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Does the ambient particle concentrator change the toxic potential of particles?

Posted on: Friday, 3 October 2003, 06:00 CDT

ABSTRACT

Inhalation exposure to urban air particles is known to increase morbidity in humans and animals. Our group utilizes the Harvard/ U.S. Environmental Protection Agency Ambient Particle Concentrator (HAPC) to generate concentrated aerosols of outdoor air particles for experimental exposures. We have reported increased pathologic responses to inhalation of concentrated urban air particles and identified silicon (as silicate) as an element associated with many of these responses. Using silicate-rich Mt. St. Helen's volcanic ash (MSHA), we exposed three groups of Sprague-Dawley rats by inhalation for 6 hr to filtered air, MSHA, or MSHA passed though the HAPC. Twenty-four hours following exposure, bronchoalveolar lavage was performed to assess total cell count, differential cell count, protein, lactate dehydrogenase, and n-[beta]-glucosaminidase levels. Peripheral blood was examined for packed cell volume, total protein, total white cells, and differential cell count. Morphologic studies localized particles in the lung and assessed pulmonary vasculature. No significant differences were observed among any of the groups in any parameter measured including morphometric analysis of pulmonary vasoconstriction. Scanning electron microscopy and X-ray analysis identified particles as silicates typical of MSHA throughout the lung. These findings suggest that particles passing through the HAPC have no change in their toxic potential in an exposure setting where particle deposition in the lung has occurred.

INTRODUCTION

Our laboratory has reported extensively on pathologic responses to inhaled concentrated ambient particles (CAPs) that include silicates. During these studies, concentrated aerosol for exposure was generated using the Harvard/U.S. Environmental Protection Agency (EPA) Ambient Particle Concentrator (HAPC), a device developed specifically to facilitate air pollution research. The HAPC delivers ambient fine particles (up to 2.5 [mu]m in size) concentrated by a factor of ~30 times for direct inhalation exposures.1-2 The optimal size range for concentration in this system is 0.1-2.5 [mu]m ultrafine particles are neither concentrated nor removed, and it is designed not to modify the physicochemical characteristics of the particles.2 Thus, the HAPC provides a method for the study of increased concentrations of ambient particulate pollution by inhalation in vivo, and it has the capacity to sample concurrently for compositional analysis of particles. The aim of this study is to provide a bioassay of the HAPC and to assess whether passage of particles through the HAPC may alter the biological behavior of a benign particle.

CAPs generated from outdoor air in Boston, MA, originate from multiple sources, including crustal elements, vehicle exhaust, power plant emissions, home heating methods, and transported aerosols.3 The air intake site for studies performed in our laboratory is located ~75 m from a busy urban avenue that is a component of several public transportation routes (bus, trolley, and van) and is within a major metropolitan medical complex.

Extensive analysis of CAP mass and composition from our site has repeatedly revealed an association between elevated levels of silicon (Si) in CAPs and biological responses. In both normal rats and rats with chronic bronchitis, elevated Si levels were significantly associated with pulmonary vasoconstriction.4 Chronic bronchitic and normal rats also demonstrated increased protein and neutrophils in bronchoalveolar lavage (BAL) fluid.5 Normal dogs exposed to CAPs had increased percentages of neutrophils in BAL, and in peripheral blood counts, increases in total white cells, neutrophils, and lymphocytes were associated with increases in the Si-associated factor.6 Dogs undergoing transient coronary artery occlusion displayed significantly elevated ST segments with transient coronary occlusion associated with Si.7 Increased chemiluminescence of the heart (indicating increased levels of oxidative stress) was associated with Si in normal rats exposed by inhalation to CAPs.8 Airway resistance was increased in asthmatic mice exposed to ozone (O^sub 3^) and particles, and was associated with the aluminum (Al)-Si particle fraction of CAPs.9

The identification of Si as an element associated with potentially pathologic changes in circulating blood parameters and pulmonary pathology was surprising. The Si identified in our studies appears to be in the form of silicate, because in all of these data sets, Si, Al, and calcium (Ca) are highly correlated, and in all cases, similar biologic correlation was associated with these elements.7,10,11 Though a more complex source may be identified for these silicates, they are associated with crustal elements such as Al and Ca, suggesting an origin in the suspension of crustal dusts.7,10,11 In source apportionment studies, we have found that Si often represents 20-30% of the mass of the Si-related factor,6 as is usually found in particles of crustal origin.12

Oxides of Si are ubiquitously abundant in the Earth's crust and can comprise a significant portion of airborne particulate matter (PM). Silicates are formed when Si and oxygen (O2) bond in tetrahedron form with other elements; most commonly silicates consist of this tetrahedron in combination with metal cations of sodium (Na), potassium (K), Ca, magnesium (Mg), iron (Fe), and Al.13 Silicon dioxide, known as silica (SiO^sub 2^), from crustal elements may occur as either crystalline SiO^sub 2^ or amorphous SiO^sub 2^ depending upon environmental factors present at the time of formation, such as temperature, pressure, cooling speed, and natural origin.13 Crystalline SiO^sub 2^, occurring in several forms characterized by three-dimensional repeating patterns, is the cause of the pulmonary disease silicosis.14,15 In contrast, amorphous SiO^sub 2^, which has no significantly repeatable pattern, has minimal health risks to people.

Amorphous SiO^sub 2^ and silicates were produced upon eruption of the Mt. St. Helen's volcano in 1980. The magma cooled very rapidly, and an amorphous silicate ash was formed. Mt. St. Helen's Ash (MSHA) has been studied extensively in natural exposures, as well as with in vivo and in vitro experiments, and has been found to be benign, even in cases involving the inhalation exposure of children with both diabetes mellitus and preexisting lung disease16 or in emphysematous rats.17 Volcanic ash from the eruption of Mt. St. Helen's was chosen for this study as our Si source precisely because of its extensive characterization as a benign amorphous silicate.

We produced Si-rich aerosols of MSHA for this study at concentration levels comparable to and above those used in CAP studies using the HAPC.4-9 We examined the ability of this concentrate to produce differential pathologic responses in animals, comparing those particles going through the HAPC versus the same aerosol at the same concentration bypassing the HAPC. During concentration, there is a hypothetical possibility of mechanical alteration of the particles with fine particle fracturing, or changes in electrostatic charge as the particles pass through the acceleration and collection nozzles in the HAPC. Alternatively, it may be that the silicates in our previous studies are actually traveling with more toxic compounds and are serving as markers of these other compounds. There has never been a bioassay study of concentrator technology to assure that these devices cannot change the toxic potential of particles.

In this experiment, HAPC-concentrated aerosolized MSHA, unconcentrated MSHA at the same exposure dose, or filtered air were delivered to normal rats for 6 hr. Four repetitions were performed: one lower dose, and three high doses. Pulmonary toxicity was assessed using measures of pulmonary inflammation as end points, and changes in peripheral blood parameters were also monitored. Pulmonary vascular morphology was assessed for vascular lumen wall ratio to assess vasoconstriction. Additionally, lung tissue was collected for electron microscopy to evaluate parenchymal deposition of ash.

EXPERIMENTAL METHODS

MSHA

Dr. Andrei M. Sarna-Wojcicki of the U.S. Geological Service generously donated the volcanic ash used in this study. The ash, collected from the Bates Ridge site in Missoula, MT, is pale gray in color, unconsolidated, and made up of a very fine grade tephra. It is considered to be typical of the ash generated by the eruption.

HAPC and Aerosol Generation

MSHA was aerosolized using a Wright Dust Feeder (BGI, Inc.) and compressed zero air (Med-Tech Gases, Inc.) supplied from a cylinder at 18 pounds per square inch (psi). The resultant aerosolized ash was passed through an elutriator that removed large particles (>7 [mu]m) by gravitational settling and mixed ash with high-efficiency particulate air (HEPA)-filtered air. At the top of the elutriator, a manifold divided the flows for different exposures. One limb went into the HAPC, where it was diluted by HEPA-filtered air coming from the concentrator inlet and then concentrated to be delivered to an exposure chamber. The second limb went directly from the elutriator to an exposure chamber. Valves controlled the flows in these two limbs so that the final concentrations foranimal exposure would be equal. The third animal exposure chamber received filtered air. Pressures in all three chambers were equivalent. The experimental setup is shown in Figure 1.

The HAPC was used to concentrate aerosolized fine ash particles of 0.1-2.5 [mu]m. The same HAPC also was used for the production of all ambient concentrated aerosols used in previous studies that suggested the association of Si with pathophysiologic change. The HAPC normally draws in ambient outdoor air and concentrates particles using a high-volume conventional impactor with a 2.5- [mu]m upper cutoff size and a series of three virtual impactors. The resulting fine particle concentrate is then delivered to animal exposure chambers.

For this study, clean airflow through the concentrator was achieved by drawing room air through a HEPA filter (Glasfloss Air Filtration Products) with an efficiency of >99.97% for particles larger than 0.3 [mu]m. The HEPA filter did not increase the total operating pressure drop of the HAPC. The concentrator was operated for several hours with the HEPA filter to verify the removal of ambient particles. Baseline mass measurements in this concentrator configuration were below the limits of detection using a tapered element oscillating microbalance (TEOM). By filtering the air going into the HAPC and then adding the MSHA aerosol just after the filter, the potential for confounding by concentrating ambient particles was eliminated from the study.

Figure 1. Aerosol generation and exposure setup.

Exposure Stability and Comparability

Concentration at the elutriator outlet was measured continuously to assess dust-generation stability using a Miniram (Monitoring Instruments for the Environment, Inc.) with external sampling flow provided at 2 L/min. A filter (47-mm Teflo Teflon filter, Gelman- Pall) was used downstream of the Miniram to obtain an integrated average concentration measurement for each limb of the exposure system; sampling flow rate was measured downstream of the filter using a calibrated rotameter. Particle size distribution was monitored using an Aerosizer (Amherst Process Instruments; see Figure 1). Stability of the particle size distribution at the elutriator was monitored using 60-sec measurements once every 5 min of the exposure. Flows of aerosolized ash were regulated to achieve equivalent concentrations for the two ash exposure groups. Mass concentration comparability between the concentrated and unconcentrated MSHA exposure chambers was assessed before and during exposure using integrated sampling techniques described later. In addition, comparability of particle size distribution between MSHA exposure groups was assessed using repeated alternating measurement with the Aerosizer.

EXPERIMENTAL GROUPS AND EXPOSURE PROTOCOL

Male Sprague-Dawley rats (239-269 g) were obtained from Charles River Laboratories and were managed in accordance with the National Institutes of Health guidelines for the care and housing of laboratory animals. Sixty animals were housed in large cages in six groups of five and allowed to accommodate to new housing conditions for three days before exposure to avoid potential confounders of new social interactions and stress. Three groups of five animals each were randomly assigned to one of the following treatments: (1) filtered air exposure; (2) aerosolized MSHA exposure; and (3) concentrated aerosolized MSHA exposure. Four repetitions of the experiment were performed. Experimenters assessing the animals were blinded to treatment parameters, and exposure chambers were only distinguishable by color-coding. Setup of the exposure chambers was uniform to avoid visual identification of the treatment protocol.

Rats were exposed unrestrained in their groups in plexiglass chambers (two exposure chambers of 8 x 12 x 15 in., one exposure chamber of 9 x 9.5 x 12 in.). They were exposed at a flow rate of 20 L/min for 6 hr and then returned to their maintenance cages until sacrifice at 24 hr postexposure. Animals were observed to sleep through the majority of the exposure period.

Particle Characterization

Animal exposures were conducted in the inhalation facility at the Harvard School of Public Health. Particles were collected on preweighed Teflon filters (collection flow rate 2 L/min). Particle size distributions were determined from Aerosizer data. (Figure 2) Concentration levels of fine particulates were determined gravimetrically. Filters were weighed using a Mettler-MT5 microbalance in a temperature- and humidity-controlled room. Start and end filter weight, sampling time, and sampling flow rate were used to calculate the particle concentration ([mu]g/m^sub 3^).

Elemental analysis of samples was performed by X-ray fluorescence (XRF; Chester LabNET) using standard techniques. One sample from each MSHA exposure group was randomly selected for analysis. Morphology of the particles was assessed from the Teflon filters using a Leo 1450VP scanning electron microscope (SEM; LEO Electron Microscopy, Inc.) operated at 20 kV. X-ray spectra of particles were assessed from the Teflon filters using Oxford's INCA Energy 300 microanalysis system (Oxford Instruments).

Figure 2. MSHA particle size distribution during repeated exposures.

Animal Assessments

Fourteen animals from each group were randomly selected for hematologic study and BAL. Twenty-four hours following the initiation of exposure, rats were euthanized with an overdose of sodium pentobarbital (Fatal Plus, Vortech). Animals were weighed. Direct cardiac puncture was performed through a thoracic incision. For complete blood count, total protein, and hematocrit, 0.5 mL of blood was collected into an EDTA tube (Sherwood Medical). Then, 3 mL were collected into a serum separator tube (Sherwood Medical), allowed to clot, and spun, and serum was frozen at -80 [degrees]C for future analyses if evidence of between-group variation was found.

BAL was performed through a tracheal incision using eight 5-mL washes with endotoxin-free Dulbecco's phosphate buffered saline (PBS). Fluid recovered was centrifuged (400 x g) at 4 [degrees]C. The supernatant from the first lavage was saved for protein analysis of BAL. Cell pellets were resuspended in 1 mL of PBS, and viability and total cell counts were determined by hemacytometer counts of 1- mL aliquots of the resuspended BAL combined with 80 [mu]L of 4% trypan blue solution. Differential cell counts were determined from modified Wright Giemsa-stained cytocentrifuge preparations, and 200 cells were counted per sample.

Biochemical Assays

Three markers of pulmonary injury were tested within the acellular BAL supernatant. Lactate dehydrogenase (LDH) levels were measured as an indicator of cytotoxicity, total protein (TP) levels were measured as a marker of pulmonary inflammation and vascular permeability, and the lysosomal enzyme [beta]-N-acetyl glucosaminidase (BNAG) was measured as a marker of phagocyte activation.18 Total protein was measured using a standard kit from Pierce. Enzymatic reagents for the measurement of BNAG and LDH were obtained from Sigma Chemical Co., and chemical reagents were obtained from Fisher Scientific Co. LDH, TP, and BNAG measurements were performed using a Beckman DU-640 spectrophotometer (Beckman Instruments). Remaining lavage fluid has been stored at -80 [degrees]C for any future biochemical analysis.

Histopathology

For assessment of particle deposition in the lung, one rat from each group in the low-dose experiment was randomly selected for intravascular fixation for histopathologic processing. Following euthanasia with an overdose of intraperitoneal sodium pentobarbital (Fatal-Plus, Vortech), rats were weighed, and vascular perfusion was performed. A tracheal incision was made for the insertion of an endotracheal tube. A cannula was introduced into the inferior vena cava below the levels of the kidneys, and tubing was threaded up to the level of the right atrium. A small incision in the liver was made to permit escape of flushed fluids. The lungs were inflated with air at constant pressure. Vascular perfusion was begun with 10 mL heparinized lactated Ringer's solution (LRS; 10,000 units heparin per 500 mL LRS) to flush the system, and completed by fixation with a constant perfusion of 2.5% glutaraldehyde in 0.03 M potassium phosphate buffer with 3% dextran over 15 min. The trachea was tied off to maintain inflation, and the lungs were excised and placed in 2.5% glutaraldehyde in 0.085 M sodium cacodylate buffer for refrigerated storage.

The lungs were sectioned horizontally into 2-mm guided sections. For assessment and identification of particles in the lung, one slice was selected randomly, cut into smaller fragments, critically point dried with a Samdri-PVT-3B (Touismis), and carbon coated with an Edwards Auto 306 carbon coater for optimal X-ray analysis or coated with a Hummer V sputter coater using a gold (Au)/palladium (Pd) target (Technics) for assessment of lung morphology. Lung tissue samples were then assessed by SEM operated at 15 kV or by INCA 300 micro-analysis to qualitatively evaluate the deposition of inhaled MSHA particles. The samples were assessed using either secondary electron (SE) mode or backscatter (BS) mode.

For assessment of lung morphology, a total of six rats from each group were randomly selected for intratracheal fixation of 2.5% glutaraldehyde in 0.1 M potassium phosphate buffer at transmural pressure of 20-22 cm of water. Lung lobes were cut horizontally into 2-mm sections with a guided razor blade. One randomly selected section from each lobe was processed for histology.

All slides were coded for blinded analysis. For morphometric assessment, each section was viewed at low magnification to select transverse sections of pulmonary arteries that were adjacent to bronchoalveolar junctions and were in true cross section, with a variation of <10% between minimum and maximum diameter. The ratio between the lumen and wall areas of the pulmonary arteries was determined morphometrically using a point grid attached to the eyepiece at high magnification (960x). Points overlying the lumen and wall were counted using an unbiased counting procedure as previously described.4,19

Table 1. Ash concentrations and particle size.

Statistical Analyses

One-way analysis of variance (ANOVA) tests were performed using Microsoft Excel to test whether there were significant differences among the three groups for each pulmonary and systemic parameter. Differences were considered significant when p < 0.05. A post-hoc power analysis was also performed. The minimum detectable difference (MDD) that would detectable between any exposure groups in a one- way ANOVA with significance level [alpha] = 0.05 was estimated. For each end point, estimates of the residual standard deviation were taken to be the square root of the mean standard error obtained from each ANOVA analysis. All power calculations were performed using the power software Nquery Advisor 4.0 (Statistical Solutions).

RESULTS

The concentration of MSHA between groups was set before passage through the HAPC. After concentration by the HAPC, an average concentration enrichment factor of 37.5 was achieved, which was within 10% of that predicted by the measured flows. The exposure concentrations and particle size distributions (geometric standard deviations [GSDs]) are summarized in Table 1. Briefly, in the four repetitions of the experiment that were performed, concentrations of ash began at a low dose of 812.9 [mu]m/m^sup 3^ concentrated ash and 859.6 [mu]m/m^sup 3^ unconcentrated ash, and were approximately doubled (to ~1600 [mu]m) in further repetitions of the experiment.

Measurement of the particle size distribution at the elutriator output demonstrated that the size distribution, presented in Figure 2 as cumulative percent undersize, was stable and reproducible over the four different exposures. Comparison of particle size distribution in the MSHA and concentrated MSHA exposure chambers demonstrates only minimal differences. Figure 3 shows the cumulative percent undersize distribution measured at the elutriator output and at each exposure chamber. Both exposure chambers show losses of larger particles as compared with elutriator output. The concentrator effectively removed particles larger than 2.5 [mu]m (as expected), and the MSHA chamber had fewer losses of larger particles. Overall, however, the median diameter and GSD of the two exposure chambers are comparable (see Table 1).

Particle size averaged 1.17 [mu]m over four days of exposure (see Table 1). Elemental analysis of composition of concentrated MSHA and unconcentrated MSHA differed by less than 10%, and most differences were within the range of uncertainty as determined by the method of analysis (Table 2).

All response analyses in the rats were performed under blinded conditions. No significant differences were found between groups in any biological parameter examined. Complete blood counts revealed no elevations or significant differences in any parameter measured among groups, at any exposure concentration, and are reported for all experiments combined (Table 3). BAL cell counts did not significantly differ between groups, and analysis of BNAG, total protein, and LDH in the acellular BAL supernatant revealed no significant differences between groups. Mean, standard deviation, and mean detectable differences are reported for all experiments combined (Table 4). Morphometric analysis of pulmonary artery lumen wall ratio revealed no significant differences in degree of vasoconstriction between groups (Table 5).

To demonstrate that there was sufficient power in the study to detect meaningful differences of interest, we performed a post hoc power analysis. In particular, given the sample sizes used in the study (14 animals per group), we estimated the MDD we would be able to detect between any two exposure groups in a one-way ANOVA with [alpha] = 0.05. For each end point, estimates of the residual standard deviation were taken to be the square root of the mean standard error obtained from each ANOVA analysis. All power calculations were performed using the power software Nquery Advisor 4.0 (Statistical Solutions). Tables 3-5 show the MDD for each outcome considered in the analysis. Because the MDDs are very small, it is concluded that the study was sufficiently powered to detect differences of scientific interest.

Figure 3. Comparison of MSHA size distribution.

Table 2. Exposure parameters as determined by XRF.

Particles with the same composition (see Table 2) and spectral signature as defined by X-ray elemental analyses were found in the lung by SEM (Figure 4a-h). Figure 4a illustrates the typical morphology of these particles collected onto a filter. The particles are irregular in shape, with numerous sharp edges. Most of these particles are less than 2.5 [mu]m in maximum dimension, and conform to the size of particles measured aerodynamically. These particles were observed in a number of locations throughout the lung, from epithelial ciliated surfaces to deep within the alveoli, and demonstrate the variable geographic deposition of the particles in Figure 4b-f. Figure 4g-h shows typical X-ray analysis spectra of particles deposited within the lung. Similar spectra were obtained from particles on filters, and these findings are very similar to the XRF analyses (see Table 2).

Table 3. Peripheral blood sample analysis mean values and standard errors (in parentheses).

Table 4. BAL parameter mean values and standard errors (in parentheses).

DISCUSSION

In studies of concentrated ambient air particles, associations of silicate with alteration of a number of biological parameters have been found.4-7,10 This experiment was designed as a biological assay for the HAPC, to be certain that toxic change in the silicate fraction of the particles was not being induced by passage through the HAPC.

MSHA was used as a source of silicate because of its extensive characterization as a benign particle. Exposure studies of loggers exposed over an extended period of time to naturally occurring ash fall after the eruption of Mt. St. Helen's showed no significant long-term changes in lung function or symptoms of disease.20 Similarly, acute exposure of children with diabetes mellitus to the ash showed no evidence of decline in lung function, even in children with additional preexisting pulmonary disease or symptoms.16 Tracheal instillation of the ash in hamsters showed a response to MSHA comparable to that of aluminum oxide, a relatively inert dust.21 Inhalation studies of normal and emphysematous rats revealed no pulmonary toxicity of MSHA.17 MSHA also has been used as a negative control particle in a number of in vitro studies, including studies on pulmonary macrophage activation by complement,22 activation of human bronchial epithelial cells,23 and induction of the lung myofibroblast PDGF receptor system.24

These results concur with previous findings that characterize MSHA as benign. More significantly, because of a lack of any significant difference between exposure groups and the fact that the study had sufficient power to detect differences of interest, we conclude that amorphous silicates are not altered by passage through the HAPC and that they remain benign upon inhalation exposure. We morphologically confirmed that the particles were inhaled into the lung and deposited at sites in the deep lung. It is demonstrated morphometrically that no significant pulmonary vasoconstriction was induced by inhalation of this silicate-rich ash (see Table 5). No significant difference in circulating hematologic parameters (see Table 3) or pulmonary inflammatory parameters (see Table 4) was demonstrated, despite evidence of translocation of the particles into the lung interstitium (see Figure 4b-f).

Concentrations of MSHA generated in this study range from 812.9 to 1762 [mu]g/m^sup 3^. These concentrations exceed concentrations of ambient air pollution that have been reported to elicit effects. In normal rats, short-term exposures with a median concentration of 182.75 [mu]g/m^sup 3^ and range of 73.5-733 [mu]g/m^sup 3^ was enough to induce vasoconstriction, with pulmonary lumen wall ratio decreasing from 2.41 in the sham-exposed animals to 1.63 in the CAP- exposed animals.4 Lung inflammation as measured by differential cell count in BAL (with variable increases in percentages of 5-10%, in both neutrophils and lymphocytes) was also induced in both normal and chronic bronchitic rats at these exposure levels.5 Mean CAP mass associated with significant changes in hematologic parameters in dogs was 203.4 [mu]g/m^sup 3^.6

Several potential mechanisms for toxic change in the silicate fraction of CAPs were considered. Passage of PM through the three stages of impactors in the HAPC was considered to be a possible source for alteration of the particles, particularly attraction of electrostatic charge as particles passed through the orifice. Recently, Nemmar et al.25 have shown differential responses with differences in particle charge. This phenomenon would be expected to be enhanced when the air is dry, because humidity dissipates electrostatic charge, and leads to increased particle losses under drier conditions.12 However, we have no evidence of electrostatic charge acquisition with the concentrator. Indeed, decreased particle enrichment was observed under humid conditions and increased particle enrichment was observed in more arid conditions.26

Table 5. Pulmonary vascular lumen/wall ratio mean values and standard deviations (in parentheses).

Figure 4. Concentrated MSHA passed through the HAPC. (a) Particles as collected on Teflon filter. (b) Particles (white arrows) are demonstrated on the surface and within the epithelial layer of an airway. (c) Particles located within the transition zone at a bifurcation of bronchioles. (d) Particles at transition from bronchiole to alveolar entrance. (e) Particles within an alveolus. (f) Particles adjacent to the pleura. (g) Spectral analysis of MSHA from particle in d. (h) Spectral analysis of MSHA from particle in c. All are assessed in BS mode except for e and f, which are in SE mode.

Fresh fracture of particles has been shown to create altered surface charge, leading to the possibility of biological activation.27 Human bronchial epithelial cells exposed to a variety of particulates evidenced biological activation in vitro associated with surface charge of the particles, which led to a predictable initiation of airway inflammation.23 It is possible, but unlikely, that sufficient force is generated in the HAPC's virtual impactors to produce this phenomenon. However, no inflammatory response was produced in our experiment by passing MSHA through the HAPC. This hypothesis is additionally unlikely because the HAPC has been shown not to distort the ambient size distribution or particle composition during concentration.2 This is confirmed by our findings (see Figure 3).

The studies reported here cannot entirely rule out the possibility of other forms of silicates, or minimum amounts of crystalline SiO^sub 2^, as being responsible for the changes observed in our CAP studies. The effect of concentration of crystalline SiO^sub 2^ through the HAPC has yet to be determined, although we hypothesize (and our data using amorphous SiO^sub 2^ suggests) that no significant enhancement of toxicity should occur.

Crystalline SiO^sub 2^ does cause significant toxic insult in biological systems. Most mining operations and many manufacturing jobs involve exposure to crystalline SiO^sub 2^ in the form of dust. Silicosis is the pulmonary disease resulting from deposition of crystalline SiO^sub 2^ in the lungs and represents a significant health risk for workers exposed by inhalation to SiO^sub 2^ dusts.28 Interactions between surface layers of the crystalline SiO^sub 2^ and interactions with lung macrophages are thought to be the major mechanisms of tissue injury in silicosis.28 Numerous animal studies confirm the pro-inflammatory capabilities of crystalline SiO^sub 2^.29,30 Crystalline silicas are of variable toxicity because of physical properties and structure.27,31,32 These factors are important in the stimulation of macrophages to produce inflammatory and fibroproliferative factors.33 Oxygen radicals are also important in the pathogenesis of SiO^sub 2^-associated lung disease and may impair the ability of the macrophages to control infection, leading to increased risk of infectious disease such as tuberculosis in patients with silicosis.34

The hypothesis, which most likely accounts for the observed association of Si with toxic changes in our CAP studies, is that the silicate travels with a more biologically active partner and serves as a surrogate marker for the presence of that partner. Previous CAP factor analysis and source apportionment studies have shown that Si represents 20-30% of the mass of the Si factor.6 Our air intake site is located ~75 m from a heavily used urban traffic route. The remaining mass of the Si factor most likely includes urban road dust, which is enriched with combustion-derived materials, organic semi-volatile compounds, and acidic inorganic gases.35 In addition, tire-and brake-derived particles and bioaerosols such as pollen are also found in road dusts.35,36 These components represent a large number of potential suspects for the initiation of adverse biologic effects and represent a rich reservoir of material for ongoing investigation in our laboratory.

We show that for the silicate used in this study, the possibility of iatrogenic change in silicate induced by the HAPC has been eliminated as an explanation for the observed pathophysiologic consequences of CAP exposure in our studies. In future studies, we will focus upon more specific characterization of PM components in our efforts to identify components that travel with Si and are responsible for toxic effects. We will also investigate what biological activity can be observed when an aerosol of proven toxicity (such as quartz) is passed through the concentrator.

ACKNOWLEDGMENTS

The assistance of Marshall Katler and Nan Fei Jiang in animal harvesting procedures is gratefully acknowledged. The expertise of G.G. Krishna Murthy was invaluable in designing the ash delivery system. Daniela Zanoni and Tatiana Arilha provided valuable assistance in morphologic analysis. Elena Gitin provided essential statistical support. The authors also thank Andrei Sarna-Wojnicki for his generosity in donating MSHA for this study. This research was supported by grants ES 08129, ES 00002, and HL 07118 from the National Institutes of Health, and Research Award R827353 from the EPA.

ISSN 1047-3289 J. Air & Waste Manage. Assoc. 53:1088-1097

Copyright 2003 Air & Waste Management Association

IMPLICATIONS

This study validates the use of the HAPC as a method of producing concentrated aerosols of fine urban air particles without altering their physical properties or potential for toxic insult.

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Sara T. Savage, Joy Lawrence, Tracy Katz, Rebecca C. Stearns, Brent A. Coull, and John J. Godleski

Harvard School of Public Health, Boston, Massachusetts

About the Authors

Sara T. Savage, DVM, is a postdoctoral fellow, Joy Lawrence, Ph.D., is a research associate, Tracy Katz is a research assistant, Rebecca C. Stearns, ALM, is a technical director and research specialist, and John J. Godleski, MD, is an associate professor in the Department of Environmental Health at the Harvard School of Public Health in Boston, MA. Brent A. Coull, Ph.D., is assistant professor in the Department of Biostatistics at the Harvard School of Public Health. Address correspondence to: John J. Godleski, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115; fax: (617) 432-4528; e-mail: jgodlesk@hsph.harvard.edu.

Copyright Air and Waste Management Association Sep 2003

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