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Toxicity of chemical components of fine particles inhaled by aged rats: Effects of concentration

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

ABSTRACT

This study tested the hypothesis that exposure to mixtures containing fine particles and ozone (O^sub 3^) would cause pulmonary injury and decrements in functions of immunological cells in exposed rats (22-24 months old) in a dose-dependent manner. Rats were exposed to high and low concentrations of ammonium bisulfate and elemental carbon and to 0.2 ppm O^sub 3^. Control groups were exposed to purified air or O^sub 3^ alone. The biological end points measured included histopathological markers of lung injury, bronchoalveolar lung fluid proteins, and measures of the function of the lung's innate immunological defenses (macrophage antigen- directed phagocytosis and respiratory burst activity). Exposure to O^sub 3^ alone at 0.2 ppm did not result in significant changes in any of the measured end points. Exposures to the particle mixtures plus O^sub 3^ produced statistically significant changes consistent with adverse effects. The low-concentration mixture produced effects that were statistically significant compared to purified air but, with the exception of macrophage Fc receptor binding, exposure to the high-concentration mixture did not. The effects of the low- and high-concentration mixtures were not significantly different. The study supports previous work that indicated that particle + O^sub 3^ mixtures were more toxic than O^sub 3^ alone.

INTRODUCTION

Human exposures to respirable fine particles (ambient particles smaller than 10 [mu]m mass median aerodynamic diameter [PM^sub 10^]) are consistently and significantly associated with increased total annual mortality rates.1,2 In addition, PM^sub 10^ exposures at levels near and below the national (150 [mu]g/m^sup 3^, 24-hr average) ambient air quality standards (NAAQS) are associated with increased hospital admissions and emergency room visits for respiratory illnesses/3-6 increased incidences of asthma attacks,7,8 increased asthma medication use,9 reduced pulmonary function,9-11 and increased daily mortality.12-15

More recently, ambient air quality standards have been promulgated for ambient particles smaller than 2.5 [mu]m mass median aerodynamic diameter (PM^sub 2.5^) in the United States and in the state of California. PM^sub 2.5^ exposures are strongly associated with health effects, and some16,17 but not all18-21 studies indicate that the fine particles are associated to a greater degree with health outcomes than are the coarse components.

It is unlikely that any one specific component of ambient fine particles will be identified as a sole causal agent for adverse health effects. Components that have been suggested include acids, sulfates, transition metals, and organics. Inhalation toxicology studies with young, healthy animals have demonstrated only subtle effects that might be mechanistically relevant but do not indicate the level of potency required to adequately explain the human mortality associated with particulate matter (PM).22,23 However, animals with chemically induced bronchitis, exposed to concentrated ambient fine particles, exhibited mortality, exacerbation of the pre- existing lung disease, and changes in cardiac rhythms.24 Kleinman et al.25 have demonstrated that inhalation of elemental carbon (EC) and ammonium bisulfate (ABS) particles, which are components of ambient fine particles, caused injury to lung epithelia and altered pulmonary macrophage functions in aged, or senescent, rats. The exposures were performed in the presence and absence of ozone (O^sub 3^), which is a common gaseous contaminant in many urban areas with increased fine particle concentrations. The results of this previous study demonstrated that (1) fine particle exposure could damage lungs and impair host defenses in aged rats; and (2) that mechanisms of injury were intensified when O^sub 3^ was administered in addition to particles. This paper reports a continuation of that earlier study. It is hypothesized that particle-induced lung injury is dose-dependent and that increasing the concentrations of EC and ABS particles would increase the intensity of biological response.

METHODS

Exposure Methodology

Atmosphere Generation and Characterization. The generation and characterization methods used in this study have been described in detail in a previous publication.25 Briefly, internally mixed particles of ABS and EC were generated by nebulization of suspensions of carbon black (Monarch 120; Cabot Corp.) in dilute aqueous solutions of ABS. The particles were diluted with dry air, brought to Boltzmann charge equilibrium by passage through a ^sup 85^Kr aerosol charge neutralizer, equilibrated to 60% relative humidity (RH), and then introduced into a nose-only exposure system. O^sub 3^ was generated by metering medical-grade oxygen through an electrical O^sub 3^ generator (Sander Ozonizer, Type III).

Size-classified aerosol samples were collected using cascade impactors (Sierra Model 226; Graseby/Andersen). A real-time aerosol monitor (RAM-1; MIE, Inc.) provided continuous mass concentration readings during the entire exposure period. Integrated 4-hr aerosol samples were collected at the rat's breathing zone on Pallflex T60A20 fluorocarbon-coated glass fiber filters (Pall Corp.). The filters were equilibrated to 60% RH and total mass was determined gravimetrically. The filters were extracted with dilute aqueous buffer (3 mM sodium bicarbonate [Na^sub 2^HCO^sub 3^]; 2 mM sodium carbonate [Na^sub 2^CO^sub 3^]), and the extracts were analyzed for sulfate (SO^sub 4^^sup 2-^) by ion chromatography. Elemental carbon was determined on samples collected on quartz-fiber filters (Microquartz, Gelman). These filters were combusted in pure oxygen and the liberated carbon dioxide (CO2) was quantified using a modified infrared absorption monitor (Model 3003, Dasibi Environmental). O^sub 3^ concentrations were measured by UV absorption (Ozone Monitor Model 1003AH, Dasibi Environmental).

Animal Housing and Exposure. Aged (22-24 months) barrier-reared and maintained F344N-NIA rats were obtained from colonies managed under contract to the National Institute on Aging. Rats were shipped to the laboratory in filter-equipped boxes and housed in laminar flow isolation units supplied with filtered air. The rats were randomly assigned to treatment groups and nose-only exposed 4 hr per day, 3 consecutive days per week, for 4 weeks. The rats were exposed to one of four atmospheres: (1) purified air; (2) O^sub 3^, 0.2 ppm; (3) Low-concentration particle mixture (EC [50 [mu]g/m^sup 3^] + ABS [70 [mu]g/m^sup 3^] + O^sub 3^ [0.2 ppm]); 0.3 [mu]m mass median aerodynamic diameter (MMAD); and (4) High-concentration particle mixture (EC [100 [mu]g/m^sup 3^] + ABS [140 [mu]g/m^sup 3^] + O^sub 3^ [0.2 ppm]); 0.3 [mu]m MMAD. Ten animals from each group were assessed for lung injury (histopathology) end points, and 12 animals from each group were assessed for host-defense-related end points (permeability, mucus production, and macrophage functions). Between exposures, rats were housed in a purified air-barrier environment and had access to clean water and dry laboratory chow ad lib. The vivarium was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and the animal protocols were reviewed and approved by an Institutional Review Board.

Biological End Points

Necropsy, Tissue Preparation, and Analysis for Lung Cell Injury. Inhalation of oxidant gases or particulate material can injure respiratory tract tissue. This injury is often followed by a wave of cellular replication in a repair response to the damage produced by the agents. Labeling with 5-bromo-2-deoxyuridine (BrdU) (Sigma) can be used to establish an index of cell replication subsequent to cell injury. BrdU is a thymidine analog that is incorporated into DNA by replicating cells during DNA synthesis. Each rat was intraperitoneally injected with BrdU (50 mg/kg body weight) a minimum of 4 hr before sacrifice. All lung tissues used for immunohistochemical localization were preserved by perfusion fixation with zinc formalin fixative (Anatech Ltd.). Airways were dehydrated in a graded series of ethanol solutions and embedded in paraffin for light microscopy according to standard procedures. Several 5-[mu]m sections were cut sagittaly from the embedded tissue along one axial pathway. The tissues were stained with hematoxylin- eosin (H&E) and evaluated by light microscopy.

For BrdU immunohistochemistry, 30-[mu]m thick paraffin sections were cut. Paraffin sections were deparaffinized in xylene, hydrated in a graded series of ethanol solutions, and treated with 0.07-N sodium hydroxide (NaOH) to denature the DNA. BrdU was detected by treating the sections with a primary monoclonal antibody directed against single-stranded DNA containing BrdU (ICN, Immnunobiologicals), using a biotinylated secondary antibody, an avidin-bound peroxidase complex (ABC Vecstatin Peroxidase mouse IgG Kit, Vector Laboratories, Inc.), and 3,3'diaminobenzidine (Sigma).26 Standard method controls included (1) substitution of phosphate buffered saline (PBS) for primary antibody, (2) normal serum for the primary antibody; or (3) series in which serial dilutions of the primary antibody were incubated with serial sections (culture media dilutions run from 1-2 to 1-10,000 in steps). Under lo\w magnification, the 5-[mu]m thick H&E sections were examined, and the terminal bronchioles were numbered in a stratified manner on the slide using NIH Image software. Four terminal bronchioles were systematically sampled for morphometry from the 10-20 terminal bronchioles sectioned per slide using a random start. A 30-[mu]m- thick section was cut from each block and an optical disector was used to count the BrdU-positive epithelial cells.27 Briefly, the number of cells per volume of epithelium was estimated using the following formula:

N^sub vBrdU/epi^ = (N^sub Brdu^/A^sub epi^)(H^sub epi^) (1)

where N^sub v BrdU/epi^ is the number of BrdU-positive cells per volume of epithelium, N^sub Brdu^ is the number of BrdU-positive cells counted using a confocal microscope in the 30-[mu]m sections excluding those that intersected the top of the section and two sides of the counting frame, A^sub epi^ is the area of epithelium in the counting frame, and H^sub epi^ is the height of the optical disector (usually 30 [mu]m). The number of BrdU-positive cells in the interstitium were computed in the same manner.

Macrophage Function and Inflammation. Bronchoalveolar lavage was performed on rats (n = 12 per atmosphere) to obtain macrophages for immunological testing and proteins for assessment of epithelial permeability.28 The rats were anesthetized, the abdominal aortas were severed, and the tracheas were exposed. A catheter was inserted into the trachea and tied in place. The lungs were lavaged with N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered (pH 7.2) Hank's Balanced Salt Solution (HBSS) without calcium (Ca^sup 2+^) or magnesium (Mg^sup 2+^) (GIBCO, BRL). The lavage volume was 7 mL and it was instilled and aspirated 3 times at a rate of ~0.5 mL/second. The lavage was repeated 3 times per animal, and the recovered fluid from each lavage was placed on ice. The lavage fluid from each animal was centrifuged at 300 g, 4 [degrees]C, for 10 min. The fluid from the first lavage was reserved for protein and biochemical assays. The cell pellets from all three lavages were pooled and resuspended in 3 mL HBSS with Ca^sup 2+^ and Mg^sup 2+^.

The cells were counted using a bright-line hemocytometer. Viability was assessed by trypan blue exclusion. The cell suspension was adjusted to 106 viable cells per mL. The yield by this lavage procedure was typically 3 million cells per rat, of which more than 95% were macrophages with an average viability of greater than 90%. A 0.1-mL aliquot of cells was plated onto a glass microscope slide using a cytocentrifuge (Shandon, Inc.). The cells were stained with Wright-Giemsa stain, and a differential cell count was made using previously described procedures.28,29 The number and percentage of polymorphonuclear lymphocytes (PMNs) in this aliquot were scored as an index of inflammation.28

Functional characteristics of alveolar macrophages that were quantified included Fc-receptor binding capacity, phagocytic activity, and production of Superoxide anion during respiratory burst activity. A rosette assay was used to measure the ability of macrophages to bind sheep red blood cells (SRBC) to Fc receptors. Adherent macrophages (10^sup 5^ cells) were incubated in 8-well microtiter chambers (Fisher Scientific) with rat anti-sheep red blood cell antibody (30 min at 37 [degrees]C) to allow the antibody to bind to the macrophage Fc receptors. The method for preparing and standardizing the antibody was presented in detail previously.20 The unbound antibody was removed by rinsing, and the macrophages were incubated with 106 SRBCs (30 min at 37 [degrees]C). Excess red blood cells were removed by rinsing, and the number of macrophages forming rosettes with three or more SRBC was counted; 300 cells were counted per sample. To correct for nonspecific binding, macrophages from each sample were analyzed, omitting the antibody incubation step; the resulting control count was subtracted from the rosette count for cells incubated with antibody.

The ability of macrophages to phagocytize particles was quantitatively measured by incubating macrophages recovered from lavage fluid, post-exposure, in vitro with polystyrene latex (PSL) particles. For this assay, 5 x 10^sup 5^ macrophages were incubated with mild agitation at 37 [degrees]C in suspension with 10(8) fluorescent polystyrene latex particles (1 [mu]m diameter) for 60 min. The suspensions were cooled to room temperature and agitated, and 1 mL aliquots were transferred to the well of a cytocentrifuge. The samples were centrifuged onto labeled microscope slides and fixed in situ with methanol. The slides were stained with H&E and treated with xylene for 6 min to quench the fluorescence of unengulfed polystyrene particles. Macrophages were viewed using a fluorescence microscope, and those containing two or more engulfed (fluorescing) particles were scored as positive. The number of positive macrophages and the numbers of particles ingested by positive macrophages were determined, using a method previously described.28

Respiratory-burst-related superoxide anion production was measured using a chemiluminescence method.28-29 Macrophages (2 x 10^sup 5^) were added to cuvettes and incubated at 37 [degrees]C for 90 min to allow adherence. Nonadherent cells were removed by rinsing, and the medium was replaced with Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10 mm glucose. Macrophages were incubated overnight to allow "relaxation" after adherence. The medium was removed and replaced with fresh RPMI, and Superoxide production was determined by lucigenin-amplified chemiluminescence (LKB-Pharmacia Model 1251 Luminometer; LKB-Wallac). Luminometer measurements were initiated immediately after adding 50 [mu]L of 200 mM bis-N-methylacridinium nitrate (lucigenin; Sigma) with or without stimulating agent (10 ng/mL opsonized zymosan). Measurements were continued until readings returned to near baseline levels (typically 30 min).

Infiltration of the lung by inflammatory cells was assessed by performing differential counts on the cells recovered from the bronchoalveolar lavage fluid (BAL). Samples were prepared using a cytocentrifuge to place cells onto microscope slides, and the slides were fixed with methanol, air-dried, and stained using Wright- Giemsa. Preparations were scored for the percent of macrophages, neutrophils, and lymphocytes.

Total Protein, Albumin, and Mucus Glycoprotein in BAL. A bicinchoninic acid (BCA) procedure30 was used for determination of total protein in the BAL. Standards were prepared by dilution from a stock solution of bovine serum albumin (BSA). Albumin and mucus glycoprotein were measured using enzyme-linked immunosorbant assays (ELISA).32

Statistics. Data were analyzed using one-way or two-way analyses of variance, as appropriate for a given end point. Tukey multiple comparison tests were used for a posteriori testing for significant differences between group means. The criterion for statistical significance was set at p < 0.05.

RESULTS

Exposures

Exposure Conditions. Average component concentrations, particle sizes, and relative humidities in the exposure atmospheres are summarized in Table 1. The aerosols were polydisperse with MMADs of ~0.3 [mu]m and geometric standard deviations (GSDs) of ~2.4.

Biological End Points

Cell Replication/BrdU Labeling. Labeling of DNA in lung epithelial and interstitial cells undergoing replication at the end of the 4-week exposure period was determined. The numbers of labeled cells per unit tissue volume were increased at both particle dose levels compared with those in tissue from either purified air or in O^sub 3^-exposed rats. As summarized in Table 2, O^sub 3^ modestly (20-40%) increased cell replication rates in interstitial and epithelial cells, but cell replication rates in particle-exposed rat's lungs were increased by between 250 and 340%, relative to purified air. The results, which are shown in Figures 1 and 2 for epithelial and interstitial cells, respectively, suggest that mechanisms of injury and cell replication may vary as a function of particle dose. Labeling of interstitial cells from rats exposed to the low-concentration particle mixture was significantly greater than that in controls. Interstitial labeling from rats exposed to the high-concentration mixture was increased to a lesser extent than that in rats exposed to the low-concentration mixture and was not significantly different from that in controls. These data demonstrate that more cell replication was induced by particle mixture exposures in the interstitial region as compared with the epithelial region relative to the baseline turnover rate (i.e., that seen after purified air exposures). Ratios of labeled interstitial/ labeled epithelial cell ratios were 50-100% greater in lungs from pollutant-exposed rats than in those from air-exposed rats (see Table 2).

Table 1. Concentrations and particle sizes of constituents of exposure atmospheres.

Table 2. Labeling of replicating epithelial and interstitial cells relative to controls as a function of concentration.

Permeability and Airway Inflammation. BAL collected 12 hr post- exposure was analyzed for albumin, mucus glycoprotein, and total protein as indicators of mucosal damage and permeability. The results are shown in Figure 3. Permeability was slightly increased by O^sub 3^ and significantly increased by exposure to the lowconcentration particle + O^sub 3^ mixture. No significant differences were seen between mucus glycoprotein concentrations in BAL from exposed versus control animals. However, for all three types of protein in BAL, there were higher concentrations in BAL from rats exposed to the low-concentration particle mixture exposure than from rats exposed to O^sub 3^ alone or the higher concentration mixture. Exposure to either high or low concentrations of the particle-containing atmosphere did not have a significant effect on \cell viability, cell yield, or the inflammatory cell (PMN) population recovered in BAL (Table 3). In general, O^sub 3^- containing atmospheres resulted in a slightly, but not significantly, higher fraction of PMNs in the BAL.

Figure 1. Bromodeoxyuridine labeling of epithelial lung cells was increased after exposure to atmospheric pollutants. Effects of the low-concentration particle mixture approached significance but the high-concentration mixture produced lesser effects. Nominal exposure atmosphere concentrations were AIR = purified air; Low Mix contained C = 50 [mu]g/m^sup 3^ carbon, ABS = 70 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0.2 ppm ozone; High Mix contained C = 100 [mu]9/m^sub 3^ carbon, ABS = 140 [mu]g/m^sub 3^ ammonium bisulfate, O^sub 3^ = 0.2 ppm ozone. Values are mean + or - SE labeled cells per unit volume of tissue.

Figure 2. Bromodeoxyuridine labeling of interstitial lung cells was significantly increased after exposure to the low-concentration mixture of particles. High-concentration exposures caused smaller increases in interstitial cell labeling. Nominal exposure atmosphere concentrations were AIR = purified air; Low Mix contained C = 50 [mu]g/m^sup 3^ carbon, ABS = 70 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0.2 ppm ozone; High Mix contained C = 100 [mu]g/m^sup 3^ carbon, ABS = 140 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0,2 ppm ozone. Bars represent mean + or - SE labeled cells per unit volume of tissue.

Macrophage Functions. Macrophages from rats exposed to both the high and low concentrations of the PM^sub 10^ component + O^sub 3^ mixtures exhibited significantly depressed ability to attack antigenic material (SRBC) via Fc receptor-mediated processes (Figure 4). This depression in Fc-receptor binding was dose-dependent. The production of superoxide during respiratory burst activity following stimulation with opsonized zymosan (Figure 5) was depressed by exposures both to concentrations of the particle-containing atmospheres relative to either purified air or to O^sub 3^. Comparisons were made between macrophage function end points in rats euthanized immediately post-exposure in a previous experiment25 and rats euthanized 12 hr post-exposure (this experiment). The results (shown in Figure 6) suggest that at the end of particle exposure, macrophages are, if anything, activated, but when macrophages are recovered 12 hr post-exposure, they are functionally depressed.

Figure 3. Epithelial permeability 12 hr post-exposure was significantly increased after exposure to low, but not to high concentrations of PM components. Mucus glycoprotein concentrations in BAL followed a pattern similar to that for total protein and albumin. Nominal exposure atmosphere concentrations were AIR = purified air; Low Mix contained C = 50 [mu]g/m^sup 3^ carbon, ABS = 70 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0.2 ppm ozone; High Mix contained C = 100 [mu]g/m^sup 3^ carbon, ABS = 140 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0,2 ppm ozone. Bars represent mean + or - SE concentrations of proteins in BAL.

Figure 4. Macrophage Fc receptor binding was significantly decreased after exposure to low and high concentrations of PM components. Nominal exposure atmosphere concentrations were AIR = purified air; Low Mix contained C = 50 [mu]g/m^sup 3^ carbon, ABS = 70 [mu]g/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0,2 ppm ozone; High Mix contained C = 100 [mu]g/m^sup 3^ carbon, ABS = 140 [mu]g/m^sup 3^ ammonium bisulfate; O^sub 3^ = 0,2 ppm ozone. Bars represent mean + or - SE labeled cells per unit volume of tissue.

DISCUSSION

The relationship between environmental PM^sub 10^ exposure and acute mortality is of continuing concern. Currently, much attention is focused on the fraction of PM^sub 10^ below 2.5 [mu]m in diameter, because these particles can penetrate to the deep lung. Also, the chemical composition of this fine particle fraction is less variable, across geographic regions, than is the composition of the large particle fraction, and previous laboratory work has demonstrated toxic effects associated with specific components of the fine particle fraction, notably acidic sulfates and combustion- related carbonaceous aerosols. The present study analyzed the effects of two PM components, ABS and EC, which were selected because they represent important fractions of ambient PM^sub 10^ and PM^sub 2.5^ aerosols. The concentrations of these components were representative of estimated peak 4-hr concentrations, based on extrapolations from ambient air data, and the sizes of the particles used were chosen based upon reported sizes of fine inorganic aerosols in ambient air.31 Exposures were performed in the presence of O^sub 3^ (0.2 ppm) because O^sub 3^ is often present in particle- contaminated atmospheres in California. It is important to note, however, that peak concentrations of particles and peak concentrations of O^sub 3^ do not necessarily occur simultaneously. Our earlier study demonstrated that mixtures of O^sub 3^ and particles were more toxic than O^sub 3^ alone but did not address whether order of exposure to these atmospheric contaminants was important. Given the lack of consistency in epidemiologic observations of O^sub 3^-particle interactions, further research is needed in this area.

Table 3. Cells recovered In BAL from rats exposed to low and high concentrations of PM components.

Figure 5. Macrophage respiratory burst activity was decreased after exposure to low and high concentrations of PM components. Nominal exposure atmosphere concentrations were: AIR = purified air; Low Mix contained C = 50 [mu]g/m^sup 3^ carbon, ABS = 70 [mu]g/ m^sup 3^ ammonium bisulfate, O^sub 3^ = 0,2 ppm ozone; High Mix contained C = 100 [mu]g/m^sup 3^ carbon, ABS = 140 [mu]9/m^sup 3^ ammonium bisulfate, O^sub 3^ = 0,2 ppm ozone. Bars represent mean + or - SE.

Figure 6. Macrophage FcR binding and respiratory burst activities were significantly decreased when measured 12 hr post-exposure, but the changes were not seen immediately after exposure. Values shown are the differences in measurements between control (AIR) exposure rats and PM exposure rats (low concentration mixture).

Several biological end points were measured in this study, each of which could be related to specific pulmonary diseases or disease processes. For example, changes in airway permeability, infiltration of the lung by inflammatory cells, and mucus glycoprotein expression were measured to examine mechanisms related to lung inflammation and edema. Histopathological alterations in the lung, as indexed by post- exposure increases in cell replication, were measured to identify sites of lung injury related to repeated exposures. Macrophage function changes, which are related to host defenses (Fc receptor binding and release of biocidal reactive oxygen species, e.g., superoxide anion), were measured because impairment of these functions can relate to increased risk of lung infection.

The mechanisms by which inhalation of ambient particles precipitate human fatalities are not clear; however, some likely possibilities have been postulated. For example, inhaled particles might impair the integrity of the lung's epithelial barrier causing increased infiltration of cells, serum, and proteins into the lung, ultimately leading to inflammatory responses, pulmonary edema, and death. Under the conditions of this study, significant permeability changes were observed in senescent rats 12 hr post-exposure, with little evidence of lung infiltration by inflammatory cells. Previous studies of younger rats, but at higher particle concentrations, had shown both permeability changes and significant PMN infiltration. Impairment of pulmonary host defenses by inhaled particles could permit development of acute respiratory infections or could promote acute inflammatory reactions with the release of excess amounts of reactive oxygen species and cytotoxic chemicals in the lung. These releases could exacerbate existing chronic pulmonary diseases. In a previous study with young adult rats exposed to ammonium sulfate ((NH^sub 4^)^sub 2^SO^sub 4^) at the same concentrations as used for ABS in this study,32 there was a tendency (which was not statistically significant) for macrophages from PM component- exposed rats to generate excess superoxide. We previously reported25 that free-radical production was increased immediately post- exposure. In this study, it was demonstrated that both respiratory burst and Fc receptor binding are depressed in macrophages sampled 12 hr post-exposure. Thus, there may be a time-dependent characteristic to the effects of inhaled particles on macrophage functions with an initial hyperactive phase followed by a depression of normal functions. There is a need for additional research to examine these mechanisms in greater detail because they could have important implications with respect to interpreting particle- associated impairment of host defenses.

This study demonstrated that more in-depth examinations of dose- response relationships are needed. For some end points, under the conditions of this study, effects were lesser, rather than greater, in rats exposed to the higher concentration of PM components. Experiment bias is unlikely to explain the observed attenuation because the exposures were simultaneous, the rats were randomized during end point evaluations, and this was a blinded study.

Some of the end points that showed a plateau, or perhaps attenuated, response with increased dose were those requiring active metabolic processes (i.e., cell replication and perhaps macrophage respiratory burst activity). It is possible that mild toxicity provoked by low-concentration exposures induced repair (e.g., cell turnover) or defense (respiratory burst) responses and that injury of responding cells at high-dose levels blocked these responses. Another possibility is that the time course of injury was altered and respons\e might have peaked at an evaluation time different from that used in the present study. Other investigators have noted nonlinearity of responses at high-dose levels. For example, Lewtas et al.33 reported that DNA-adduct formation in humans exposed to coal-tar aerosols and rats exposed to coal-tar pitch aerosols was decreased under high-level exposure conditions. These nonlinear responses could be caused by saturation of metabolic enzymes, induction of repair mechanisms, or induction of other detoxification mechanisms. These possibilities should be further explored. Additional studies of intermediate concentrations that bracket those relevant to ambient exposures and a range of exposure/evaluation times could help elucidate injury mechanisms and their dose- response relationships.

ACKNOWLEDGMENTS

This study was supported by the California Air Resources Board, Contract No. 93-152. Aged rats were obtained with partial support from the National Institute on Aging. The authors acknowledge the support of Dr. Glen Cass, who provided advice on atmosphere selection, and Gary Devillez, Richard Mannix, and Laura Huffman for their assistance in conducting this project. The statements and conclusions in this report are not necessarily those of the California Air Resources Board. The mention of commercial products, their source, or their use in connection with material reported herein is not to be construed as actual or implied endorsement of such products.

ISSN 1047-3289 J. Air & Waste Manage. Assoc. 53:1080-1087

Copyright 2003 Air & Waste Management Association

IMPLICATIONS

Current air quality standards deal with one pollutant at a time. This study demonstrates that pollutant mixtures containing acidic particles and an oxidant gas can have greater effects than do the individual pollutants. Thus, it may be important in the future to develop strategies for regulating air pollution mixtures. This study also demonstrates that dose responses may not be monotonic and that exposures at lower concentrations may be more toxic than predicted from studies in which higher exposure levels were used.

REFERENCES

1. Ozkaynak, H.; Thurston, G.D. Associations between 1980 U.S. Mortality Rates and Alternative Measures of Airborne Particle Concentration; Risk Anal. 1987, 7, 449-461.

2. Ozkaynak, H.; Spengler, J.D. Analysis of Health Effects Resulting from Population Exposures to Acid Precipitation Precursors; Environ. Health Perspect. 1985, 63, 45-55.

3. Martin, A. E. Mortality and Morbidity Statistics and Air Pollution; Proc. R. Soc. Med. 1964, 57, 969-975.

4. Greenburg, L.; Field, F.; Erhardt, C.; Glasser, M.; Reed, J. Air Pollution, Influenza and Mortality in New York City; Arch. Environ. Health 1967, 15, 430-438.

5. Samet, J.M.; Bishop, Y.; Speizer, F.E.; Spengler, J.D.; Ferris, B.G., Jr. The Relationship between Air Pollution and Emergency Room Visits in an Industrial Community; J. Air Pollut. Control. Assoc. 1981, 31, 236-240.

6. Knight, K.; Leroux, B.; Millar, J.; Petkau, A.J. Air Pollution and Human Health: A Study Based on Data from Prince George, British Columbia; Tech Report #85; Department of Statistics, University of British Columbia: Vancouver, British Columbia, Canada, 1989.

7. Whittermore, A.; Korn, E. Asthma and Air Pollution in the Los Angeles Area; Am. J. Public Health 1980, 70, 687-696.

8. Schenker, M. Air Pollution and Mortality (editorial); N. Engl. J. Med. 1993, 329, 1807-1808.

9. Pope, C.A., III; Dockery, D.W.; Spengler, JD.; Raizenne, M.E. Respiratory Health and PM^sub 10^ Pollution. A Daily Time Series Analysis; Am. Rev. Respir. Dis. 1991, 144, 668-674.

10. Stern, B.; Jones, E.; Raizenne, M.; Burnett, R.; Meranger, J.C.; Franklin, C.A. Respiratory Health Effects Associated with Ambient Sulfates and Ozone in Two Rural Canadian Communities; Environ. Res. 1989, 49, 20-39.

11. Pope, C.A.; Kanner, R.E. Acute Effects of PM^sub 10^ Pollution on Pulmonary Function of Smokers with Mild to Moderate Chronic Obstructive Pulmonary Disease; Am. Rev. Respir. Dis. 1993, 147, 1336-1340.

12. Schwartz, J. Paniculate Air Pollution and Daily Mortality: A Synthesis; Public Health Rev. 1991, 19, 39-60.

13. Schwartz, J.; Dockery, D.W. Increased Mortality in Philadelphia Associated with Daily Air Pollution Concentrations; Am. Rev. Respir. Dis. 1992, 145, 600-604.

14. Schwartz, J. Particulate Air Pollution and Chronic Respiratory Disease; Environ. Res. 1993, 62, 7-13.

15. Dockery, D.W.; Pope, C.A., III, Xiping, X.; Spengler, J.D.; Ware, J.H.; Fay, M.E.; Ferris, B.G., Jr.; Speizer, F.E. An Association between Air Pollution and Mortality in Six U.S. Cities; N. Engl. J. Med. 1993, 329, 1753-1759.

16. Fairley, D. Daily Mortality and Air Pollution in Santa Clara County, California, 1989-1996; Environ. Health Perspect. 1999, 307, 637-641.

17. Burnett, R.T.; Brook, J.R.; Dann, T.; Delocla, C.; Philips, O.; Calmak, S. Associations between Particulate-Phase and Gas-Phase Components of Urban Air Pollution and Daily Mortality in Eight Canadian Cities; Inhal. Toxicol. 2000, 12 (Suppl. 4), 15-39.

18. Ostro, B.D.; Broadwin, R.; Lipsett, M.J. Coarse and Fine Particles and Daily Mortality in the Coachella Valley, California: A Follow-Up Study; J. Expo. Anal. Environ. Epidemiol. 2000, 10, 412- 429.

19. Lippmann, M.; Ito, K.; Nadas, A.; Burnett, R.T. Association of Particulate Matter Components with Daily Mortality and Morbidity in Urban Populations; Res. Rep. Health Eff. Inst. 2000, 95, 5-72.

20. Castillejos, M.; Borja-Aburto, V.H.; Dockery, D.W.; Gold, D.R.; Loomis, D. Airborne Coarse Particles and Mortality; Inhal. Toxicol. 2000, 12 (Suppl. 1), 67-72.

21. Pope, C.A., III. Paniculate Air Pollution and Health: A Review of the Utah Valley Experience; J. Expo. Anal. Environ. Epidemiol. 1996, 6, 23-34.

22. Schlesinger, R. Toxicologic Evidence for Health Effects from Inhaled Particulate Pollution. In Proceedings of the Colloquium on Particulate Air Pollution and Human Mortality and Morbidity, Phalen, R.F., Ed.; Irvine, CA, 1994.

23. Lippmann, M.; Schlesinger, R.B. Toxicological Bases for the Setting of Health-Related Air Pollution Standards; Anna. Rev. Public Health 2000, 21, 309-333.

24. Godlewski, J.J.; Sioutas, C.; Katler, M.; Catalano, P.; Koutrakis, P. Death from Inhalation of Concentrated Ambient Air Particles in Animal Models of Pulmonary Disease. In Proceedings of the Second Colloquium on Particulate Air Pollution and Human Health, Lee, J., Phalen, R.F., Eds.; Park City, UT, 1996.

25. Kleinman, M.T.; Bufalino, C.; Rasmussen, R.; Hyde, D.; Bhalla, D.K.; Mautz, W.J. Toxicity of Chemical Components of Ambient Fine Particulate Matter (PM^sub 2.5^) Inhaled by Aged Rats; J. Appl. Toxicol. 2000, 20, 357-364.

26. Nowakowski, R.S.; Lewin, S.B.; Miller, M.W. Bromodeoxyuridine Immunohistochemical Determination of the Length of the Cell Cycle and the DNA-Synthetic Phase for an Anatomically Defined Population; J. Neurocytol., 1989, 18, 311-317.

27. Bolender, R.P.; Hyde, D.M.; Dehoff, R.T. Lung Morphometry: A New Generation of Tools and Experiments for Organ, Tissue, Cell, and Molecular Biology; Am. J. Physiol. Lung Cell Mol. Physiol. 2 1993, 65, L521.

28. Kleinman, M.T.; Bhalla, O.K.; Ziegler, B.; Bucher-Evens, S.; McClure, T.R. Effects of Inhaled Fine Particles and Ozone on Pulmonary Macrophages and Epithelia; Inhal. Toxicol. 1993, 5, 371- 388.

29. Nadziejko, C.E.; Nansen, L.; Mannix, R.C.; Kleinman, M.T.; Phalen, R.F. The Effect of Nitric Acid on the Response to Ozone; Inhal. Toxicol. 1992, 4, 343-358.

30. Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fugimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of Protein Using Bicinchoninic Acid; Anal. Biochem. 1985, 150, 76-85.

31. John, W.; Wall, S.M.; Ondo, J.L.; Winklmayr, W. Modes in the Size Distribution of Atmospheric Inorganic Aerosol; Atmos. Environ. 1990, 24, 2349-2360.

32. Kleinman, M.T.; Bhalla, O.K.; Mautz, W.J.; Phalen, R.F. Cellular and Immunologic Injury with PM^sub 10^, Inhalation; Inhal. Toxicol. 1995, 7, 589-602.

33. Lewtas, J.; Walsh, D.; Williams, R.; Dobias, L. Air Pollution Exposure-DNA Adduct Dosimetry in Humans and Rodents: Evidence for Nonlinearity at High Doses; Mutat. Res. 1997, 378, 51-63.

Michael T. Kleinman

Department of Community and Environmental Medicine, University of California, Irvine, California

Dallas M. Hyde

School of Veterinary Medicine, University of California, Davis, California

Charles Bufalino

University of California, Riverside, California

Carol Basbaum

University of California, San Francisco, California

Deepak K. Bhalla

Division of Allied Health Professions, Wayne State University, Detroit, Michigan

William J. Mautz

Department of Biology, University of Hawaii, Hilo, Hawaii

About the Authors

Michael T. Kleinman is the codirector of the Air Pollution Health Effects Laboratory and a professor in the Department of Community and Environmental Medicine at the University of California, Irvine. Dallas M. Hyde is a professor of anatomy, physiology, and cell biology and director of the California National Primate Research Center in the School of Veterinary Medicine at the University of California, Davis. Charles Bufalino is a research specialist at the University of California, Riverside. Carol Basbaum is a professor of anatomy at the University of California, San Francisco. Deepak K. Bhalla is a professor in the Division of Allied Health Professions at Wayne State University in Detroit, MI. William J. Mautz is an associate professor and chair of the Department of Biology at the University of Hawaii, Hilo. Address correspondence to: Michael T. Kleinman, Department of Community and Environmental Medicine, University of California, Irvine, CA 92697; e-mail: mtkleinm@uci.edu.

Copyright Air and Waste Management Association Sep 2003

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