitial spaces, and in the lung-associated lymph nodes following the inhalation exposures (SH > WKY = SD). There were strain-related differences in peripheral white blood cell counts and plasma fibrinogen with no major EPM inhalation effect. The present study demonstrated the critical differences in pulmonary responsiveness to EPM between IT and inhalation exposures, probably attributable to the dose of bioavailable zinc. EPM IT exposures, but not acute and long-term inhalation of up to 10 mg/m3, caused neutrophilic inflammation. Inhalation exposures may result in particle accumulation and macrophage recruitment with potential strain differences in EPM clearance.

Key Words: zinc-containing emission particles; lung injury; inflammation; spontaneously hypertensive rats; plasma fibrinogen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal toxicological and occupational studies support the contention that the health effects of particulate matter (PM) are associated with the physicochemical characteristics of the particle. While the roles of specific physicochemical characteristics, such as size and composition of model or combustion-derived PM, have been addressed in many toxicological studies (Dreher et al., 1997Go; Hatch et al., 1985Go; Kodavanti et al., 1997aGo, 1998bGo; Oberdorster, 2001Go; Pritchard et al., 1996Go; Tran et al., 2000Go), ambient PM are less well studied in this regard. Moreover, the manner in which different components interact with healthy and compromised biological systems is even less understood. Bioavailable metals derived from combustion source residual oil fly ash (ROFA) contribute to ambient PM and have been postulated to play a role in lung injury (Hatch et al., 1985Go; Kodavanti et al., 1997aGo; Pritchard et al., 1996Go). Further, we have shown that bioavailable vanadium and nickel, which are found in many combustion source PM, both play a role in toxicity, however, these metals likely employ different mechanisms to cause pulmonary injury and inflammation (Kodavanti et al., 1998bGo).

Although regional differences exist in the composition of PM within United States (Walsh and Gilliland, 2001Go), combustion-derived elemental metals are detectable in most PM samples, and frequently these metals are readily traceable to nearby industrial sources (Balachandran et al., 2000Go; Dye et al., 2001Go; Harrison and Yin, 2000Go). It is likely that the metal composition of such samples can vary significantly based on the type of combustion materials and products; thus, the use of these samples in toxicological studies can provide critical information needed for source apportionment. Zinc is one such metal that has been associated with industrial emissions and is detected in relatively higher levels than most metals in many ambient air samples (Adamson et al., 2000Go; Balachandran et al., 2000Go; Dye et al., 2001Go; Harrison and Yin, 2000Go; Kodavanti et al., 2000aGo). A widely studied bag-house total suspended particulate sample collected from ambient air in Ottawa, Canada also contained significant levels of leachable zinc (5–10 µg/mg mass) and has been shown to cause lung injury in animals (Adamson et al., 1999Go, 2000Go). Similarly, Utah Valley PM extracts contained several metals, with notable levels of zinc. The health effects of these extracts were, in large part, attributable to the levels of zinc, which changed depending on the operational status of a nearby steel mill (Dye et al., 2001Go; Soukup et al., 2000Go). Occupationally, exposures to zinc fumes are known to cause acute pulmonary inflammation as well as a significant systemic response (Fine et al., 2000Go; Gordon et al., 1992Go). Thus, combustion PM samples with zinc may be appropriate surrogates of ambient PM for toxicological studies.

The purpose of this study was to characterize the chemical composition of and the biological responses to an emission-derived PM (EPM, collected from the stack of an oil burning power plant in Boston, MA) containing low levels of leachable zinc, similar to selected samples of ambient PM derived from different locations (Adamson et al., 1999Go, 2000Go; Dye et al., 2001Go; Harrison and Yin, 2000Go). As susceptibility is thought to be integral to the epidemiological findings of PM health effects, we elected to study the EPM in both healthy and PM-susceptible rat models, e.g., the spontaneously hypertensive (SH) rat (Kodavanti and Costa, 1999Go, 2001Go; Kodavanti et al., 1998aGo, 2000bGo). To determine the impact of this EPM relative to other combustion source PM in causing pulmonary injury and to ascertain the role of bioavailable zinc, pulmonary injury responses were initially characterized in Sprague-Dawley (SD) rats using intratracheal (IT) instillations of EPM suspension, saline-leachable fraction of EPM, particulate fraction of EPM, and also zinc sulfate (ZnSO4). Subsequently, to compare exposure method-related differences in both pulmonary and systemic effects and to characterize exposure time and concentration effects in healthy and susceptible rat models, multiple inhalation exposures were conducted using SD, Wistar-Kyoto (WKY), and SH rats. Since, the EPM elemental composition resembled that of ambient samples, especially involving zinc as the primary leachable metal, the inhalation exposure scenarios were selected to mimic studies we have previously conducted using real-time concentrated ambient air PM (Kodavanti et al., 2000aGo).


    METHODS AND MATERIALS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Healthy, male SD rats, 10–17 weeks old (Charles River, Raleigh, NC), were used for IT studies to determine the dose-response relationship and to characterize the role of leachable zinc in pulmonary toxicity. The inhalation studies included 11–13-week-old healthy, male SD, normotensive WKY and SH (SHR/NCrlBR) rats (derived from WKY rats by phenotypic segregation of the hypertensive trait and inbreeding). Rats for inhalation studies were also purchased from Charles River Laboratories, Raleigh, NC, with the exception of WKY rats in groups D, E, F (Harlan Sprague Dawley, Indianapolis, IN), and group G (Taconic, Germantown, NY; Table 1Go) due to nonavailability of age-matched WKY rats at that time. Rats used in IT studies were double-housed, whereas for inhalation studies, rats were singly housed to minimize stress and to remain consistent with the nose-only exposure environment. All rats were maintained in an isolated animal room in an AAALAC approved animal facility (21 ± 1°C, 50 ± 5% relative humidity, 12 h light/dark cycle) for one to two weeks quarantine and nonexposure periods. The rats were housed in plastic cages with beta chip bedding that was changed three times per week. All animals received standard (5001) Purina rat chow (Brentwood, MO) and water ad libitum except during the nose-only exposure periods of 6 h. The protocol for the use of rats in nose-only inhalation and IT instillation studies was approved by the Institution’s Animal Care and Use Committee.


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TABLE 1 Experimental Protocol for Inhalation Study
 
EPM source and composition analysis.
The EPM used in this study was collected off the stack, from the outside surface of the emission device of a power plant burning residual oil (#6) during 1997 (located in Boston, MA), and stored in a sterile 50 ml polyethylene tube. This bulk sample was ground and sieved according to the method described by Kodavanti et al. (1998b)Go. The bulk sample was analyzed for size using a TSI Model 3310A Aerodynamic Particle Sizer (TSI, Inc., St. Paul, MN) assuming a log normal distribution according to the method described in Kodavanti et al. (1998b)Go. The particles were made airborne with a TSI Model 3433 small scale powder dispenser. Samples of bulk ground particles and gravimetric filters, collected during the inhalation exposure (see below), were extracted in double distilled water or 1.0 M HCl as described in Kodavanti et al. (1998b)Go. Supernatants were analyzed for the presence of sulfate (SO4), zinc (Zn), nickel (Ni), vanadium (V), iron (Fe), manganese (Mn), and copper (Cu), which were presumed to be the predominant metals of this combustion source emission based on previous studies (Kodavanti et al., 1997aGo, 1998bGo). The elemental analysis of the extracts was done using Inductively Coupled Plasma–Atomic Emission Spectroscopy (ICP-AES; as described in Kodavanti et al., 1998bGo). Analysis closely followed EPA Method 200.7 (U.S. EPA Publication, 1994). Calibration standards were obtained from Spex Certiprep (Metuchen, NJ; p/n QC21 for metals, p/n AS-SO49-2-1 for sulfur); quality assurance standards were obtained from VHG Labs (Manchester, NH; p/n LCAL6020-100 for metals, p/n ASW-100). The concentrations of metals were calculated based on the original weight of particle samples suspended in distilled water or 1 M HCl.

Total carbon, nitrogen, and hydrogen contents of bulk ground EPM were determined by Galbraith Laboratories, Inc. (Knoxville, TN) using standard combustion techniques where the sample is heated to 1000°C in an O2 atmosphere, the resulting CO2 and H2O are measured by infrared absorption, and N2 is measured by thermal conductivity. EPM was also analyzed for endotoxin content using the gel-clot method (Cape Cod, Inc., Falmouth, MA).

Randomization of animals.
SD, WKY, and SH rats in each inhalation exposure were randomized into air control and EPM groups of equal mean weights (Table 1Go) from a group previously ordered from low weight to high weight. The rats used for the IT instillation studies were not randomized on a weight basis but were arbitrarily assigned to a group.

IT instillation studies.
Since the EPM composition was unique in that its elemental concentration of bioavailable zinc was similar to that of a number of ambient PM samples (Adamson et al., 2000Go; Dye et al., 2001Go; Harrison and Yin, 2000Go), and was very distinct in overall metal composition from previously used complex vanadium-, nickel-, and iron-loaded ROFAs (Kodavanti et al., 1997aGo,bGo, 1998bGo, 1999Go), we wanted to do a thorough investigation of its cardiopulmonary impact, as this sample may be a better substitute for ambient surrogates. Initially, to relate the effectiveness of this EPM in causing pulmonary injury to what has been observed using other ROFA samples, IT instillations were performed. Because it is likely that different PM samples may induce pulmonary injury with different kinetics, we wanted to determine EPM responses at two time points. We have shown temporal differences in pulmonary injury/inflammation caused by combustion PM and their individual metal components (Kodavanti et al., 1997bGo, 2001Go), The EPM sample was weighed and suspended in sterile saline at concentrations of 0.0, 0.83, 3.3, or 8.3 mg/ml, briefly vortexed, and mixed for 20 min prior to IT instillation. Male SD rats (n = 5/group) were IT-instilled with either sterile saline or a saline suspension of EPM at 0.8, 3.3, or 8.3 mg/ml/kg under halothane anesthesia (Costa et al., 1986Go), and necropsies/lavages were performed at 24 or 96 h postinstillation.

Additional IT studies were performed to ascertain if IT EPM-induced injury can be accounted for by the leachable fraction containing zinc, and to determine if injury can be reproduced by soluble zinc sulfate (ZnSO4). Firstly, to prepare leachable and particulate fractions of EPM for IT instillation 8.3 mg/ml aliquots of EPM were suspended in sterile saline in two tubes and mixed as stated above. One tube was used for the whole suspension instillation while the other tube was centrifuged at 14000 x g, 4°C, for 10 min. The resulting supernatant was filtered through 0.45 µm sterile syringe filter and used as the EPM leachable fraction. The pellet fraction was resuspended in sterile saline, vortex mixed, and centrifuged again. The supernatant was discarded and pellet was again resuspended in saline. A total of three washes were performed to remove any remaining leachable material from the particulate fraction. This particulate fraction was then resuspended in an equivalent original volume of saline and vortexed for IT instillation. Male SD rats were IT-instilled with either saline, whole EPM suspension, leachable fraction of EPM, or pellet fraction of EPM (8.3 mg/kg). In this study a separate group of rats was also instilled with Mount St. Helen’s (MSH) ash suspension (prepared in a similar manner to EPM at 8.3 mg/ml/kg concentration) as a negative control. MSH ash is known not to cause significant lung injury, and historically has been used as negative control PM (Hatch et al., 1985Go). Necropsies were performed 24 h later, as this time point gave maximum injury response.

Since the leachable fraction of EPM contained primarily zinc (14.5 µg/mg EPM), the purpose of the last instillation study was to ascertain if lung injury can be accounted for by soluble ZnSO4 when instilled at concentrations of zinc that occur in EPM instillates. Separate groups of male SD rats were also instilled with either 0.0, 145.0, or 290.0 µg/ml/kg ZnSO4.7H2O reflecting 0.0, 33.0, or 66.0 µg/ml/kg zinc, respectively. Twenty-four hour later, bronchoalveolar lavages were performed as stated below.

All IT instillation studies included characterization of pulmonary injury by analysis of biochemical and inflammatory components of bronchoalveolar lavage fluid (BALF). Pathology or systemic responses were not included in these studies.

Nose-only EPM inhalation exposure study.
There were two complementary purposes of the inhalation studies; first to compare acute and long-term inhalation exposure responses to the IT pulmonary toxicity, and second to investigate possible acute versus long-term systemic and pulmonary effects of EPM relative to our previous studies with compositionally different ROFAs (Kodavanti et al., 1997bGo, 1998bGo, 2002Go). In those studies, ROFA caused differential and commutative rat strain-dependent inflammation as well as systemic effects, but without apparent fibrosis in WKY and SH rats (Kodavanti et al., 2002Go); ROFA exposure, however, was previously associated with distinct fibrosis in SD rats (Kodavanti et al., 1997bGo). To address this issue, three rat strains exhibiting differential susceptibilities to acute systemic/pulmonary and long-term fibrosis effects were employed. Male SD, WKY, and SH rats were exposed (see Table 1Go for exposure groups and experimental design) by nose-only inhalation (Ledbetter et al., 1998Go) to either filtered air or EPM (2, 5, or 10 mg/m3, 6 h/day x 4 day/week x 1 week) to assess concentration-response relationship and to 10 mg/m3 (6 h/day x 1 day/week for 1, 4, or 16 weeks) to assess cumulative nature of injury. The responses were determined 2 days postexposure as 1–4 days postexposure time points seemed to give steady inflammatory response in our previous studies (Kodavanti et al., 2002Go).

Aerosolized particles were aerodynamically size separated through a cyclone impactor at < 2.5 µm and introduced into the mixing chamber. Each exposed animal was restrained in a conical, nose-only exposure plastic restrainer (Lab Products, Seaford, DE). Chamber aerosol concentrations were determined real time by a Real Time Aerosol monitor (RAM-1, GCA Corp., Bedford, MA). Actual chamber concentrations were determined gravimetrically approximately once per h during each exposure day using 47 mm Teflon filters (Gelman Sciences, Ann Arbor, MI) via an unused animal port at a sample flow rate of approximately 0.4 l/min. Each filter was weighed just prior to and immediately after sampling using a computer-controlled Cahn 33 microbalance (Thermo Cahn, Madison, WI). Chamber concentration was then determined by dividing the increase of filter mass by the total volume sampled (mg/m3). Determination of aerosol-size distribution was performed at least once per exposure using a 7-stage cascade impactor (Intox Products, Albuquerque, NM). Chamber temperature, relative humidity, airflow, and pressure were monitored continuously and maintained at constant levels (Ledbetter et al., 1998Go). All rats were acclimatized for 1 h to the nose-only inhalation tubes, 1 day prior to the start of exposure.

To evaluate the effect of tube restraint and exposure on WKY and SH rats, micro temperature transducers (IPTT-100, DAS-5001 Data System, BioMedic Data System Inc. [BMDS], Seaford, DE) were implanted in 8 SH (four air and four EPM) and eight WKY (four air and four EPM) in group A (Table 1Go). The sterile transducers were implanted under the skin between the shoulder blades using a 16-gauge needle. A receiver, held next to the rat, recorded the body temperature from the transmitter into an Excel Spreadsheet (BMDS.exe, developed in house). Body temperatures were recorded at least hourly during each exposure. In addition, periodic body temperature data were collected during nonexposure periods while the rats were in their home cages singly housed.

Blood collection, tissue isolation, bronchoalveolar lavage (BAL), and lung histology.
At designated time points rats were weighed and anesthetized with sodium pentobarbital (50–100 mg/kg, ip). Blood was collected (inhalation study only) through cardiac puncture directly into vacuutainers containing EDTA (for complete blood counts) or citrate as anticoagulants (for fibrinogen analysis). The trachea was cannulated and the left lung was tied. The right lung (the whole lung in some IT studies) was lavaged with Ca++/Mg++ free phosphate buffered saline (pH 7.4) with a volume equal to 28 ml/kg body weight (approximately 75% total lung capacity) x 0.6 (right lung representing 60% of total lung mass). Three in-and-out washes were performed with the same buffer aliquot. After the right bronchus was severed, the left lung was untied and then inflated with filtered 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2). The trachea was tied and the left lung was placed in 4% paraformaldehyde for 1–7 days at 4°C. The volume of fixative used to inflate the left lungs was based on 28 ml/kg body weight total lung capacity with the left lung representing 40% of the total lung mass. After fixation, the lung tissues were embedded in paraffin and 4 µm thick transverse sections were mounted on slides and stained with hematoxylin and eosin (Experimental Pathology Laboratory, Research Triangle Park, NC). Lung sections were examined under light microscopy for qualitative evaluation of lesions.

Bronchoalveolar lavage fluid (BALF) analysis.
One aliquot of whole lavage fluid was used for determining total cell counts (Coulter Counter, Coulter, Inc., Miami, FL), and a second aliquot was centrifuged (Shandon 3 Cytospin, Shandon, Pittsburgh, PA) to prepare cell differential slides. The slides were dried at room temperature and stained with LeukoStat (Fisher Scientific Co., Pittsburgh, PA). Macrophages, neutrophils, eosinophils, and lymphocytes were counted using light microscopy (over 200 cells counted per slide). The remaining BALF was centrifuged (1500 x g) to remove cells, and the supernatant fluids were analyzed for protein, albumin, and n-acetyl glucosaminidase (NAG) activity. These assays were modified and adapted for use on a Hoffmann-La Roche Cobas Fara II clinical analyzer (Roche Diagnostics, Branchburg, NJ). Total protein content was determined (Coomassie Plus Protein Assay Kit, Pierce, Rockford, IL) with bovine serum albumin as a standard. BALF samples were analyzed for albumin content using a commercially available kit and controls from ICN Star Corporation (Stillwater, MN). NAG activity (U/l) was determined using a kit and controls from Boehringer Mannheim Corporation Products (Indianapolis, IN).

An aliquot of BALF was mixed with an equal volume of 6% perchloric acid and vortexed. After standing on ice for 10 min, it was centrifuged (14,000 x g) for 10 min (4°C), and then the supernatants were stored at –80° C. Total glutathione was measured using 5,5‘-dithio-bis-(2-nitrobenzoic) acid-glutathione disulfide reductase recycling assay (Anderson, 1985Go) adapted for the Hoffmann-La Roche Cobas Fara II clinical analyzer.

Plasma and blood analysis.
Plasma fibrinogen and complete blood counts were performed by the University of North Carolina Hospitals, Core Facility (Chapel Hill, NC). Each blood sample containing citrate anticoagulant was centrifuged at 4500 rpm for 10 min at 4°C. Plasma was then collected and diluted with imidazole buffer. Thrombin was added (warmed to 37°C) to warmed diluted plasma samples and the time required for clot formation was measured using a coagulation analyzer (MDA 180, Oreganon Teknika, Durham, NC). A reference curve was generated using reference plasma samples (MDA Verify Reference Plasma, Organon Teknika) and sample values were calculated based on the reference curve (Organon Teknika Corp., 1996).

The complete blood counts were performed on a Technicon H-2 hematology analyzer (Bayer Corp., Tarrytown, NY) using Bayer Technicon reagents.

Statistics.
The data from the IT instillation and inhalation studies were analyzed using a multivariate ANOVA model. The independent variables for the inhalation studies were rat strains (SD, WKY, or SH), exposures (saline/air or EPM), and exposure durations, while for the IT studies, either concentration and time point, or PM fraction types were used. Pair-wise comparisons were performed as subtests of the overall ANOVA. EPM effects and comparisons were considered significant if the p-values were less than 0.05 in comparison to matching controls. Adjustments in the significance levels for multiple comparison were made using a modified Bonferroni type correction (Legendre and Legendre, 1998Go). The modification addresses the overly conservative effect of the ordinary Bonferroni correction when numerous comparisons are made.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
EPM Composition
Analysis of bulk ground EPM indicated the predominant presence of water-leachable zinc and sulfate with a small amount of nickel and iron (Table 2Go). Iron and vanadium concentrations were higher in the 1M HCl-leachable fraction of this EPM. The sulfur concentration in the leachate fraction, probably representing sulfates, was ~10% of the total mass and was similar in both the water and acid leachable fractions, as was zinc and nickel. Thus, this EPM differed from the ROFAs used in previous studies (Dreher et al., 1997Go; Kodavanti et al., 1998bGo) in that it contained low levels of iron, vanadium, and nickel but relatively higher levels of zinc. Organic analysis indicated ~19% carbon on a mass basis, and thus, this EPM also had significantly higher levels of organic carbon relative to the ROFAs used earlier (Dreher et al., 1997Go; Kodavanti et al., 1998bGo). The mass median aerodynamic diameter (MMAD) was 1.2 µm and the geometric standard deviation (GSD, {sigma}g) was 2.6. The endotoxin content of this EPM was negligible and would likely not result in lung inflammation (0.26 EU/mg EPM).


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TABLE 2 Elemental Constituents of EPM
 
Body Weights and Temperatures
There were no major exposure- or housing-related effects on body weights during IT (data not shown) or inhalation studies (Table 3Go). It was observed that SH rats do appear to react differently from SD or WKY during confinement in that they did not appear to adapt to the nose-only tubes as evidenced by the percentage of body weight lost during each exposure. Moreover, the loss in body weight during tube confinement and next day recovery did not modulate like non-SH rats as they acclimatized to the nose-only tubes; this was demonstrated by lower net body weight gains in SH rats relative to SD and WKY rats (Table 3Go). It was also observed that the body temperature of the SH rats had larger daily variation than WKY rats with no apparent EPM exposure effects.


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TABLE 3 Body Weights of Rats Used in EPM Inhalation Study
 
BALF Markers of Pulmonary Injury and Inflammation (IT Instillation)
IT instillation of the whole EPM suspension resulted in dose-dependent increases in BALF albumin (Fig. 1Go) and protein (data not shown) at Days 1 and 4 postinstillation (Fig. 1Go). Marked increases in total lavageable cells (due primarily to neutrophils) occurred in a dose-dependent manner at Day 1 following instillation. However, the neutrophilic influx was largely reversed by Day 4, which was also reflected in the reversal in total cell numbers (Fig. 1Go). Macrophage numbers did not increase at either time point (data not shown).



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FIG. 1. Concentration-dependent effects of IT EPM exposure on bronchoalveolar lavage fluid (BALF) albumin, total cells, and neutrophils in Sprague Dawley (SD) rats. Twenty-four or 96 h post-IT instillation of whole EPM suspension, whole lungs were lavaged and lavage fluid analyzed for pulmonary injury markers (protein and albumin; protein data not shown but it followed a trend similar to albumin changes in the BALF). *Significant EPM effect (p < 0.05).

 
To determine if EPM toxicity was associated with the saline leachable or particulate fraction, SD rats were IT-instilled with either whole EPM suspension or its fractions. MSH PM containing minimal leachable metal was used as a negative control (Hatch et al., 1985Go; Fig. 2Go). In contrast to MSH, IT instillation of whole EPM suspension or saline leachable fraction resulted in marked increases in BALF albumin, NAG activity, total cells, and neutrophils. The effects produced by whole EPM suspension were largely accounted by leachable fraction. The insoluble particulate fraction, while causing minimal pulmonary leakage, was associated with significant neutrophilic inflammation—considerably more than that induced by MSH suspension, but markedly less than the increase resulting from leachate instillation. The increases in NAG activity suggest that the macrophage cell population, while not increased, might have been activated by the EPM.



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FIG. 2. Pulmonary injury determined by bronchoalveolar lavage fluid (BALF) analysis following intratracheal (IT) instillations of EPM fractions or ZnSO4 in Sprague Dawley (SD) rats. Upper panel: Saline leachable (EPM-L) and particulate fractions (EPM-P) were prepared from whole EPM suspension (EPM-S) at 8.3 mg/ml concentration as described in the Methods section. Whole suspension of Mount St. Helen’s ash (MSH), 8.3 mg/ml/kg was used for an additional negative control. Lower panel: In a separate experiment, ZnSO4 was dissolved in saline at a concentration of 0.0, 145.0 (Zn-LD), or 290.0 µg/ml (Zn-HD); representing 0.0, 33.0, or 66.0 µg/ml zinc, respectively, and IT-instilled in SD rats at 1 ml/kg. Twenty-four h later, lungs were lavaged and BALF analyzed for pulmonary injury markers. *Indicates significant effect of whole EPM, its fractions, or ZnSO4 (p < 0.05) relative to saline and/or MSH controls.

 
To determine if the injury caused by the leachable fraction or whole suspension could be reproduced by IT instillation of soluble zinc, rats were IT-instilled with a low (33.0 µg zinc/kg) or high (66.0 µg zinc/kg) dose of ZnSO4. The calculated leachable zinc concentration of EPM suspensions at 0.8, 3.3, and 8.3 mg/kg are 12, 48, and 120 µg/ml/kg, respectively. The doses of ZnSO4 for this comparison were selected such that the levels would fall within the range of zinc concentrations achieved in the EPM suspension study. IT instillation of rats with ZnSO4 was associated with concentration-dependent increases in BALF protein (data not shown), albumin, NAG activity, total cells, and neutrophils (Fig. 2Go, lower panel) suggesting that EPM injury can be reproduced by soluble ZnSO4.

BALF Markers of Pulmonary Injury and Inflammation (Nose-Only Inhalation)
BALF protein, albumin (data not shown), and NAG activity were measured as markers of pulmonary capillary leakage and macrophage phagocytic ability, respectively. EPM exposure for 4 consecutive days at 10 mg/m3 was associated with slight, but significant increases in BALF protein in the SD and WKY rats. An increase in protein was also apparent in SH rats, but it did not reach significance because of the high and variable background values (Fig. 3AGo). One day per week exposures for a total of 16 weeks revealed no differences in BALF protein values (Fig. 3BGo). There appeared to be no strain-related differences in BALF protein leakage following exposure to this EPM.



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FIG. 3. Concentration and exposure duration-dependent effects of EPM inhalation on BALF protein in SD, WKY, and SH rats. (A) Rats were exposed to EPM, 6 h/day, for four consecutive days and analyzed two days postexposure. (B) Rats were exposed to 10 mg/m3 EPM, 6 h/day, one day/week for 1, 4, or 16 weeks and analyzed two days following last exposure. Values represent means ± SE of 6–8 rats per group. *Significant EPM effect (p < 0.05) for a given concentration or exposure duration.

 
BALF NAG activity was increased following 4 day EPM exposure at all concentrations in SH and at high concentration in SD and WKY rats (Fig. 4AGo). In some exposure groups, however, this increase was not significant. Increases in NAG activities in the exposure duration study were highly variable within control and exposed animals with no specific pattern-related to EPM effects (Fig. 4BGo). There were strain-related variations in the basal levels of BALF total glutathione but no significant exposure-related effects were evident in any of the inhalation exposed groups (data not shown).



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FIG. 4. Concentration and exposure duration-dependent effects of EPM inhalation on BALF n-acetyl glucosaminidase (NAG) activity in rats. (A) Rats were exposed to EPM, 6 h/day, for four consecutive days and analyzed two days postexposure. (B) Rats were exposed to 10 mg/m3 EPM, 6 h/day, one day/week for 1, 4, or 16 weeks and analyzed two days following last exposure. Values represent means ± SE of 6–8 rats per group. *Significant EPM effect (p < 0.05) for a given concentration or exposure duration.

 
Accumulation of particle laden macrophages was the only prominent and consistent effect of EPM inhalation in all three rat strains (Fig. 5Go). These increases in macrophages were largely concentration-dependent (Fig. 5AGo) and reflected increases in total lavageable cell counts (data not shown). Although increases were noted in SD and WKY rats at all concentrations, the values reached statistical significance only at higher concentrations in SD rats. SH rats seemed to have the largest increase in macrophage numbers following 4 days exposure (Fig. 5AGo). The exposure scenario of 1 day/week exposures for 1, 4, or 16 weeks revealed increases in macrophages in all three stains at 4 weeks (Fig. 5BGo). This increase was persistent at 16 weeks in SD and WKY, but because of the higher macrophage numbers in 16 week control SH rats the EPM effect was not apparent. Unlike the IT instillation experiments, significant increases in BALF neutrophils were not associated with inhalation exposure of rats to EPM regardless of inhalation concentration, duration of exposure or strain (data not shown).



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FIG. 5. EPM concentration and exposure duration-dependent increases in BALF macrophages in rats. (A) Rats were exposed to EPM, 6 h/day, for four consecutive days and analyzed two days postexposure. (B) Rats were exposed to 10 mg/m3 EPM, 6 h/day, one day/week for 1, 4, or 16 weeks and analyzed two days following last exposure. Values represent means ± SE of 6–8 rats per group. *Significant EPM effect (p < 0.05) for a given concentration or exposure duration.

 
Lung Histology
Representative areas of the lung tissue of control and EPM-exposed (1 day/week x 16 weeks) SD (upper panel), WKY (middle panel), and SH rats (lower panel) are shown in Figure 6Go. Light microscopic examination of lung sections revealed dose- and time-dependent accumulation of particles in alveolar macrophages. Particle laden macrophages were more readily apparent in focal areas of the parenchyma (Figs. 6B, 6E, and 6GGo). Careful examination of the bronchus-associated lymph nodes also revealed the presence of inflammatory cells loaded with EPM particles in all three rat strains. It appeared that there were more particle containing inflammatory cells in the lymph nodes of SH (Fig. 6IGo) than that of SD (Fig. 6CGo) and WKY rats (Fig. 6FGo). Lung sections of control 16 week SH rats also revealed more macrophages but without the accumulation of particles, similar to what was observed in BALF.



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FIG. 6. Lung histology revealing EPM accumulation in macrophages within parenchyma and in lung-associated lymph nodes following 16 exposures of one day per week. A representative photomicrograph from each rat strain is given. Arrows indicate location of particle-laden macrophages. Note that at 16 weeks, the predominant EPM-associated effect was accumulation of EPM-laden alveolar macrophages. (A), (D), and (G) are air controls. Exposure duration-dependent accumulation of particles within alveolar macrophages was readily apparent in parenchyma (B, E, and H) and in lung-associated lymph nodes (C, F, I). It appeared that there were more particle-laden macrophages found in lymph nodes of SH rats in comparison to SD and WKY rats.

 
Hematology
Plasma fibrinogen and complete blood counts (hemoglobin, hematocrit, red blood cell count, platelets, total and differential white blood cell count) were performed in air control and nose-only EPM exposed rats. There were minimal alterations in all the blood parameters measured following EPM exposure. It appeared that there was higher plasma fibrinogen in rats exposed to EPM for one week, especially in SH rats, however, this increase was not significant (data not shown). There appeared to be a slight decrease in blood lymphocytes of all three strains following one week of EPM exposure, however, this trend again was not statistically significant (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present study was to characterize pulmonary and systemic toxicity of a combustion EPM containing bioavailable zinc. We selected this EPM for three primary reasons:

Multiple IT and inhalation exposure scenarios, dose-response, and exposure durations were evaluated using three rat strains, one with systemic hypertension and underlying cardiovascular disease. A single IT instillation of EPM was associated with dose-dependent lung injury and neutrophilic inflammation attributable to the leachable or bioavailable fraction of EPM. The pulmonary injury caused by the EPM (whole suspension or leachate) could be reproduced by instillation of ZnSO4, suggesting the role of leachable zinc. Interestingly, no neutrophilic inflammation was evident following EPM inhalation exposures of up to 10 mg/m3. However, following EPM inhalation there was a concentration- and time-dependent accumulation of particle-laden macrophages. SH rats behaved differently in that their lymph nodes appeared to have more particle-laden macrophages than the lymph nodes in WKY or SD rats at 16 weeks.

Industrially generated combustion and ambient PM differ significantly in the type and the quantity of bioavailable elements. Because of these differences, the mechanisms or targets of toxicity may also vary (Gavett et al., 1997Go; Kodavanti et al., 1997aGo; 1998bGo; Nadadur and Kodavanti, 2002Go). Recently it has been shown that the toxicity of ambient-derived PM from Utah Valley (Dye et al., 2001Go; Soukup et al., 2000Go) and Ottawa, Canada (Adamson et al., 2000Go; Vincent et al., 1997Go) could in part be related to the level of bioavailable zinc when rats were exposed to the aqueous extracts of these particles by IT instillation. However, the mechanism by which zinc may induce cell signaling, nuclear translocation of nuclear factors, and downstream transcription of inflammatory genes is not clear. It is postulated that zinc-induced pulmonary inflammation may involve EGF receptor-mediated activation of MAP kinases (Huang et al., 2002Go; Silbajoris et al., 2000Go).

To determine if EPM toxicity was associated with significant neutrophilic inflammation/lung injury, and if the water soluble, bioavailable fraction containing zinc was responsible for pulmonary injury, we carried out two IT exposure experiments in SD rats. The dose-dependent acute neutrophilic inflammation and pulmonary protein leakage in BALF following IT instillation of whole EPM suspension was reproducible by using the leachable fraction of EPM or ZnSO4. This response is analogous to the previously reported studies using zinc-rich Ottawa particle leachate (Adamson et al., 1999Go) and Utah Valley PM-extracts (Dye et al., 2001Go) and suggest a potential role for zinc in pulmonary injury/inflammation caused by EPM when IT instilled. In contrast to the other studies, however, the particulate fraction of EPM was also associated with significant (but less dramatic) inflammation that could not be produced by the inert negative control particles (MSH). This residual activity would suggest that the reactive components on the surface of washed EPM particles are still active in biological systems.

Unlike the IT instillation, acute or long-term inhalation exposures were not associated with neutrophilic inflammation as evidenced by analysis of BALF and histology. This is in contrast to inhalation exposures using different ROFAs that contained high levels of vanadium and nickel (Kodavanti et al., 1999Go, 2000bGo, 2002Go). The differences in the neutrophilic inflammation following inhalation exposure to these two different combustion particles may be due to the type and the concentration of metal needed to initiate neutrophil recruitment in the lung. It is likely that the inhalation concentration of metals was not sufficient to cause neutrophilic inflammation following EPM exposure.

The hallmark effect of EPM inhalation exposure in rats was a dose- and a time-dependent accumulation of particle-laden alveolar macrophages with no apparent neutrophil increase. Activity of NAG, a marker for macrophage activation (Henderson et al., 1985Go), was also elevated in BALF and followed a similar pattern of increase as macrophages with acute four-day exposure scenarios, suggesting that phagocytosis of particles may have triggered macrophage activation. Thus, NAG activity in BALF may serve as an important marker for acute pulmonary changes without apparent neutrophilic inflammation. However, the significance and impact of the NAG activity increase remains to be investigated. No changes in LDH activity were noted with inhalation exposures (data not shown). It is also unclear how particle phagocytosis and macrophage accumulation may have occurred without consequent or associated neutrophilic inflammation in the inhalation protocol. Macrophage accumulation has been noted following exposure of rats to other combustion source particles, but generally followed an acute spike of neutrophilic inflammation (Kodavanti et al., 1999Go, 2001Go). With vanadium- and nickel-rich ROFA we have previously shown that this sequence of events leads to fibrosis in SD rats (Kodavanti et al., 1997bGo) suggesting a role for neutrophil/macrophage interactions in the development of fibrosis (Kumar and Lykke, 1995Go). Although fibrotic changes in the lungs have been reported in animals exposed to relatively high concentrations of zinc (Brown et al., 1990Go; Gordon et al., 1992Go; Lam et al., 1985Go), the lack of fibrosis in the present study in any of the rat strains suggests that the concentrations of EPM used were not sufficient to trigger a significant neutrophilic inflammation and matrix alterations. This finding may have an impact on the health risk involving this outcome associated with air pollution PM exposure.

While no neutrophilic inflammation was apparent following acute or long-term inhalation of EPM in any rat strain, there was a small acute increase in BALF protein (and albumin, data not shown) in all rat strains exposed for four days at 10 mg/m3. This increase was neither apparent with lower concentrations nor with longer exposure durations. While the IT instillation of the leachable fraction, and ZnSO4 caused a marked protein increase in BALF, these findings suggest a role for leachable zinc in inhaled EPM-induced acute protein/albumin leakage. Inhalation exposure to zinc oxide in human clinical studies has been associated with tolerance to subsequent zinc exposures (Fine et al., 2000Go). Similarly, rats may develop tolerance that may, in part, account for the lack of BALF protein or neutrophil increases with long-term exposures to EPM.

Particle clearance by alveolar macrophage phagocytosis and migration to the bronchus-associated lymph tissue is one of the many different mechanisms of host defense (Lehnert et al., 1986Go). Histological examination of bronchus-associated lymph nodes in rats exposed for 16 days (one day/week) revealed the presence of particle clusters, possibly associated with mononuclear cells, suggesting that EPM may be cleared by this route, in addition to ciliary clearance and interstitial uptake. Surprisingly, the SH rats appear to have more prominent populations of particle-laden cells in bronchus-associated lymph nodes. However, it is not clear how this may relate to differential susceptibility of SH rats to EPM exposure. In general, larger and more pronounced bronchus-associated lymph nodes in SH rats compared to SD or WKY rats may be due to chronic systemic and baseline pulmonary inflammation as a consequence of their genetic predisposition (Schmid-Schonbein et al., 1991Go). How this may relate to EPM clearance in SH rats, or how effective these clearance mechanisms may be in removing more toxic metal-laden particles is not known.

Epidemiological and clinical studies have shown associations between systemic changes in plasma fibrinogen and inflammatory cell populations as well as cardiac conductivity and myocardial infarction with PM (Dockery, 2001Go; Morris, 2001Go; Peters et al., 2001Go; Schwartz, 2001Go). Ours and other studies have shown that exposure to ROFA or ambient PM can cause plasma fibrinogen increases and imbalance of circulating lymphocytes and neutrophils, along with cardiac physiology changes (Campen et al., 2001Go; Gardner et al., 2000Go; Kodavanti et al., 2000bGo, 2002Go; Schladweiler et al., 2002Go), supporting the hypothesis that systemic endothelial activation may be one of the mechanisms responsible for the cardiovascular effects of PM. To determine if EPM also caused similar changes in healthy and cardiovascular compromised SH rats, we analyzed systemic hematological parameters, including fibrinogen. There was an insignificant but consistent small increase in plasma fibrinogen following acute exposures, which coincided with an acute increase in lung permeability following EPM inhalation. The significance of this finding can not be ascertained as the increase was not significant. A higher concentration of EPM may be required to induce fibrinogen increase in rats.

Unlike the studies done in the past using different combustion-derived ROFAs (Dreher et al., 1997Go; Kodavanti et al., 1998bGo, 2000bGo), the unique aspect of this study was to include a dose-response evaluation with acute inhalation exposures. The concentrations used for the inhalation study ranged from 2–10 mg/m3, a range frequently achievable in studies done using concentrated ambient particle exposures (Kodavanti et al., 2000aGo; Schladweiler et al., 2002Go). While these concentrations are unlikely to be encountered in ambient situations, they are comparable to occupational exposures (Hauser et al., 1995Go), and are lower than used in previous ROFA studies. Our hypothesis was that the lower dose should not induce detectable lung injury because this EPM contained lower levels of endotoxin and soluble metals. However, it was apparent that even 2 mg/m3 resulted in small but often significant increases in lavageable total cells, macrophages, and NAG activity, a marker for macrophage phagocytosis. EPM-laden alveolar macrophages were readily apparent histologically at this concentration. Thus, pulmonary responses can be generated without apparent neutrophilic inflammation by EPM at occupationally relevant concentrations.

In summary, our study shows that IT instillation of zinc containing EPM in rats caused marked and dose-dependent neutrophilic inflammation; 70–80% of which was attributable to the soluble fraction and 20–30% to the particulate fraction of EPM. Pulmonary injury and neutrophilic inflammation caused by IT instillation of whole suspension or leachate could be reproduced by instillation of ZnSO4 in rats. However, inhalation of this EPM was not associated with neutrophilic inflammation in any of the exposure scenarios, but caused a modest lung permeability increase with a high dose and an acute 4-day exposure. The only prominent effect of EPM inhalation was a dose- and time-dependant particle uptake with a concomitant increase in alveolar macrophages in all three rat strains: SD, WKY, and SH. SH rats exhibited marked accumulation of particle-laden macrophages within bronchus-associated lymph nodes with long-term exposure. Thus, pulmonary injury from IT instillation of EPM was not very predictive of inhalation effects. This study demonstrated that the exposure concentration of zinc during IT instillation may be critical in initiating neutrophilic inflammatory responses in the lung. Thus, in real atmospheric exposures pulmonary changes are likely with or without neutrophilic inflammation, depending upon the concentration and the chemical composition of ambient particles.


    ACKNOWLEDGMENTS
 
We thank Mr. James R. Lehmann (U.S. EPA) for intratracheal instillations and Mr. Donald L. Doerfler (U.S. EPA) for statistical evaluation of the data and Drs. William P. Watkinson, M. Ian Gilmour, MaryJane Selgrade, and Linda S. Birnbaum of the U.S. EPA for their critical reviews of the manuscript.


    NOTES
 
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and the policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at Pulmonary Toxicology Branch, MD-82, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 86 T. W. Alexander Dr., Research Triangle Park, NC 27711. Fax: (919) 541-0026. E-mail: kodavanti.urmila{at}epa.gov. Back


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