Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Dosimetry of beryllium in cultured canine pulmonary alveolar macrophages A. F. Eidson , A. Taya , G. L. Finch , M. D. Hoover & Cindy Cook To cite this article: A. F. Eidson , A. Taya , G. L. Finch , M. D. Hoover & Cindy Cook (1991) Dosimetry of beryllium in cultured canine pulmonary alveolar macrophages, Journal of Toxicology and Environmental Health, 34:4, 433-448, DOI: 10.1080/15287399109531581 To link to this article: http://dx.doi.org/10.1080/15287399109531581
Published online: 20 Oct 2009.
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DOSIMETRY OF BERYLLIUM IN CULTURED CANINE PULMONARY ALVEOLAR MACROPHAGES A. F. Eidson, A. Taya, G. L. Finch, M. D. Hoover, Cindy Cook
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International Technology, Albuquerque, New Mexico
This study was designed to determine the dosimetry within macrophages of beryllium compounds administered at sublethal doses. Information on the dosimetry of beryllium within macrophages is required to guide further efforts to isolate and characterize beryllium-containing haptens. Inhalation of beryllium aerosols can cause chronic berylliosis, a progressive, granulomatous fibrosis of the lung. Studies in laboratory animals indicate that alveolar macrophages take up beryllium compounds and participate in a hypersensitivity immune response to beryllium-containing antigen. Beagle dog macrophage cultures were incubated with 7BeSO4 in solution or with suspensions of 7BeO particles that had been calcined at 500 or 1000°C. Beryllium-7 was measured in fractions collected from cultures after successive centrifugation and filtration steps at 2, 6, 20, and 48 h after addition. An insignificant percentage of BeSO4 was taken up by the cells and did not cause cytotoxicity. Maximum BeO uptake occurred within 6 h, was 60 ± 6% of added BeO, and was independent of BeO calcination temperature or specific surface area. Approximately 22% of 500°C BeO dissolved within 48 h after addition to cell culture, concurrent with 39% cell killing. Dissolved beryllium remained associated with cells until a cytotoxic concentration was reached (2.2 × 10-5 M, 15 nmol Be/106 cells), when the beryllium was released into the medium. There was no significant dissolution of the 1000°C BeO within 48 h, and no significant cell killing. The results indicate that beryllium dissolved from phagocytized BeO was more cytotoxic than soluble beryllium added extracellularly. The data support an interactive mechanism in which phagocytized BeO particles were dissolved, and dissolved beryllium remained associated with the macrophage until a cytotoxic concentration accumulated, whereupon the beryllium was released to the medium and not appreciably taken up by viable cells.
INTRODUCTION Beryllium metal is used in the aerospace and nuclear industries because of its low density, high heat capacity, mechanical strength, and good neutronic properties (Harmsen et al., 1984). Beryllium oxide is used The authors acknowledge many helpful discussions with Dr. Janet Benson, Dr. Bruce Muggenburg, Dr. R. F. Henderson, and Dr. Alan R. Dahl. We also express our appreciation to the many skilled technicians assisting with the experiments. This study was supported by the U.S. Department of Energy Office of Health and Environmental Research under contract DE-AC04-76EV01013, in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Cindy Cook participated under the Associated Western Universities Summer Student Research Program. Requests for reprints should be sent to A. F. Eidson, International Technology Corporation, 5301 Central Avenue, Suite 700, Albuquerque, NM 87108. 433 Journal of Toxicology and Environmental Health, 34:433-448, 1991
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A. F. E1DSON ET AL.
directly as an insulator and heat conductor in the semiconductor and ceramics industries. Fabrication of BeO components can release respirable aerosols, and fabrication of beryllium metal components can release aerosols that contain a surface coating of BeO (Hoover et al., 1989). Inhalation of beryllium aerosols can cause chronic berylliosis, a progressive, granulomatous fibrosis of the lung (Freiman and Hardy, 1970; Kriebel et al., 1988). As new cases are reported (Johnson, 1983; Cullen et al., 1987), there is continuing concern about beryllium exposure. Studies of disease induction mechanisms in laboratory animals support the hypothesis that alveolar macrophages take up beryllium compounds and participate in a hypersensitivity immune response to a berylliumcontaining antigen (Hanifin et al., 1970; Kriebel et al., 1988). A number of observations indicate that the solubility of BeO particles is an important factor in chronic berylliosis induction. The in vitro dissolution rate of BeO particles calcined at 500°C is greater than that of BeO calcined at 1000°C, in direct proportion to the greater specific surface area of the 500°C particles (Finch et al., 1988a). As a result of its greater solubility, the toxicity of 500°C-calcined BeO particles to cultured canine pulmonary macrophages (PAM) is greater than that of the 1000°C form (Finch et al., 1988b). BeO particles calcined at 500°C clear more rapidly from lungs of beagle dogs and cause an increased lymphocyte response and greater histopathology than BeO calcined at 1000°C (Haley, 1989). Epidemiological evidence indicates that the incidence of chronic berylliosis is higher among individuals exposed to dusts of BeO fired at approximately 500°C rather than at 1000°C (Eisenbud and Lisson, 1983). Beryllium metal is cytotoxic to macrophages (Camner et al., 1974; Andre et al., 1987). Soluble beryllium is incorporated by rat and guinea pig alveolar macrophages as particles (Hart and Pittman, 1980), probably as beryllium hydroxide formed in the culture medium (Skilleter and Paine, 1979). Soluble beryllium has been reported to be cytotoxic to alveolar macrophages within 26 h when added at 9.5 x 10~5 M (Kang and Salvaggio, 1976; Kang et al., 1977) and to rat liver macrophage (Kupffer) cells within 24 h when 1 nmol Be/106 cells was incorporated (Skilleter and Price, 1981). We hypothesize that pulmonary alveolar macrophages interact with beryllium to form a beryllium-containing hapten that acts as an antigen. Previous quantitative studies of macrophage response to beryllium compounds have centered primarily on beryllium uptake and cytotoxicity. To understand the role of beryllium in hypersensitivity immune response and in macrophage cytotoxicity, one must investigate the ways in which living macrophages interact with beryllium administered in sublethal doses. The objectives of this study were to investigate the internal dosimetry of beryllium within living macrophages and to guide future efforts to isolate and characterize a beryllium-containing antigen.
DOSIMETRY OF BERYLLIUM
435
MATERIALS AND METHODS
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Experimental Design Pulmonary alveolar macrophages (PAM) from beagle dogs were cultured and dosed with 7BeSO4 solution or 7BeO particles that had been calcined at 500 or 1000°C. The amounts of each beryllium compound added to cultured PAM (Table 1) were chosen to maintain cell viability (Kang et al., 1977; Finch et al., 1988b), yet provide detectable 7Be radioactivity. Cultures were also prepared that contained beryllium compounds, but without cells (acellular controls). Separate cultures were prepared and dosed in the same way for cell viability determinations (viability controls). At selected times after beryllium addition, these cultures were treated as described below to measure viability and to isolate dissolved Be and undissolved BeO in subfractions of the cells and the culture medium. All cell cultures were prepared in duplicate, and then the experiment was repeated in duplicate using cells from a different animal. Beryllium Compounds Aqueous solutions of 7BeCI2 (Los Alamos National Laboratory, Los Alamos, N. Mex.) and BeSO4-4H2O (Aldrich, Milwaukee, Wis.) were mixed using distilled, deionized water to prepare a solution containing 2.6 fig Be2+/ml and 3.96 MBq 7Be27ml. This stock solution contained negligible amounts of CI" and was used for all 7BeSO4 experiments. A stock suspension for preparing the 7BeO powders was prepared by precipitating 7 Be(OH)2 from 7BeC2O4 solution. The resulting suspension was nebulized to form an aerosol, which was passed through a high-temperature column, collected by filtration, and then calcined for 16 h at 500 or 1000°C (Hoover et al., 1988). X-ray diffractometry was used to confirm that the material was BeO. The particle size distribution of both forms was measured using cascade impactors and was lognormally distributed with a mass median physical particle diameter of 0.43 /im and a geometric standard deviation of 2.3. The BeO specific activity was 13.3 MBq 7BeO/g. Specific surface areas of the aerosol particles were measured using a TABLE 1. Beryllium Compounds Added to Pulmonary Alveolar Macrophage Cultures Be compound
Be molarity
Moles Be/106 cells
7
Be added (Bq)
Available surface area/106 cells (m2)
BeSO4 BeO (500°C) BeO (1000°C)
2.8 x 10" 6 1.0 x 10"" 3.0 x 10" 4
1.9 x 10~9 6.7 x 10~8 2.0 x 10" 7
7.7 x 10 s 6.7 x 102 2.0 X 103
NAa 3.2 X 1 0 " 4 b 1.8 X 1 0 " 4 c
a
Not applicable. Specific surface area - 189.3 m2/g. Specific surface area - 35.8 m2/g.
fc c
436
A. F. EIDSON ET AL.
Quantasorb model QS-10 instrument (Quantachrome Corp., Syossett, N.Y.) (Rothenberg et al., 1982), and found to be 189.3 and 35.8 m2/g, for the 500°C-calcined and 1000°C-calcined BeO particles, respectively. Beryllium-7 was quantitated using a Beckman-8000 gamma counter (Beckman Instruments, Inc., Fullerton, Calif.), which was calibrated with a 7Be primary radioactivity standard (Amersham Corp., Arlington Heights, III.). The detection limit of the gamma counter was 16 Bq, at a counting efficiency of 3.3%.
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Cell Culture Techniques Alveolar macrophages were obtained by bronchopulmonary lavage of beagle dogs (7-8 yr of age) from the Institute's colony (Muggenburg and Mauderly, 1975). The lavage fluids were centrifuged at 150 x g for 10 min at 4°C. The supernatant was removed, and the cells were resuspended in saline solution and counted using a Levy hemocytometer (Hauser Scientific, Blue Bell, Pa.). Cell viability was determined by trypan blue dye exclusion (0.4% in saline; Gibco, Grand Island, N.Y.). For differential cell counts, cytocentrifuge preparations were prepared and stained with Diff-Quik stain (Gibco). All cell populations contained 93 ± 2% macrophages, 3 ± 1% lymphocytes, and 4 ± 2% neutrophils. Macrophages were >95% viable. The cells were washed twice in medium (RPM11640, Gibco) and then resuspended in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah) and 50 /ig/ml gentamycin sulfate (Sigma Chemical Co., St. Louis, Mo.). Twenty milliliters of a cell suspension containing 1.5 x 106 viable cells/ml were added to sterile plastic cell culture dishes (100 mm diameter, Costar, Van Nuys, Calif.), which were incubated for 2 h at 37°C in a 5% CO2/95% air atmosphere at 85% relative humidity to allow cell attachment. Using this technique, 80-90% of the cells are adherent after 2 h (Mueller et al., 1989). Acellular control cultures and viability control cultures were prepared at the same time. Before being added to cell cultures, paniculate 7BeO suspensions or 7 BeSO4 solutions were subjected to ultrasonic agitation for 20-30 min to suspend the BeO particles. The suspensions were then diluted to 20 ml with supplemented medium. The medium was removed from the cultured cells and replaced with 20 ml of supplemented medium containing the test compound. The cell cultures were returned to the incubator under the same conditions. Dosimetry of Beryllium in Cultured Cells The dosimetry of beryllium in the cultured cells was determined by measurements of 7Be in soluble or particulate forms, which were either associated or not associated with the cells. The 20-ml cell cultures were
DOSIMETRY OF BERYLLIUM
437
fractionated at 2, 6, 20, and 48 h after addition of one of the beryllium compounds (Figs. 1 and 2). Successive centrifugation and filtration steps described below were used to separate cell cultures into subfractions containing beryllium. The radioactivity measured in each subfraction was expressed as a percentage of the total amount of radioactivity added to the culture dish. Statistical inferences were made by using the Student's t test at p < .05.
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Fractionation of Cultured Cells Containing BeSO4 Beryllium administered as BeSO4 was isolated according to the flow chart shown in Figure 1. The medium was removed from the culture dish and centrifuged. The supernatant was filtered through a 0.1 /*m pore-size polycarbonate membrane filter (Nucleopore, Pleasanton, Calif.). Radioactivity measured on the filter was designated as F1, and that in the filtrate was designated as F2. The centrifuge cell pellet was set aside. Fresh medium was added to the culture dish and the adherent cells were harvested by gentle scraping with a rubber spatula. The suspension was centrifuged, and the supernatant was filtered to collect fractions F3 and F4. The two centrifuge pellets were combined with the previous pellet and washed by resuspending in saline solution. The saline suspension was centrifuged, and the supernatant was filtered to collect F5 and F6. The pellet of washed cells was resuspended again in saline and filtered (F7 and F8). CELL CULTURE REMOVE MEDIUM MEDIUM
CELLS
CENTRIFUGE (150 g)
HARVEST WITH FRESH MEDIUM CENTRIFUGE (150 g)
SUPERNATANT
CELL PELLET
CELL PELLET
SUPERNATANT
FILTER
FILTER F1
FILTER
I
I
I
COMBINED PELLETS
< | FILTER I p'3
FILTRATE RESUSPEND AND CENTRIFUGE (150 g)
F2 SUPERNATANT
CELL PELLET (FIG. 2)
F|.' FIL
RESUSPEND AND FILTER
|
I
I
FILTER
FILTRATE
I F5 7
I FILTRATE I pj,
I FILTER
I FILTRATE
I
I
I
F6
F7
F8
FIGURE 1. Scheme for isolating Be from cultures of canine alveolar macrophages incubated with BeSO4 solution.
7
438
A. F. EIDSON ET AL.
CELL PELLET (FROM FIG. 1) SUSPEND MIX WITH ALBUMIN CENTRIFUGE (1000 X g)
PELLET
ALBUMIN LAYER
SALINE LAYER SUSPEND
AF7
AF8
DISRUPT CELLS FILTER FILTER
FILTRATE
AF10
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7
FIGURE 2. Scheme for isolating Be from canine cultures of canine alveolar macrophages incubated with 7BeO particles. This scheme begins with the step in Figure 1 where the final cell pellet is resuspended.
Beryllium that remained in the supernatant after centrifugation of cells, and isolated in supernatant filtrates, was defined as dissolved beryllium (F2, F4, F6, F8). Beryllium collected on filters F1, F3, or F5 was defined as free beryllium precipitates formed by hydrolysis at physiological pH. Beryllium collected on filter F7 was defined as beryllium that was associated with cells, either as Be2+ or as cell-associated hydrolysis products. Filtration efficiency was determined by using a solution of 7BeSO4 in supplemented medium. The results showed that 93.4 ± 4.4% of 7Be was collected in the filtrate (mean ± SD, n = 18). Each measurement of beryllium was corrected to reflect filtration efficiency. Note that F7 might include dissolved beryllium associated with any lysed cell debris, as well as dissolved beryllium associated with viable cells and hydrolysis products. A separate experiment was done to test whether dissolved beryllium would bind to dead cell debris. Cells (30 x 106) were lysed ultrasonically and incubated with 8.3 kBq 7BeSO4 for 20 h, and the mixtures were filtered through a 0.1 ^m pore size filter. The 7Be collected on the filter was 27% of the added amount, indicating that 27% of the dissolved 7Be available to a population of lysed cells was filterable. In a culture of 90% viable and 10% lysed cells incubated with dissolved beryllium, the expected contribution to F1, F3, and F7 would total 10% x 27%, or 2.7% of the dissolved beryllium. This expected value represents the upper bound of the dissolved beryllium percentage bound to dead cell debris in a typical culture, because phagocytosis of cell debris by viable cells occurs. In later experiments using relatively insoluble BeO, the upper bound of the contribution was expected to be 1% of the total beryllium added and was considered to be negligible. Fractionation of Cultured Cells Containing BeO When BeO particles were added to cell cultures, the initial scheme for isolating 7Be was identical to that shown in Figure 1. Additional steps
DOSIMETRY OF BERYLLIUM
439
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(Fig. 2) were used to fractionate the cell pellet containing 7BeO particles. The final cell pellet (Fig. 1) was resuspended in saline, layered on top of a saline solution of 35% bovine serum albumin, and the solution was centrifuged at 1000 x g for 10 min (Robinson and Schneider, 1980). Free BeO particles were isolated in the pellet (AF7, Fig. 2). The albumin supernatant layer could not be filtered through a 0.1-/xm filter; therefore, the total volume was counted as fraction AF8. The saline layer and cells collected at the top of the albumin layer were disrupted with 1% sodium dodecylsulfate dissolved in supplemented culture medium, and the cell lysate was filtered (fractions AF9 and AF10). Classification of BeO-Treated Cell Culture Subfractions BeO particles that were taken up by phagocytosis can be described as intracellular BeO, and particles that were not phagocytized can be described as extracellular. Dissolved beryllium bound to cations, or cationic sites of biomolecules, could either be intracellular, extracellular, or bound to the cell membrane, depending on the location of the binding site. Designation of dissolved beryllium as intracellular or extracellular wrongly implies that the location of the beryllium within the cell is known. Therefore, the terms cell-associated and non-cell-associated Be are used here to categorize all beryllium subfractions, whether they contain BeO particles or a dissolved beryllium complex. Beryllium identified in each of the subfractions was assigned to one of four categories [Eqs. (1)-(4)]. Non-cell-associated paniculate Be = F1 + F3 + F5 + AF7
(1)
Non-cell-associated dissolved Be = F2 + F4 + F6 + AF8
(2)
Cell-associated particulate Be = AF9
(3)
Cell-associated dissolved Be = AF10
(4)
The percentages of BeO particles in the non-cell-associated particulate Be category [Eq. (1)] were isolated by filtration of supernatants (F1, F3, F5) and by centrifugation (AF7). Dissolved beryllium in the non-cell-associated dissolved Be category [Eq. (2)] was isolated in supernatant filtrates (F2, F4, F6) and in the albumin layer (AF8). BeO particles isolated by filtration of the disrupted cell suspension (AF9) were placed in the cell-associated particulate Be category [Eq. (3)]. Beryllium isolated in the filtrate of the disrupted cells (AF10) was placed in the cell-associated dissolved Be category [Eq. (4)].
440
A. F. EIDSON ET AL.
RESULTS Cytotoxicity
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Cell viability in control cultures was 95 ± 4% after 48 h. Cultures containing test concentrations of the beryllium compounds (Table 1) were shown to maintain >97% cell viability, relative to viability controls, through 20 h (Fig. 3). Addition of 2.8 x 10"6 M BeSO4 (1.9 nmol BeSO4/105 cells) did not result in significant cytotoxicity. Addition of 500°C BeO at 35 times the BeSO4 concentration (67 nmol BeO/106 cells) reduced viability to 61 ± 4% within 48 h, but no significant cell killing occurred after addition of 1000°C BeO at 105 times the BeSO4 dose.
Distribution of BeSO4 in Cell Cultures Beryllium sulfate added as a solution remained dissolved and did not associate with cells during the 48 h incubation (Table 2). Added beryllium was isolated predominantly in F2 (—85%), with fractions F4, F6, and F8 totaling ~ 8 % of the beryllium added. Filterable beryllium totaled 7.4% of that added to cell cultures, and 6.2% of that added to acellular culture medium. These latter values are not statistically different and were as110 100903
80-
m >
70-
O
pE 60 H o O
li.
SO-
o m o ffj
30-)
500 C BeO
1000 C BeO
BeS04
500 C BeO
1000 C BeO
201020 hr
48 hr
TIME (hr) FIGURE 3. Viability (mean ± SD) of canine alveolar macrophages incubated with BeSO 4 , 500°Ccalcined BeO, or 1000°C-calcined BeO for 48 h at the concentrations listed in Table 1. Asterisk indicates statistically significant cell killing (p < .05).
DOSIMETRY OF BERYLLIUM
441
TABLE 2. Distribution of BeSO4 in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium (Mean ± SD, n - 4) 3
b Be on filter (%)
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Be in filtrate (%) Time (h)
Acellular medium
PAM culture
2 6 20 48
96.7 96.6 96.3 93.5
92.0 93.3 91.8 94.5
Mean
95.8 ± 1.5
± ± ± ±
10.3 17.3 3.1 7.1
± ± ± ±
3.7 2.7 2.6 2.4
92.9 ± 1 . 3
Acellular medium
PAM culture
7.0 2.9 8.3 6.6
8.0 6.6 8.1 7.0
± ± ± ±
3.8 3.5 5.5 4.5
6.2 ± 2.3
± ± ± ±
2.7 3.6 2.8 2.4
7.4 ± 0.7
a
Sum of F2, F4, F6, F8, as described in Figure 1. Sum of F1, F3, F5, F7, as described in Figure 1.
6
sumed to represent the formation of colloidal beryllium hydroxides in culture medium (Skilleter and Paine, 1979). If F7 included dissolved beryllium associated with living cells, it was a small fraction that was not distinguishable from that isolated from acellular controls. Distribution of BeO in Cell Culture The recovery of beryllium from oxides added to the cultures was 84 ± 6%. Distribution of BeO among the four categories [Eqs. (1)-(4)] is shown in Tables 3 and 4 and Figures 4 and 5. The non-cell-associated particulate Be category [Eq. (1)] was dominated by fractions F1 and AF7. Means of fractions F3 and F5 were usually not statistically different from zero for viable cell cultures, indicating that the percentages of added BeO that might have been weakly associated with the cells was minor. For the cultures in which 39% cell killing occurred at 48 h (Table 3 and Fig. 4), F3 contributed 11.1 ± 3.5% and F5 contributed 10.9 ± 1.1%, indicating some release of particles from killed cells. The mean percentage of 500°C BeO in the cell-associated particulate Be category [Eq. (3), Table 3, and Fig. 4] showed an increasing trend from 33 to 66% between 2 and 20 h. The values at 6 and 20 h suggest continued uptake after 6 h, but the values are not significantly different, indicating that particle uptake occurred predominantly during the first 6 h. The percentages measured at 48 h were significantly less than at 20 h. This reduction in cell-associated particulate Be was accompanied by 39% cell killing, as reflected by the increase in non-cell-associated particulate Be between 20 and 48 h. The percentage of 1000°C BeO in the cellassociated particulate Be fraction Gable 4 and Fig. 5) did not change significantly between 2 and 48 h, indicating that uptake occurred predominantly within 2 h. There were no significant differences between
442
A. F. EIDSON ET AL.
TABLE 3. Distribution of 500°C-Calcined BeO in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium
Percentage added 7BeO Category
Time (h)
Acellular control medium
SD (n - 4)
48
0.0 0.0 4.5 1.6
0.0 0.0 5.7 3.2
Non-cell-associated particulate Be
2 6 20 48
47.8 28.4 21.1 38.2
9.2 4.4 5.5 6.5
Non-cell-associated dissolved Be6
2 6 20 48
4.7 1.6 7.9
14.1
2.4 0.4 2.7 1.6
2 6 20 48
32.6 52.1 65.6 42.1
2 6 20 48
5.1 4.4 7.5 7.7
2 6
20 a
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Mean
c
Cell-associated particulate Be
Cell-associated dissolved Bed
5.7
12.1 7.8 7.6 8.8 6.3
11.0 2.6
a
Sum of F1, F3, F5, AF7, as described in Figures 1 and 2. Sum of F2, F4, F6, AF8, as described in Figures 1 and 2. Traction AF9, as described in Figure 2. "^Fraction AF10, as described in Figure 2. b
the cell-associated particulate Be values measured for the 500°C BeO and 1000°C BeO forms at 6 and 20 h (Tables 3 and 4). The cell-associated particulate Be percentage for both forms at 6 and 20 h was 60 ± 6% (mean ± standard deviation, n = 16). The non-cell-associated dissolved Be percentage of 500°C BeO [Eq. (2), Table 3, and Fig. 4] was not significantly greater than zero and greater than the percentage dissolved in acellular medium until 48 h, when it was 14.1 ± 1.6% of added BeO. The cell-associated dissolved Be percentage was also significantly greater at 48 h (7.7 ± 2.6%). The percentages of 1000°C BeO in either the non-cell-associated dissolved Be or cellassociated dissolved Be categories were never significantly greater than zero and the percentage dissolved in acellular medium (Table 4 and Fig. 5).
443
DOSIMETRY OF BERYLLIUM
DISCUSSION
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Beryllium Uptake and Dissolution Macrophages take up a variety of particulate metals by phagocytosis (Witschi and Aldridge, 1968). Uptake of beryllium compounds is an active process that requires Ca2+ and Mg2+, and that can be blocked by a variety of metabolic inhibitors, by inhibitors of microfilament formation, such as cytochalasin B (Skilleter and Pain, 1979; Skilleter and Price, 1979; Hart and Pittman, 1980). Particulates are further taken up by macrophage lysosomes (Marafante, 1987; Nilsen, 1988), and cell damage by beryllium compounds is accompanied by release of enzymes indicative of lysosomal damage (Skilleter and Price, 1979,1981; Kang and Salvaggio, 1976). The amount oi BeO particles taken up in our first experiments was TABLE 4. Distribution of 1000cC-Calcined BeO in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium Percentage added 7BeO Category
Time (h)
Acellular control medium
2 6 20 48
3.1 0.7 0.0 2.5
3.6 0.8 0.0 3.2
Non-cell-associated particulate Bea
2 6 20 48
38.0 30.6 36.1 34.3
12.0 9.7 9.9 8.6
Non-cell-associated dissolved Befc
2 6 20 48
5.7 4.3 5.3 4.5
4.8 1.7 2.6 1.1
Cell-associated particulate Bec
2 6 20 48
53.2 62.2 58.6 56.7
14.1 7.3 18.0 10.0
Cell-associated dissolved Bed
2 6 20 48
2.9 0.7 0.0 4.8
5.3 0.8 0.0 0.5
a
Sum of F1, F3, F5, AF7, as described in Figures 1 and 2. Sum of F2, F4, F6, AF8, as described in Figures 1 and 2. c Fraction AF9, as described in Figure 2. ^Fraction AF10, as described in Figure 2. 6
Mean
SD (n - 4)
A. F. EIDSON ET AL.
444
80
70IUO
3
u
60Q UI
so40-
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m O
if ii
3§ ? 8o
g 20-
10-
2 hr
6 hr
ii
20 hr
48 hr
TIME (hr)
FIGURE 4. Distribution of 500°C-calcined BeO in canine alveolar macrophage cultures [see Eqs. (1)-(4)]. Mean percentages indicate dissolved Be in acellular control medium (no shading), cellassociated dissolved Be (vertical shading), non-cell-associated dissolved Be (horizontal shading), cell-associated particulate Be (right shading), and non-cell-associated paniculate Be (left shading). Error bars indicate ±1 standard deviation (n — 4). Significant differences (p < .05) between the fractions of 500°C-calcined BeO and the fraction dissolved in acellular control media are indicated by asterisk.
independent of BeO temperature history or specific surface area and occurred predominantly within 2-6 h. Similar uptake by bovine PAM phagocytosis of (U,Pu)O2 particles has been reported by Mueller et al. (1986), and canine PAM phagocytosis of latex particles has been reported by Mueller et al. (1989). The 500°C BeO was more soluble than 1000°C BeO in the presence of canine PAM, and the 500°C BeO was the more cytotoxic form. The greater dissolution of the 500°C BeO can be attributed to its greater available surface area (Table 1). This observation agrees with theory (Mercer, 1967) and with the increased 500°C BeO dissolution observed in vitro (Hoover et al., 1988; Finch et al., 1988a) and in vivo (Haley et al., 1989). Although BeSO4 is much more soluble than BeO, cells exposed to low BeSO4 concentrations remained viable by excluding the beryllium, in agreement with previous results (Skilleter and Paine, 1979; Hart and Pittman, 1980). It cannot be determined from our results whether the beryllium was actively excluded, or whether surface binding or internalization by pinocytosis was inefficient.
DOSIMETRY OF BERYLLIUM
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Accumulation of Dissolved Be Although 500°C BeO probably began dissolving immediately upon phagocytosis (Finch et al., 1988b), statistically significant amounts were not detected until 48 h (Table 3 and Fig. 4), concurrent with 39% cell killing. The total percentage of added BeO dissolved at 48 h (cellassociated dissolved Be + non-cell-associated dissolved Be) was 21.8 ± 3.1%. These results suggest that viable PAM sequestered and dissolved BeO particles within lysosomes (Witschi and Aldridge, 1968; Camner et al., 1974) until a cytotoxic concentration of dissolved beryllium was reached, at which time dissolved beryllium and particulate beryllium were released. The portion of dissolved beryllium released from the 39% of cells that were killed would account for 8.5% of the added beryllium, which is somewhat less than the 14.1% measured in the non-cellassociated dissolved Be fraction, suggesting that remaining viable PAM might also release dissolved beryllium under these conditions. The form of the dissolved beryllium released is not known from these measurements. However, the beryllium released from dead cells was not appre-
2hr
6hr
20 hr
48 hr
TIME (hr)
FIGURE 5. Distribution of 1000°C-calcined BeO in canine alveolar macrophage cultures [see Eqs. (1)-(4)]. Mean percentages indicate dissolved Be in acellular control medium (no shading), cellassociated dissolved Be (vertical shading), non-cell-associated dissolved Be (horizontal shading), cell-associated particulate Be (right shading), and non-cell-associated particulate Be (left shading). Error bars indicate ±1 standard deviation (n - 4).
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A. F. EIDSON ET AL.
ciably taken up by remaining viable cells, similar to the fate of soluble beryllium added extracellularly. The cytotoxic concentration at 48 h can be estimated as the concentration of BeO added (1.0 x 10~4 M, Table 1) times the total percentage dissolved (21.8%) = 2.2 x 10~5 M Be. The EC40 is, therefore, estimated at 2.2 x 10"5 M Be. This BeO concentration is less than the 1.1 x 10~4 M soluble Be concentration required to cause 50% cell killing of canine PAM (Finch et a!., 1988b), and it shows that beryllium dissolved from phagocytized BeO particles is more toxic to PAM than is soluble beryllium added extracellularly. This 2.2 x 10~5 M dissolved beryllium is greater than the 1 x 10~6 M soluble beryllium concentration reported to cause maximum transformation of lymphocytes from beryllium-sensitive humans (Hanifin et al., 1970), indicating that canine PAM might dissolve sufficient BeO to produce a beryllium-containing antigen, provided the BeO is of a form that can be dissolved by phagolysosomes. The cytotoxic concentration at 48 h can also be expressed relative to the number of cells in the culture. The amount of BeO added to the culture (67 nmol Be/106 cells) times the total percentage dissolved (21.8%) = 15 nmol/105 cells. Rat liver epithelial cells remain viable until 25 nmol Be/106 cells are accumulated (Skilleter and Paine, 1979), whereas macrophage (Kupffer) cell killing occurs at 1 nmol Be/106 cells (Skilleter and Price, 1981). Our results indicate that canine PAM are less sensitive to beryllium toxicity than rat epithelial cells or liver macrophages. Our results support the following proposed mechanism for alveolar macrophage cytotoxicity from BeO particles: 1. BeO particles are phagocytized and sequestered in phagolysosomes where dissolved as a function of available BeO surface area. This dissolution is more likely for particles calcined at 500 than at 1000°C. It is quite likely that the particles are sequestered in phagolysosomes, as shown previously for beryllium and a variety of other particles. 2. The beryllium dissolved in association with the cell is more cytotoxic than extracellular soluble beryllium and accumulates until cell killing occurs, whereupon the beryllium is released to the medium, perhaps as an extracellular beryllium-containing hapten. 3. The dissolved beryllium released upon cell death is not appreciably taken up by viable cells. In the light of these results, future efforts to isolate and characterize a beryllium-containing hapten from viable PAM exposed to BeO might concentrate on dissolved beryllium that is associated with the cells. It is possible, though not demonstrated here, that the beryllium-containing hapten might be bound to the macrophage membrane. This suggestion is consistent with reports that direct contact between monocytes and lymphocytes is required for consistent lymphocyte transformation (Hani-
DOS1METRY OF BERYLLIUM
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fin, 1968). If cell killing occurs, the beryllium-containing hapten might be more readily isolated from culture medium or lung lavage fluid.
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REFERENCES André, S., Metevier, H., Lantenois, G., Boyer, M., Nolibé, D., and Masse, R. 1987. Beryllium metal solubility in the lung, comparison of metal and hot-pressed forms by in vivo and in vitro dissolution bioassays. Hum. Toxicol. 6:233-240. Camner, P., Lundborg, M., and Hellstrom, P. 1974. Alveolar macrophages and 5 µm particles coated with different metals. Arch. Environ. Health 29:211-213. Cullen, M., Kominsky, J., Rossman, M., Cherniak, M., Rankin, J., Balmes, J., Kern, J., Daniele, R., Palmer, L., Naegel, G., McManus, K., and Cruz, R. 1987. Chronic beryllium disease in a precious metal refinery. Am. Rev. Respir. Dis. 135:201-208. Eisenbud, M., and Lisson, J. 1983. Epidemiological aspects of beryllium-induced nonmalignant lung disease: A 30-year update. J. Occup. Med. 25:196-202. Finch, G. L., Mewhinney, J. A., Eidson, A. F., Hoover, M. D., and Rothenberg, S. J. 1988a. In vitro dissolution characteristics of beryllium oxide and beryllium metal aerosols. J. Aerosol Sci. 19:333-342. Finch, G. L., Verburg, R. J., Mewhinney, J. A., Eidson, A. F., and Hoover, M. D. 1988b. The effect of beryllium compound solubility on in vitro canine alveolar macrophage cytotoxicity. Toxicol. Lett. 41:97-105. Freiman, D. G., and Hardy, H. L. 1970. Beryllium disease: The relation of pulmonary pathology to clinical course and prognosis based on a study of 130 cases from the U.S. beryllium case registry. Hum. Pathol. 1:25-44. Haley, P. J., Finch, G. L., Mewhinney, J. A., Harmsen, A. G., Hahn, F. F., Hoover, M. D., Muggenburg, B. A., and Bice, D. E. 1989. A canine model of beryllium-induced granulomatous lung disease. Lab. Invest. 61:219-227. Hanifin, J. M., Epstein, W. L., and Cline, M. J. 1970. In vitro studies of granulomatous hypersensitivity to beryllium. J. Invest. Dermatol. 55:284-288. Harmsen, A. G., Hoover, M. D., and Seiler, F. A. 1984. Health risk implications of using beryllium in fusion reactors. J. Nucl. Mater. 122 & 123:821-826. Hart, B. A., and Pittman, D. G. 1980. The uptake of beryllium by the alveolar macrophage. J. Reticuloendothel. Soc. 27:49-58. Hoover, M. D., Castorina, B. T., Finch, G. L., and Rothenberg, S. J. 1989. Determination of the oxide layer thickness on beryllium metal particles. Am. Ind. Hyg. J. 50:550-553. Hoover, M. D., Eidson, A. F., Mewhinney, J. A., Finch, G. L., Greenspan, B. J., and Cornell, C. A. 1988. Generation and characterization of respirable beryllium oxide aerosols for toxicity studies. Aerosol Sci. Technol. 9:83-92. Johnson, N. R. 1983. Beryllium disease among workers in a spacecraft manufacturing plantCalifornia. Morbid. Mortal. Weekly Rep. 32:419-420. Kang, K., Bice, D., Hoffmann, E., D'Amato, R., and Salvaggio, J. 1977. Experimental studies of sensitization to beryllium, zirconium, and aluminum compounds in the rabbit. J. Allergy Clin. Immunol. 59:425-436. Kang, K., and Salvaggio, J. 1976. Effects of asbestos and beryllium compounds on the alveolar macrophages. Med. J. Osaka Univ. 27:47-58. Kriebel, D., Brain, J., Sprince, N., and Kazemi. 1988. The pulmonary toxicity of beryllium. Am. Rev. Respir. Dis. 137:464-473. Marafante, E., Lundborg, M., Vahter, M., and Camner, P. 1987. Dissolution of two arsenic compounds by rabbit alveolar macrophages in vitro. Fundam. Appl. Toxicol. 8:382-388. Mercer, T. T. 1967. On the role of particle size in the dissolution of lung burdens. Health Phys. 13:1211-1221. Mueller, H.-L., Guilmette, R. A., and Muggenburg, B. A. 1989. Uptake of inert particles by dog
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alveolar macrophages in vitro—A comparison of monolayer and suspension techniques. J. Appl. Toxicol. 9:135-143. Mueller, H.-L., Hotz, G., Seidel, A., Thiele, H., and Ray, I. L. 1986. Uptake of uranium-plutonium mixed-oxides by alveolar macrophages in vivo and in vitro: Electron microscopic studies. In Aerosols: Formation and Reactivity, pp. 311-317. Oxford: Pergamon Journals. Muggenburg, B. A., and Mauderly, J. L. 1975. Lung lavage using a single-lumen endotracheal tube. J. Appl. Physiol. 25:471-475. Nilsen, A., Nyberg, K., and Camner, P. 1988. Intra-phagolysosomal pH in alveolar macrophages after phagocytosis in vivo and in vitro of fluorescein-labeled yeast particles. Exp. Lung Res. 14:197207. Robinson, A. V, and Schneider, R. P. 1980. Phagocytosis, toxicity and solubility of AmO2 in alveolar macrophages. In Pulmonary Toxicology of Respirable Particles, eds. C. L. Sanders and F. T. Cross, CONF-791002, pp. 325-337. Rothenberg, S. J., DeNee, P. B., Cheng, Y. S., Hanson, R. L., Yeh, H. C., and Eidson, A. F. 1982. Methods for the measurement of surface areas of aerosols by absorption. Adv. Colloid Interface Sci. 15:223-249. Skilleter, D. N., and Paine, A. J. 1979. Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake. Chem. Biol. Interact. 24:19-33. Skilleter, D. N., and Price, R. J. 1981. Effects of beryllium compounds on rat liver kupffer cells in culture. Toxicol. Appl. Pharmacol. 59:279-286. Witschi, H. P., and Aldridge, W. N. 1968. Uptake, distribution and binding of beryllium to organelles of the rat liver cell. Biochem. J. 106:811-820. Received February 11, 1991 Accepted July 4, 1991
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Dosimetry of beryllium in cultured canine pulmonary alveolar macrophages A. F. Eidson , A. Taya , G. L. Finch , M. D. Hoover & Cindy Cook To cite this article: A. F. Eidson , A. Taya , G. L. Finch , M. D. Hoover & Cindy Cook (1991) Dosimetry of beryllium in cultured canine pulmonary alveolar macrophages, Journal of Toxicology and Environmental Health, 34:4, 433-448, DOI: 10.1080/15287399109531581 To link to this article: http://dx.doi.org/10.1080/15287399109531581
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DOSIMETRY OF BERYLLIUM IN CULTURED CANINE PULMONARY ALVEOLAR MACROPHAGES A. F. Eidson, A. Taya, G. L. Finch, M. D. Hoover, Cindy Cook
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International Technology, Albuquerque, New Mexico
This study was designed to determine the dosimetry within macrophages of beryllium compounds administered at sublethal doses. Information on the dosimetry of beryllium within macrophages is required to guide further efforts to isolate and characterize beryllium-containing haptens. Inhalation of beryllium aerosols can cause chronic berylliosis, a progressive, granulomatous fibrosis of the lung. Studies in laboratory animals indicate that alveolar macrophages take up beryllium compounds and participate in a hypersensitivity immune response to beryllium-containing antigen. Beagle dog macrophage cultures were incubated with 7BeSO4 in solution or with suspensions of 7BeO particles that had been calcined at 500 or 1000°C. Beryllium-7 was measured in fractions collected from cultures after successive centrifugation and filtration steps at 2, 6, 20, and 48 h after addition. An insignificant percentage of BeSO4 was taken up by the cells and did not cause cytotoxicity. Maximum BeO uptake occurred within 6 h, was 60 ± 6% of added BeO, and was independent of BeO calcination temperature or specific surface area. Approximately 22% of 500°C BeO dissolved within 48 h after addition to cell culture, concurrent with 39% cell killing. Dissolved beryllium remained associated with cells until a cytotoxic concentration was reached (2.2 × 10-5 M, 15 nmol Be/106 cells), when the beryllium was released into the medium. There was no significant dissolution of the 1000°C BeO within 48 h, and no significant cell killing. The results indicate that beryllium dissolved from phagocytized BeO was more cytotoxic than soluble beryllium added extracellularly. The data support an interactive mechanism in which phagocytized BeO particles were dissolved, and dissolved beryllium remained associated with the macrophage until a cytotoxic concentration accumulated, whereupon the beryllium was released to the medium and not appreciably taken up by viable cells.
INTRODUCTION Beryllium metal is used in the aerospace and nuclear industries because of its low density, high heat capacity, mechanical strength, and good neutronic properties (Harmsen et al., 1984). Beryllium oxide is used The authors acknowledge many helpful discussions with Dr. Janet Benson, Dr. Bruce Muggenburg, Dr. R. F. Henderson, and Dr. Alan R. Dahl. We also express our appreciation to the many skilled technicians assisting with the experiments. This study was supported by the U.S. Department of Energy Office of Health and Environmental Research under contract DE-AC04-76EV01013, in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Cindy Cook participated under the Associated Western Universities Summer Student Research Program. Requests for reprints should be sent to A. F. Eidson, International Technology Corporation, 5301 Central Avenue, Suite 700, Albuquerque, NM 87108. 433 Journal of Toxicology and Environmental Health, 34:433-448, 1991
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A. F. E1DSON ET AL.
directly as an insulator and heat conductor in the semiconductor and ceramics industries. Fabrication of BeO components can release respirable aerosols, and fabrication of beryllium metal components can release aerosols that contain a surface coating of BeO (Hoover et al., 1989). Inhalation of beryllium aerosols can cause chronic berylliosis, a progressive, granulomatous fibrosis of the lung (Freiman and Hardy, 1970; Kriebel et al., 1988). As new cases are reported (Johnson, 1983; Cullen et al., 1987), there is continuing concern about beryllium exposure. Studies of disease induction mechanisms in laboratory animals support the hypothesis that alveolar macrophages take up beryllium compounds and participate in a hypersensitivity immune response to a berylliumcontaining antigen (Hanifin et al., 1970; Kriebel et al., 1988). A number of observations indicate that the solubility of BeO particles is an important factor in chronic berylliosis induction. The in vitro dissolution rate of BeO particles calcined at 500°C is greater than that of BeO calcined at 1000°C, in direct proportion to the greater specific surface area of the 500°C particles (Finch et al., 1988a). As a result of its greater solubility, the toxicity of 500°C-calcined BeO particles to cultured canine pulmonary macrophages (PAM) is greater than that of the 1000°C form (Finch et al., 1988b). BeO particles calcined at 500°C clear more rapidly from lungs of beagle dogs and cause an increased lymphocyte response and greater histopathology than BeO calcined at 1000°C (Haley, 1989). Epidemiological evidence indicates that the incidence of chronic berylliosis is higher among individuals exposed to dusts of BeO fired at approximately 500°C rather than at 1000°C (Eisenbud and Lisson, 1983). Beryllium metal is cytotoxic to macrophages (Camner et al., 1974; Andre et al., 1987). Soluble beryllium is incorporated by rat and guinea pig alveolar macrophages as particles (Hart and Pittman, 1980), probably as beryllium hydroxide formed in the culture medium (Skilleter and Paine, 1979). Soluble beryllium has been reported to be cytotoxic to alveolar macrophages within 26 h when added at 9.5 x 10~5 M (Kang and Salvaggio, 1976; Kang et al., 1977) and to rat liver macrophage (Kupffer) cells within 24 h when 1 nmol Be/106 cells was incorporated (Skilleter and Price, 1981). We hypothesize that pulmonary alveolar macrophages interact with beryllium to form a beryllium-containing hapten that acts as an antigen. Previous quantitative studies of macrophage response to beryllium compounds have centered primarily on beryllium uptake and cytotoxicity. To understand the role of beryllium in hypersensitivity immune response and in macrophage cytotoxicity, one must investigate the ways in which living macrophages interact with beryllium administered in sublethal doses. The objectives of this study were to investigate the internal dosimetry of beryllium within living macrophages and to guide future efforts to isolate and characterize a beryllium-containing antigen.
DOSIMETRY OF BERYLLIUM
435
MATERIALS AND METHODS
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Experimental Design Pulmonary alveolar macrophages (PAM) from beagle dogs were cultured and dosed with 7BeSO4 solution or 7BeO particles that had been calcined at 500 or 1000°C. The amounts of each beryllium compound added to cultured PAM (Table 1) were chosen to maintain cell viability (Kang et al., 1977; Finch et al., 1988b), yet provide detectable 7Be radioactivity. Cultures were also prepared that contained beryllium compounds, but without cells (acellular controls). Separate cultures were prepared and dosed in the same way for cell viability determinations (viability controls). At selected times after beryllium addition, these cultures were treated as described below to measure viability and to isolate dissolved Be and undissolved BeO in subfractions of the cells and the culture medium. All cell cultures were prepared in duplicate, and then the experiment was repeated in duplicate using cells from a different animal. Beryllium Compounds Aqueous solutions of 7BeCI2 (Los Alamos National Laboratory, Los Alamos, N. Mex.) and BeSO4-4H2O (Aldrich, Milwaukee, Wis.) were mixed using distilled, deionized water to prepare a solution containing 2.6 fig Be2+/ml and 3.96 MBq 7Be27ml. This stock solution contained negligible amounts of CI" and was used for all 7BeSO4 experiments. A stock suspension for preparing the 7BeO powders was prepared by precipitating 7 Be(OH)2 from 7BeC2O4 solution. The resulting suspension was nebulized to form an aerosol, which was passed through a high-temperature column, collected by filtration, and then calcined for 16 h at 500 or 1000°C (Hoover et al., 1988). X-ray diffractometry was used to confirm that the material was BeO. The particle size distribution of both forms was measured using cascade impactors and was lognormally distributed with a mass median physical particle diameter of 0.43 /im and a geometric standard deviation of 2.3. The BeO specific activity was 13.3 MBq 7BeO/g. Specific surface areas of the aerosol particles were measured using a TABLE 1. Beryllium Compounds Added to Pulmonary Alveolar Macrophage Cultures Be compound
Be molarity
Moles Be/106 cells
7
Be added (Bq)
Available surface area/106 cells (m2)
BeSO4 BeO (500°C) BeO (1000°C)
2.8 x 10" 6 1.0 x 10"" 3.0 x 10" 4
1.9 x 10~9 6.7 x 10~8 2.0 x 10" 7
7.7 x 10 s 6.7 x 102 2.0 X 103
NAa 3.2 X 1 0 " 4 b 1.8 X 1 0 " 4 c
a
Not applicable. Specific surface area - 189.3 m2/g. Specific surface area - 35.8 m2/g.
fc c
436
A. F. EIDSON ET AL.
Quantasorb model QS-10 instrument (Quantachrome Corp., Syossett, N.Y.) (Rothenberg et al., 1982), and found to be 189.3 and 35.8 m2/g, for the 500°C-calcined and 1000°C-calcined BeO particles, respectively. Beryllium-7 was quantitated using a Beckman-8000 gamma counter (Beckman Instruments, Inc., Fullerton, Calif.), which was calibrated with a 7Be primary radioactivity standard (Amersham Corp., Arlington Heights, III.). The detection limit of the gamma counter was 16 Bq, at a counting efficiency of 3.3%.
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Cell Culture Techniques Alveolar macrophages were obtained by bronchopulmonary lavage of beagle dogs (7-8 yr of age) from the Institute's colony (Muggenburg and Mauderly, 1975). The lavage fluids were centrifuged at 150 x g for 10 min at 4°C. The supernatant was removed, and the cells were resuspended in saline solution and counted using a Levy hemocytometer (Hauser Scientific, Blue Bell, Pa.). Cell viability was determined by trypan blue dye exclusion (0.4% in saline; Gibco, Grand Island, N.Y.). For differential cell counts, cytocentrifuge preparations were prepared and stained with Diff-Quik stain (Gibco). All cell populations contained 93 ± 2% macrophages, 3 ± 1% lymphocytes, and 4 ± 2% neutrophils. Macrophages were >95% viable. The cells were washed twice in medium (RPM11640, Gibco) and then resuspended in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah) and 50 /ig/ml gentamycin sulfate (Sigma Chemical Co., St. Louis, Mo.). Twenty milliliters of a cell suspension containing 1.5 x 106 viable cells/ml were added to sterile plastic cell culture dishes (100 mm diameter, Costar, Van Nuys, Calif.), which were incubated for 2 h at 37°C in a 5% CO2/95% air atmosphere at 85% relative humidity to allow cell attachment. Using this technique, 80-90% of the cells are adherent after 2 h (Mueller et al., 1989). Acellular control cultures and viability control cultures were prepared at the same time. Before being added to cell cultures, paniculate 7BeO suspensions or 7 BeSO4 solutions were subjected to ultrasonic agitation for 20-30 min to suspend the BeO particles. The suspensions were then diluted to 20 ml with supplemented medium. The medium was removed from the cultured cells and replaced with 20 ml of supplemented medium containing the test compound. The cell cultures were returned to the incubator under the same conditions. Dosimetry of Beryllium in Cultured Cells The dosimetry of beryllium in the cultured cells was determined by measurements of 7Be in soluble or particulate forms, which were either associated or not associated with the cells. The 20-ml cell cultures were
DOSIMETRY OF BERYLLIUM
437
fractionated at 2, 6, 20, and 48 h after addition of one of the beryllium compounds (Figs. 1 and 2). Successive centrifugation and filtration steps described below were used to separate cell cultures into subfractions containing beryllium. The radioactivity measured in each subfraction was expressed as a percentage of the total amount of radioactivity added to the culture dish. Statistical inferences were made by using the Student's t test at p < .05.
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Fractionation of Cultured Cells Containing BeSO4 Beryllium administered as BeSO4 was isolated according to the flow chart shown in Figure 1. The medium was removed from the culture dish and centrifuged. The supernatant was filtered through a 0.1 /*m pore-size polycarbonate membrane filter (Nucleopore, Pleasanton, Calif.). Radioactivity measured on the filter was designated as F1, and that in the filtrate was designated as F2. The centrifuge cell pellet was set aside. Fresh medium was added to the culture dish and the adherent cells were harvested by gentle scraping with a rubber spatula. The suspension was centrifuged, and the supernatant was filtered to collect fractions F3 and F4. The two centrifuge pellets were combined with the previous pellet and washed by resuspending in saline solution. The saline suspension was centrifuged, and the supernatant was filtered to collect F5 and F6. The pellet of washed cells was resuspended again in saline and filtered (F7 and F8). CELL CULTURE REMOVE MEDIUM MEDIUM
CELLS
CENTRIFUGE (150 g)
HARVEST WITH FRESH MEDIUM CENTRIFUGE (150 g)
SUPERNATANT
CELL PELLET
CELL PELLET
SUPERNATANT
FILTER
FILTER F1
FILTER
I
I
I
COMBINED PELLETS
< | FILTER I p'3
FILTRATE RESUSPEND AND CENTRIFUGE (150 g)
F2 SUPERNATANT
CELL PELLET (FIG. 2)
F|.' FIL
RESUSPEND AND FILTER
|
I
I
FILTER
FILTRATE
I F5 7
I FILTRATE I pj,
I FILTER
I FILTRATE
I
I
I
F6
F7
F8
FIGURE 1. Scheme for isolating Be from cultures of canine alveolar macrophages incubated with BeSO4 solution.
7
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A. F. EIDSON ET AL.
CELL PELLET (FROM FIG. 1) SUSPEND MIX WITH ALBUMIN CENTRIFUGE (1000 X g)
PELLET
ALBUMIN LAYER
SALINE LAYER SUSPEND
AF7
AF8
DISRUPT CELLS FILTER FILTER
FILTRATE
AF10
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7
FIGURE 2. Scheme for isolating Be from canine cultures of canine alveolar macrophages incubated with 7BeO particles. This scheme begins with the step in Figure 1 where the final cell pellet is resuspended.
Beryllium that remained in the supernatant after centrifugation of cells, and isolated in supernatant filtrates, was defined as dissolved beryllium (F2, F4, F6, F8). Beryllium collected on filters F1, F3, or F5 was defined as free beryllium precipitates formed by hydrolysis at physiological pH. Beryllium collected on filter F7 was defined as beryllium that was associated with cells, either as Be2+ or as cell-associated hydrolysis products. Filtration efficiency was determined by using a solution of 7BeSO4 in supplemented medium. The results showed that 93.4 ± 4.4% of 7Be was collected in the filtrate (mean ± SD, n = 18). Each measurement of beryllium was corrected to reflect filtration efficiency. Note that F7 might include dissolved beryllium associated with any lysed cell debris, as well as dissolved beryllium associated with viable cells and hydrolysis products. A separate experiment was done to test whether dissolved beryllium would bind to dead cell debris. Cells (30 x 106) were lysed ultrasonically and incubated with 8.3 kBq 7BeSO4 for 20 h, and the mixtures were filtered through a 0.1 ^m pore size filter. The 7Be collected on the filter was 27% of the added amount, indicating that 27% of the dissolved 7Be available to a population of lysed cells was filterable. In a culture of 90% viable and 10% lysed cells incubated with dissolved beryllium, the expected contribution to F1, F3, and F7 would total 10% x 27%, or 2.7% of the dissolved beryllium. This expected value represents the upper bound of the dissolved beryllium percentage bound to dead cell debris in a typical culture, because phagocytosis of cell debris by viable cells occurs. In later experiments using relatively insoluble BeO, the upper bound of the contribution was expected to be 1% of the total beryllium added and was considered to be negligible. Fractionation of Cultured Cells Containing BeO When BeO particles were added to cell cultures, the initial scheme for isolating 7Be was identical to that shown in Figure 1. Additional steps
DOSIMETRY OF BERYLLIUM
439
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(Fig. 2) were used to fractionate the cell pellet containing 7BeO particles. The final cell pellet (Fig. 1) was resuspended in saline, layered on top of a saline solution of 35% bovine serum albumin, and the solution was centrifuged at 1000 x g for 10 min (Robinson and Schneider, 1980). Free BeO particles were isolated in the pellet (AF7, Fig. 2). The albumin supernatant layer could not be filtered through a 0.1-/xm filter; therefore, the total volume was counted as fraction AF8. The saline layer and cells collected at the top of the albumin layer were disrupted with 1% sodium dodecylsulfate dissolved in supplemented culture medium, and the cell lysate was filtered (fractions AF9 and AF10). Classification of BeO-Treated Cell Culture Subfractions BeO particles that were taken up by phagocytosis can be described as intracellular BeO, and particles that were not phagocytized can be described as extracellular. Dissolved beryllium bound to cations, or cationic sites of biomolecules, could either be intracellular, extracellular, or bound to the cell membrane, depending on the location of the binding site. Designation of dissolved beryllium as intracellular or extracellular wrongly implies that the location of the beryllium within the cell is known. Therefore, the terms cell-associated and non-cell-associated Be are used here to categorize all beryllium subfractions, whether they contain BeO particles or a dissolved beryllium complex. Beryllium identified in each of the subfractions was assigned to one of four categories [Eqs. (1)-(4)]. Non-cell-associated paniculate Be = F1 + F3 + F5 + AF7
(1)
Non-cell-associated dissolved Be = F2 + F4 + F6 + AF8
(2)
Cell-associated particulate Be = AF9
(3)
Cell-associated dissolved Be = AF10
(4)
The percentages of BeO particles in the non-cell-associated particulate Be category [Eq. (1)] were isolated by filtration of supernatants (F1, F3, F5) and by centrifugation (AF7). Dissolved beryllium in the non-cell-associated dissolved Be category [Eq. (2)] was isolated in supernatant filtrates (F2, F4, F6) and in the albumin layer (AF8). BeO particles isolated by filtration of the disrupted cell suspension (AF9) were placed in the cell-associated particulate Be category [Eq. (3)]. Beryllium isolated in the filtrate of the disrupted cells (AF10) was placed in the cell-associated dissolved Be category [Eq. (4)].
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A. F. EIDSON ET AL.
RESULTS Cytotoxicity
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Cell viability in control cultures was 95 ± 4% after 48 h. Cultures containing test concentrations of the beryllium compounds (Table 1) were shown to maintain >97% cell viability, relative to viability controls, through 20 h (Fig. 3). Addition of 2.8 x 10"6 M BeSO4 (1.9 nmol BeSO4/105 cells) did not result in significant cytotoxicity. Addition of 500°C BeO at 35 times the BeSO4 concentration (67 nmol BeO/106 cells) reduced viability to 61 ± 4% within 48 h, but no significant cell killing occurred after addition of 1000°C BeO at 105 times the BeSO4 dose.
Distribution of BeSO4 in Cell Cultures Beryllium sulfate added as a solution remained dissolved and did not associate with cells during the 48 h incubation (Table 2). Added beryllium was isolated predominantly in F2 (—85%), with fractions F4, F6, and F8 totaling ~ 8 % of the beryllium added. Filterable beryllium totaled 7.4% of that added to cell cultures, and 6.2% of that added to acellular culture medium. These latter values are not statistically different and were as110 100903
80-
m >
70-
O
pE 60 H o O
li.
SO-
o m o ffj
30-)
500 C BeO
1000 C BeO
BeS04
500 C BeO
1000 C BeO
201020 hr
48 hr
TIME (hr) FIGURE 3. Viability (mean ± SD) of canine alveolar macrophages incubated with BeSO 4 , 500°Ccalcined BeO, or 1000°C-calcined BeO for 48 h at the concentrations listed in Table 1. Asterisk indicates statistically significant cell killing (p < .05).
DOSIMETRY OF BERYLLIUM
441
TABLE 2. Distribution of BeSO4 in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium (Mean ± SD, n - 4) 3
b Be on filter (%)
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Be in filtrate (%) Time (h)
Acellular medium
PAM culture
2 6 20 48
96.7 96.6 96.3 93.5
92.0 93.3 91.8 94.5
Mean
95.8 ± 1.5
± ± ± ±
10.3 17.3 3.1 7.1
± ± ± ±
3.7 2.7 2.6 2.4
92.9 ± 1 . 3
Acellular medium
PAM culture
7.0 2.9 8.3 6.6
8.0 6.6 8.1 7.0
± ± ± ±
3.8 3.5 5.5 4.5
6.2 ± 2.3
± ± ± ±
2.7 3.6 2.8 2.4
7.4 ± 0.7
a
Sum of F2, F4, F6, F8, as described in Figure 1. Sum of F1, F3, F5, F7, as described in Figure 1.
6
sumed to represent the formation of colloidal beryllium hydroxides in culture medium (Skilleter and Paine, 1979). If F7 included dissolved beryllium associated with living cells, it was a small fraction that was not distinguishable from that isolated from acellular controls. Distribution of BeO in Cell Culture The recovery of beryllium from oxides added to the cultures was 84 ± 6%. Distribution of BeO among the four categories [Eqs. (1)-(4)] is shown in Tables 3 and 4 and Figures 4 and 5. The non-cell-associated particulate Be category [Eq. (1)] was dominated by fractions F1 and AF7. Means of fractions F3 and F5 were usually not statistically different from zero for viable cell cultures, indicating that the percentages of added BeO that might have been weakly associated with the cells was minor. For the cultures in which 39% cell killing occurred at 48 h (Table 3 and Fig. 4), F3 contributed 11.1 ± 3.5% and F5 contributed 10.9 ± 1.1%, indicating some release of particles from killed cells. The mean percentage of 500°C BeO in the cell-associated particulate Be category [Eq. (3), Table 3, and Fig. 4] showed an increasing trend from 33 to 66% between 2 and 20 h. The values at 6 and 20 h suggest continued uptake after 6 h, but the values are not significantly different, indicating that particle uptake occurred predominantly during the first 6 h. The percentages measured at 48 h were significantly less than at 20 h. This reduction in cell-associated particulate Be was accompanied by 39% cell killing, as reflected by the increase in non-cell-associated particulate Be between 20 and 48 h. The percentage of 1000°C BeO in the cellassociated particulate Be fraction Gable 4 and Fig. 5) did not change significantly between 2 and 48 h, indicating that uptake occurred predominantly within 2 h. There were no significant differences between
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TABLE 3. Distribution of 500°C-Calcined BeO in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium
Percentage added 7BeO Category
Time (h)
Acellular control medium
SD (n - 4)
48
0.0 0.0 4.5 1.6
0.0 0.0 5.7 3.2
Non-cell-associated particulate Be
2 6 20 48
47.8 28.4 21.1 38.2
9.2 4.4 5.5 6.5
Non-cell-associated dissolved Be6
2 6 20 48
4.7 1.6 7.9
14.1
2.4 0.4 2.7 1.6
2 6 20 48
32.6 52.1 65.6 42.1
2 6 20 48
5.1 4.4 7.5 7.7
2 6
20 a
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Mean
c
Cell-associated particulate Be
Cell-associated dissolved Bed
5.7
12.1 7.8 7.6 8.8 6.3
11.0 2.6
a
Sum of F1, F3, F5, AF7, as described in Figures 1 and 2. Sum of F2, F4, F6, AF8, as described in Figures 1 and 2. Traction AF9, as described in Figure 2. "^Fraction AF10, as described in Figure 2. b
the cell-associated particulate Be values measured for the 500°C BeO and 1000°C BeO forms at 6 and 20 h (Tables 3 and 4). The cell-associated particulate Be percentage for both forms at 6 and 20 h was 60 ± 6% (mean ± standard deviation, n = 16). The non-cell-associated dissolved Be percentage of 500°C BeO [Eq. (2), Table 3, and Fig. 4] was not significantly greater than zero and greater than the percentage dissolved in acellular medium until 48 h, when it was 14.1 ± 1.6% of added BeO. The cell-associated dissolved Be percentage was also significantly greater at 48 h (7.7 ± 2.6%). The percentages of 1000°C BeO in either the non-cell-associated dissolved Be or cellassociated dissolved Be categories were never significantly greater than zero and the percentage dissolved in acellular medium (Table 4 and Fig. 5).
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DOSIMETRY OF BERYLLIUM
DISCUSSION
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Beryllium Uptake and Dissolution Macrophages take up a variety of particulate metals by phagocytosis (Witschi and Aldridge, 1968). Uptake of beryllium compounds is an active process that requires Ca2+ and Mg2+, and that can be blocked by a variety of metabolic inhibitors, by inhibitors of microfilament formation, such as cytochalasin B (Skilleter and Pain, 1979; Skilleter and Price, 1979; Hart and Pittman, 1980). Particulates are further taken up by macrophage lysosomes (Marafante, 1987; Nilsen, 1988), and cell damage by beryllium compounds is accompanied by release of enzymes indicative of lysosomal damage (Skilleter and Price, 1979,1981; Kang and Salvaggio, 1976). The amount oi BeO particles taken up in our first experiments was TABLE 4. Distribution of 1000cC-Calcined BeO in Pulmonary Alveolar Macrophage (PAM) Cultures and in Acellular Medium Percentage added 7BeO Category
Time (h)
Acellular control medium
2 6 20 48
3.1 0.7 0.0 2.5
3.6 0.8 0.0 3.2
Non-cell-associated particulate Bea
2 6 20 48
38.0 30.6 36.1 34.3
12.0 9.7 9.9 8.6
Non-cell-associated dissolved Befc
2 6 20 48
5.7 4.3 5.3 4.5
4.8 1.7 2.6 1.1
Cell-associated particulate Bec
2 6 20 48
53.2 62.2 58.6 56.7
14.1 7.3 18.0 10.0
Cell-associated dissolved Bed
2 6 20 48
2.9 0.7 0.0 4.8
5.3 0.8 0.0 0.5
a
Sum of F1, F3, F5, AF7, as described in Figures 1 and 2. Sum of F2, F4, F6, AF8, as described in Figures 1 and 2. c Fraction AF9, as described in Figure 2. ^Fraction AF10, as described in Figure 2. 6
Mean
SD (n - 4)
A. F. EIDSON ET AL.
444
80
70IUO
3
u
60Q UI
so40-
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m O
if ii
3§ ? 8o
g 20-
10-
2 hr
6 hr
ii
20 hr
48 hr
TIME (hr)
FIGURE 4. Distribution of 500°C-calcined BeO in canine alveolar macrophage cultures [see Eqs. (1)-(4)]. Mean percentages indicate dissolved Be in acellular control medium (no shading), cellassociated dissolved Be (vertical shading), non-cell-associated dissolved Be (horizontal shading), cell-associated particulate Be (right shading), and non-cell-associated paniculate Be (left shading). Error bars indicate ±1 standard deviation (n — 4). Significant differences (p < .05) between the fractions of 500°C-calcined BeO and the fraction dissolved in acellular control media are indicated by asterisk.
independent of BeO temperature history or specific surface area and occurred predominantly within 2-6 h. Similar uptake by bovine PAM phagocytosis of (U,Pu)O2 particles has been reported by Mueller et al. (1986), and canine PAM phagocytosis of latex particles has been reported by Mueller et al. (1989). The 500°C BeO was more soluble than 1000°C BeO in the presence of canine PAM, and the 500°C BeO was the more cytotoxic form. The greater dissolution of the 500°C BeO can be attributed to its greater available surface area (Table 1). This observation agrees with theory (Mercer, 1967) and with the increased 500°C BeO dissolution observed in vitro (Hoover et al., 1988; Finch et al., 1988a) and in vivo (Haley et al., 1989). Although BeSO4 is much more soluble than BeO, cells exposed to low BeSO4 concentrations remained viable by excluding the beryllium, in agreement with previous results (Skilleter and Paine, 1979; Hart and Pittman, 1980). It cannot be determined from our results whether the beryllium was actively excluded, or whether surface binding or internalization by pinocytosis was inefficient.
DOSIMETRY OF BERYLLIUM
445
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Accumulation of Dissolved Be Although 500°C BeO probably began dissolving immediately upon phagocytosis (Finch et al., 1988b), statistically significant amounts were not detected until 48 h (Table 3 and Fig. 4), concurrent with 39% cell killing. The total percentage of added BeO dissolved at 48 h (cellassociated dissolved Be + non-cell-associated dissolved Be) was 21.8 ± 3.1%. These results suggest that viable PAM sequestered and dissolved BeO particles within lysosomes (Witschi and Aldridge, 1968; Camner et al., 1974) until a cytotoxic concentration of dissolved beryllium was reached, at which time dissolved beryllium and particulate beryllium were released. The portion of dissolved beryllium released from the 39% of cells that were killed would account for 8.5% of the added beryllium, which is somewhat less than the 14.1% measured in the non-cellassociated dissolved Be fraction, suggesting that remaining viable PAM might also release dissolved beryllium under these conditions. The form of the dissolved beryllium released is not known from these measurements. However, the beryllium released from dead cells was not appre-
2hr
6hr
20 hr
48 hr
TIME (hr)
FIGURE 5. Distribution of 1000°C-calcined BeO in canine alveolar macrophage cultures [see Eqs. (1)-(4)]. Mean percentages indicate dissolved Be in acellular control medium (no shading), cellassociated dissolved Be (vertical shading), non-cell-associated dissolved Be (horizontal shading), cell-associated particulate Be (right shading), and non-cell-associated particulate Be (left shading). Error bars indicate ±1 standard deviation (n - 4).
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A. F. EIDSON ET AL.
ciably taken up by remaining viable cells, similar to the fate of soluble beryllium added extracellularly. The cytotoxic concentration at 48 h can be estimated as the concentration of BeO added (1.0 x 10~4 M, Table 1) times the total percentage dissolved (21.8%) = 2.2 x 10~5 M Be. The EC40 is, therefore, estimated at 2.2 x 10"5 M Be. This BeO concentration is less than the 1.1 x 10~4 M soluble Be concentration required to cause 50% cell killing of canine PAM (Finch et a!., 1988b), and it shows that beryllium dissolved from phagocytized BeO particles is more toxic to PAM than is soluble beryllium added extracellularly. This 2.2 x 10~5 M dissolved beryllium is greater than the 1 x 10~6 M soluble beryllium concentration reported to cause maximum transformation of lymphocytes from beryllium-sensitive humans (Hanifin et al., 1970), indicating that canine PAM might dissolve sufficient BeO to produce a beryllium-containing antigen, provided the BeO is of a form that can be dissolved by phagolysosomes. The cytotoxic concentration at 48 h can also be expressed relative to the number of cells in the culture. The amount of BeO added to the culture (67 nmol Be/106 cells) times the total percentage dissolved (21.8%) = 15 nmol/105 cells. Rat liver epithelial cells remain viable until 25 nmol Be/106 cells are accumulated (Skilleter and Paine, 1979), whereas macrophage (Kupffer) cell killing occurs at 1 nmol Be/106 cells (Skilleter and Price, 1981). Our results indicate that canine PAM are less sensitive to beryllium toxicity than rat epithelial cells or liver macrophages. Our results support the following proposed mechanism for alveolar macrophage cytotoxicity from BeO particles: 1. BeO particles are phagocytized and sequestered in phagolysosomes where dissolved as a function of available BeO surface area. This dissolution is more likely for particles calcined at 500 than at 1000°C. It is quite likely that the particles are sequestered in phagolysosomes, as shown previously for beryllium and a variety of other particles. 2. The beryllium dissolved in association with the cell is more cytotoxic than extracellular soluble beryllium and accumulates until cell killing occurs, whereupon the beryllium is released to the medium, perhaps as an extracellular beryllium-containing hapten. 3. The dissolved beryllium released upon cell death is not appreciably taken up by viable cells. In the light of these results, future efforts to isolate and characterize a beryllium-containing hapten from viable PAM exposed to BeO might concentrate on dissolved beryllium that is associated with the cells. It is possible, though not demonstrated here, that the beryllium-containing hapten might be bound to the macrophage membrane. This suggestion is consistent with reports that direct contact between monocytes and lymphocytes is required for consistent lymphocyte transformation (Hani-
DOS1METRY OF BERYLLIUM
447
fin, 1968). If cell killing occurs, the beryllium-containing hapten might be more readily isolated from culture medium or lung lavage fluid.
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REFERENCES André, S., Metevier, H., Lantenois, G., Boyer, M., Nolibé, D., and Masse, R. 1987. Beryllium metal solubility in the lung, comparison of metal and hot-pressed forms by in vivo and in vitro dissolution bioassays. Hum. Toxicol. 6:233-240. Camner, P., Lundborg, M., and Hellstrom, P. 1974. Alveolar macrophages and 5 µm particles coated with different metals. Arch. Environ. Health 29:211-213. Cullen, M., Kominsky, J., Rossman, M., Cherniak, M., Rankin, J., Balmes, J., Kern, J., Daniele, R., Palmer, L., Naegel, G., McManus, K., and Cruz, R. 1987. Chronic beryllium disease in a precious metal refinery. Am. Rev. Respir. Dis. 135:201-208. Eisenbud, M., and Lisson, J. 1983. Epidemiological aspects of beryllium-induced nonmalignant lung disease: A 30-year update. J. Occup. Med. 25:196-202. Finch, G. L., Mewhinney, J. A., Eidson, A. F., Hoover, M. D., and Rothenberg, S. J. 1988a. In vitro dissolution characteristics of beryllium oxide and beryllium metal aerosols. J. Aerosol Sci. 19:333-342. Finch, G. L., Verburg, R. J., Mewhinney, J. A., Eidson, A. F., and Hoover, M. D. 1988b. The effect of beryllium compound solubility on in vitro canine alveolar macrophage cytotoxicity. Toxicol. Lett. 41:97-105. Freiman, D. G., and Hardy, H. L. 1970. Beryllium disease: The relation of pulmonary pathology to clinical course and prognosis based on a study of 130 cases from the U.S. beryllium case registry. Hum. Pathol. 1:25-44. Haley, P. J., Finch, G. L., Mewhinney, J. A., Harmsen, A. G., Hahn, F. F., Hoover, M. D., Muggenburg, B. A., and Bice, D. E. 1989. A canine model of beryllium-induced granulomatous lung disease. Lab. Invest. 61:219-227. Hanifin, J. M., Epstein, W. L., and Cline, M. J. 1970. In vitro studies of granulomatous hypersensitivity to beryllium. J. Invest. Dermatol. 55:284-288. Harmsen, A. G., Hoover, M. D., and Seiler, F. A. 1984. Health risk implications of using beryllium in fusion reactors. J. Nucl. Mater. 122 & 123:821-826. Hart, B. A., and Pittman, D. G. 1980. The uptake of beryllium by the alveolar macrophage. J. Reticuloendothel. Soc. 27:49-58. Hoover, M. D., Castorina, B. T., Finch, G. L., and Rothenberg, S. J. 1989. Determination of the oxide layer thickness on beryllium metal particles. Am. Ind. Hyg. J. 50:550-553. Hoover, M. D., Eidson, A. F., Mewhinney, J. A., Finch, G. L., Greenspan, B. J., and Cornell, C. A. 1988. Generation and characterization of respirable beryllium oxide aerosols for toxicity studies. Aerosol Sci. Technol. 9:83-92. Johnson, N. R. 1983. Beryllium disease among workers in a spacecraft manufacturing plantCalifornia. Morbid. Mortal. Weekly Rep. 32:419-420. Kang, K., Bice, D., Hoffmann, E., D'Amato, R., and Salvaggio, J. 1977. Experimental studies of sensitization to beryllium, zirconium, and aluminum compounds in the rabbit. J. Allergy Clin. Immunol. 59:425-436. Kang, K., and Salvaggio, J. 1976. Effects of asbestos and beryllium compounds on the alveolar macrophages. Med. J. Osaka Univ. 27:47-58. Kriebel, D., Brain, J., Sprince, N., and Kazemi. 1988. The pulmonary toxicity of beryllium. Am. Rev. Respir. Dis. 137:464-473. Marafante, E., Lundborg, M., Vahter, M., and Camner, P. 1987. Dissolution of two arsenic compounds by rabbit alveolar macrophages in vitro. Fundam. Appl. Toxicol. 8:382-388. Mercer, T. T. 1967. On the role of particle size in the dissolution of lung burdens. Health Phys. 13:1211-1221. Mueller, H.-L., Guilmette, R. A., and Muggenburg, B. A. 1989. Uptake of inert particles by dog
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alveolar macrophages in vitro—A comparison of monolayer and suspension techniques. J. Appl. Toxicol. 9:135-143. Mueller, H.-L., Hotz, G., Seidel, A., Thiele, H., and Ray, I. L. 1986. Uptake of uranium-plutonium mixed-oxides by alveolar macrophages in vivo and in vitro: Electron microscopic studies. In Aerosols: Formation and Reactivity, pp. 311-317. Oxford: Pergamon Journals. Muggenburg, B. A., and Mauderly, J. L. 1975. Lung lavage using a single-lumen endotracheal tube. J. Appl. Physiol. 25:471-475. Nilsen, A., Nyberg, K., and Camner, P. 1988. Intra-phagolysosomal pH in alveolar macrophages after phagocytosis in vivo and in vitro of fluorescein-labeled yeast particles. Exp. Lung Res. 14:197207. Robinson, A. V, and Schneider, R. P. 1980. Phagocytosis, toxicity and solubility of AmO2 in alveolar macrophages. In Pulmonary Toxicology of Respirable Particles, eds. C. L. Sanders and F. T. Cross, CONF-791002, pp. 325-337. Rothenberg, S. J., DeNee, P. B., Cheng, Y. S., Hanson, R. L., Yeh, H. C., and Eidson, A. F. 1982. Methods for the measurement of surface areas of aerosols by absorption. Adv. Colloid Interface Sci. 15:223-249. Skilleter, D. N., and Paine, A. J. 1979. Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake. Chem. Biol. Interact. 24:19-33. Skilleter, D. N., and Price, R. J. 1981. Effects of beryllium compounds on rat liver kupffer cells in culture. Toxicol. Appl. Pharmacol. 59:279-286. Witschi, H. P., and Aldridge, W. N. 1968. Uptake, distribution and binding of beryllium to organelles of the rat liver cell. Biochem. J. 106:811-820. Received February 11, 1991 Accepted July 4, 1991
