Inhalation of acid coated carbon black particles impairs alveolar macrophage phagocytosis

Toxicology Letters Toxicology Letters 88 (1996) 243-248

Inhalation (of acid coated carbon black particles impairs alveolar macrophage phagocytosis George J. Jakab

*a ,

Robert W. Clarke”, David R. Hemenwayb, Malinda V. Longphre”, Steven R. Kleeberger”, Robert Frank=

of Environmentul

‘Depurtment

He&h

615 North “Depurtment

Sciences, The Johns Hopkins School of Hygiene und Public Health, Wove Street, Bultimore.

of Civil & Environmentul

Engineering,

MD 21205.

USA

University of Vermont, Burlington,

VT 05405,

USA

Abstract A flow-past nose-only inhalation system was used for the co-exposure of mice to carbon black aerosols (CBA) and sulfur dioxide (SO,) at varying relative humidities (RH). The conversion of SO2 to sulfate (SO,-*) on the CBA, at a fixed aerosol concentration, was dependent on RH and SO, concentration. The effect of the aerosol-gas mixture on alveolar macrophage (AM) phagocytosis was assessed three days following exposure for 4 h. Exposure to 10 mg/m3 CBA alone att low RH (10%) and high RH (SF/,), to 10 ppm SO, alone at both RH, and to the mixture at low RH had no effect on AM phagocytosis. In contrast, AM phagocytosis was significantly suppressed following co-exposure at 85%, RH, the only circumstance in which significant chemisorption of the gas by the aerosol and oxidation to SO,-* occurred. The results suggest that fine carbon particles can be an effective vector for the delivery of toxic amounts of SO,-* to the periphery of the lung. Keywords: Particles; Aerosol; Carbon black; Sulfate; Sulfuric acid; Lung; Alveolar macrophages; Phagocytosis

1. Introduction

Sulfur dioxide (SO,) and carbonaceous particulate matter arc primary effluents of fossil fuel combustion. Due to its hydrophilicity, gas phase SO, is almost completely absorbed in the upper respiratory tract, thereby causing little toxicity in the lung parenchyma. However, depending on atmospheric conditions, SO, is converted to acid sulfates (SO,-*) in the air by photo- or catalytic *Corresponding

author.

037%4274/96/$15.00 PII

0

SO378-4274(96)03745-9

oxidation. Field and laboratory studies have shown that carbon particles act as nuclei for the conversion of SO, to SO,-* [1,2] and that this process is accelerated by high relative humidity (W C3,41. In recent years, increasing attention has focused on acid aerosols and their potential role in acute and chronic respiratory health effects. In the studies described herein, a flow-past noseonly inhalation chamber was used to generate an experimental carbon black aerosol (CBA). The CBA was co-generated with SO, under varying

1996 Elsevier Ireland Ltd. All rights reserved

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G. J. Jukuh PI ul. I Toxicology

RH for the conversion of SO, to SO,-‘. Peripheral lung response following exposure to CBA alone, SO, alone, and the mixture were assessed through studies of alveolar macrophage (AM) phagocytosis.

2. Methods 2.1. Carbon black

Carbon black Regal 660 with a specific surface area of 90 m2/g (composition: 96.90% carbon, 0.30% hydrogen, 1.42% oxygen; empirical formula C910H3401d a generous gift of Cabot Corporation, Billerica, MA) was used for these studies. 2.2. Exposure system The chamber characteristics, aerosol and SO, generation and monitoring, particle size analysis, and particle-associated SObe analysis have been detailed previously [5,6]. Briefly, a flow-past nose-only inhalation chamber was modified for the exposure of animals to a mixture of CBA and SO, at RH that ranged from 10% to 85%. The CBA was generated with a Wright dust feed (BGI Inc., Waltham, MA) and monitored with a realtime monitor (RAM-l; M.I.E. Technologies, Inc., Bedford, MA). Time-weighted CBA concentrations were measured using 25 mm, 0.2 pm pore size membrane filters (HT200, Gelman Sciences, Ann Arbor, MI) held in an electrically conductive 25 mm diameter open-faced filter cassette (#Ol038-1, Fisher Scientific Co., Pittsburgh, PA). Aerodynamic aerosol size distributions were measured using a lo-stage Sierra cascade impactor (Anderson, Inc., Atlanta, GA). Metered SO, from a pressurized tank containing 1.5% SO, in air (Matheson Gas Co., E. Rutherford, NJ) was mixed with diluent air before entering the exposure chamber. The concentration of SO, was monitored continuously (Model #43H, Therm0 Environmental Instruments, Inc., Franklin, MA). Particle-associated SObm2 analysis was performed by a modification of ASTM Method No. 4500-SO,- 2 E [7].

Letters 88 (I 9%) 243-248

2.3. Animals and exposures

Five-week-old pathogen-free white female Swiss mice weighing 20-23 g (Hilltop Laboratory Animals Inc., Scottsdale, PA) were used. The animals were housed in filter-topped stainless steel cages with wood shaving bedding and provided with food (Agway Prolab Animal Diet, Syracuse, NY) and water ad libitum. The animals were maintained and observed for a minimum of 1 week before exposure. Mice were exposed for 4 h at 10% and 85% RH to target concentrations of either 10 mg/m3 of CBA alone, 10 ppm SO, alone, or a mixture of the agents. In another set of experiments, mice were exposed for 4 h at 85% RH to 10 mg/m3 of CBA mixed with either 5 ppm, 10 ppm or 20 ppm SO,. During exposure, a minimum of four aerosol samples were taken at the exposure ports for analysis of time-weighted concentrations of CBA and CBA-associated SOdw2. 2.4. Bronchoalveolar lavage Cells were collected by inserting a Pasteur pipette into the trachea of surgically removed lungs and introducing and withdrawing 1.5 ml of sterile lavage solution (0.85% NaCl, 0.1% glucose, 0.1% ethylenediamine tetraacetic acid (EDTA), and 20 mM N-2-hydroxyethylpiperadine-N’-2-ethansulfonic acid (HEPES) three times (total = 4.5 ml). The total number of ceils was quantitated with a hemocytometer. Differential cell counts were performed on cytocentrifuged preparations stained with Diff-Quick. Cell viability was determined with a “live/dead stain” (Molecular Probes Inc., Eugene, OR). 2.5. Alveolar macrophage Fc-receptor mediated phagocytosis AM phagocytosis was determined as previously described [8]. Following in vivo exposure, the lavage fluid was centrifuged (200 x g, 10 min) and the cell pellet resuspended at a concentration of 5 x lo5 cells/ml in RPM1 tissue culture media supplemented with 10% newborn calf serum (NCS). Three to four 200 ~1 aliquots of the celf

G.J. Jakab et al. I Toxicology Letters88 (19%) 243-248

suspensions were allowed to adhere to 22-mm2 albumin-coated glasscoverslips in 35 x 10 mm plastic Petri dishes for 45 min (37”C, 5% CO,, 95% RH). After the monolayering, the fluid was removed and immediately replaced with 1.5 ml of 0.5% sensitized sheep red blood cells (RBC) in RPM1 medium and the resulting suspension incubated at 37°C for 45 min. After removal of the RBC by aspiration and washing of the monolayers with RPM1 medium, non-ingested RBC were hypotonically lysed for 10 s, followed by several rinses with culture medium. The monolayers were then dried, fixed with methanol, and stained with Wright-Giemsa. The stained monolayers were read microscopically at 1000 x to quantify the percentage of AM containing RBC and the number of RBC ingested per phagocytic AM. The phagocytic index (total number of RBC ingested by 100 AM) was calculated by multiplying the percent of phagocytic AM by the mean number of RBC ingested per phagocytic AM. One hundred AM were counted on each monolayer with 3 to 4 monolayers counted per animal. For each exposure group, an identical number of non-exposed animals was tested. 2.6. Statistical analysis Data were anal:yzed by Student’s t-test. All statements of significance are made at P < 0.05.

3. Results

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generated at 85% RH, or in the presence of SO, at 10% and 85% RH. Insofar as the ventilatory pattern and pulmonary mechanics of the mice were not selectively altered by one or another experimental condition, the similarity of size distribution suggests that the regional deposition of the inhaled CBA was also similar for the different experimental conditions. 3.2. Carbon black-associated sulfate The amount of SObm2 formed on the CBA as a function of RH is presented in Fig. 1. The SObm2 mass concentration on the CBA was independent of RH over a range of 10% to 60%. At 85% RH, CBA-associated SObe mass concentration increased more than three-fold over previous levels: 13.7 pg SO,-‘/mg CBA, compared to 4.0 pg SO,-‘/mg CBA. We believe that this non-linearity is the result of a relatively rapid sorption of SO, in the water monolayer (on the surface of the CBA) that is rapidly oxidized and hydrolyzed to H,SO,. Because H,SO, is highly hygroscopic, more water is sorbed, leading to the additional sorption of SO,. Below 50%-60% RH, there may be insufficient sorbed water to develop this self-growth process during the residence time in the inhalation system. To determine the relationship between gas phase SO, concentration and CBA-associated SObV2 at 85% RH, 10 mg/m3 of CBA was

l

16r

3.1. Chamber performance For each experiment, the exposure system was allowed to equilibrate to steady-state conditions prior to sampling or exposure of animals. The performance characteristics of the exposure system were as follows: (1) the CBA concentrations achieved fell within + 8% of the target concentration; (2) the mass median aerodynamic diameter (MMAD) of the CEBA,generated at 10% RH, was 0.3 ,um with a geometric standard deviation (GSD) of 2.7; (3) neither the MMAD or GSD changed significantly when the CBA alone was

10.0

30.0

60.0

85.0

Percent Relative Humidity

Fig. 1. The effect of RH on CBA-associated SO,-’ mass concentrations. Each value represents the mean + SE of 5 experiments with each consisting of 7 samples (P < 0.05).

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G.J. Jakab et al. I Toxicology Letters 88 (1996) 243-248

co-generated with 5 ppm, 10 ppm, and 20 ppm of SO,. The CBA-associated S04-’ concentration was 6 pg SO,-‘/mg CBA and 13.7 pg SO,-*/mg CBA, respectively, at co-exposure to 5 ppm and 10 ppm SO,. Increasing the gas phase SO, concentration from 10 ppm to 20 ppm while holding the CBA concentration constant at 10 mg/m3 increased CBA-bound SO,-* four-fold to 48.7 pg SO,-*/mg CBA. This is consistent with a selfgrowth or repetitive sorption process with the higher concentrations providing more SO,-* on the surface of the particle, which causes more water to be sorbed which, in turn, further enhances additional SO, sorption. 3.3. Ceil count and cell viability None of the six exposure regimes (CBA alone, SO, alone, CBA plus SO, at 10% and 85% RH) affected total lavage cell counts, differential cell counts or cell viability at any time interval between 1 and 14 days post-exposure (data not shown). In unexposed control mice, the total cell count was 5.2 + 0.5 x lo5 (mean f SEM), of which 98% were AM, 1% were polymorphonuclear leukocytes (PMN) and 1% were lymphocytes. The viability of AM exceeded 97% for all experimental circumstances.

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Ca-bm Black Aerosol (CSA)

Fig. 2. Comparison of Fc-receptor mediated phagocytosis by AM retrieved from mice 3 days after exposure for 4 h to either 10 mg/m” of CBA at 10% and 85% relative humidity, 10 ppm of SO, at 10% or 85% relative humidity, or co-exposure to both agents at 10% and 85% relative humidity. Each value represents the mean k SE of 3 experiments consisting of 5 exposed animals and 5 air-matched controls (P < 0.05 from all other values).

mediately, 1, 3, 7, and 14 days thereafter. Fig. 4 shows a slight suppression of AM phagocytosis immediately after exposure, with significant suppression at days 1, 3, and 7, followed by a full re-establishment of phagocytic potential by day 14.

3.4. Alveolar macrophage phagocytosis The effect of exposure on AM Fc-receptormediated phagocytosis was determined 3 days following exposure, as presented in Fig. 2. Phagocytosis was depressed only following co-exposure to CBA plus SO, at 85% RH, suggesting that particle-bound SO,-* was the responsible agent. This notion is further supported by the doseresponse relationship seen between increasing concentrations of CBA-bound SO,-* and decrements in AM phagocytosis (Fig. 3). Following this initial demonstration of depressed AM phagocytosis associated with co-exposure to CBA and SO, at 85% RH, we plotted the time-course for the defect over a two-week period. Mice were exposed for 4 h to the effective mixture and AM phagocytosis was assessed im-

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Fig. 3. Comparison of Fc-receptor mediated phagocytosis by AM retrieved from mice 3 days after exposure for 4 h to 10 mg/m3 of CBA at 85% RH and co-exposure to either 5 ppm, 10 ppm, or 20 ppm of SO,. Each value represents the mean f SE of 2 experiments consisting of 5 exposed animals and 5 air-matched controls (P < 0.05 from air exposed value).

G.J. Jakah et al. I Toxicology Letters 88 (19%) 243-248

Fig. 4. Alveolar macroiphage phagocytosis immediately after and at 1, 3, 7, and 14 days after 4 h of co-exposure to 10 mg/m, of CBA and 10 ppm SO’ at 85% RH. Each value represents the mean rt SE of 2 experiments consisting of 5 exposed animals and 5 air-matched controls (P c 0.05 from control value).

4. Discussion

In recent years, increasing attention has focused on acid aerosols and their potential role in acute and chronic respiratory health effects. Toxicologic studies have used several approaches to generate acid aierosols for animal inhalation exposures. One approach is the seminal work of Amdur [9] who demonstrated that gas phase SO, is sorbed to particles and rapidly converted to acid SO,-* [lo:]. In this system, ZnO particles and SO, were combined to mimic metal-rich flyash produced by coal combustion. Exposure of animals to the mixture of ZnO and SO, clearly resulted in enhanced pulmonary effects as compared to exposure to either agent alone [ll]. No effect resulted from exposure to SO, alone. However, pulmonary effects developed following ZnO exposure [12], indicating that the particle carrier itself was toxic. Another approach is to expose animals directly to aerosols of sulfuric acid (H,SO,; [13]). These studies have also demonstrated dysfunctions of a variety of pulmonary parameters following exposure of animals to the H,SO, aerosol [14,15]. Herein, we exposed animals to a mixture of atmospheric particles and SO,. The public health relevance derives from the following consider-

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ations: SO, is ubiquitous, carbon comprises about half of the total particulate emissions in urban air [16], about 85% of the carbon mass is present in particles under 2.5 pm in diameter [17], while studies have shown that ambient carbon particulates act as nuclei for the catalytic oxidation of SO, to H,SO, [l-4]. Relative to inhalation of H,SO, aerosols, inhalation of acid-coated particles may result in different pulmonary effects, the same effects but at different concentration, or both. The large surface area of the carbon particle offers many crevices for binding and, therefore, the sorbed acid may be less readily neutralized then when present in droplet form. This may mean slower rates of neutralization of the carbon-bound acid by endogenous ammonia in the airways while in transit [18] and, upon deposition, less accessibility to the buffering capacity of the epithelial lining fluid. Then, depending on the binding forces, the carbon particle may retain the acid sulfate until ingestion by AM, resulting in the potential delivery of a higher concentration of acid to the phagocyte then would be achieved through the alveolar deposition of droplet H,SO,. The present studies used carbon black particles, with a size distribution and surface properties similar to those present in ambient air [19], to generate an experimental carrier aerosol that is considered to be biologically “inert” [20-221. The CBA was then generated in the presence of varying concentrations of SO, at low and high RH. Significant chemisorption of the gas and oxidation to SO,-* occurred only at high RH and was dependent on gas phase SO, concentrations. The peripheral lung response, assessed by impairments of AM phagocytosis following inhalation exposure, was dose-dependent with increasing concentrations of CBA-bound SO,-*. The data indicate that respirable carbon particles can act as vectors for the delivery of toxic amounts of acid SO,-* to the distal lung.

Acknowledgements

This study was supported in part by Grant P30 ES03819 from the NIEHS Center, NIH

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grant HL51697, and NIEHS Training Grant 07141 for support of R.C. and NHLBI Training Grant HL07534 for support of M.L.

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