ENVIRONMENTALRESEARCH54, 24--38 (1991)
Lung Lesions after Combined Inhalation of Cobalt and Nickel ANNE JOHANSSON*'t'~ TORE CURSTEDT,§ AND PER CAMNER*'t *Section of Lung Medicine, Unit of Environmental Medicine, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden; ~Department of Environmental Hygiene, Karolinska Institute, Stockholm, Sweden; eThe Wenner-Gren Institute, Biology Building, University of Stockholm, Stockhohn, Sweden; and §Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden
Received December 14, 1989 Rabbits inhaled 0.5 mg/m3 Co2+ a s CoCl2, a combination of 0.5 mg/m3 Co2+ as CoC12 and 0.5 mg/m3 of Ni2+ as NiCI2, or filtered air (controls) for 4 months, 5 days/week, 6 hr/day. The pattern of morphological effects on lung tissue and alveolar macrophages after the simultaneous exposure to Co2+ and Ni2+ was a combination of the patterns seen after exposure to Co2+ and Niz+ alone. However, nickel seemed to potentiate the specific effect of cobalt, i.e., the formation of noduli of type II cells. Further, the increase in phospholipids, especially in 1,2-dipalmitoylphosphatidylcholine, appeared more pronounced after the combined exposure than after the additive combination. © 1991AcademicPress,Inc.
INTRODUCTION Cobalt is regarded as the main causative factor for hard-metal p n e u m o c o n i o s i s (Coates and Watson, 1971; K i t a m u r a et al., 1978; H a r t u n g et aI., 1982; Balmes, 1987). In the process of hard-metal production, c e m e n t e d tungsten carbide with cobalt as a binder is used. Also titanium, tantalum, chromium, and nickel are sometimes added. Fibrotic changes in the lung have b e e n d e m o n s t r a t e d in animals after cobalt exposure. W e h n e r et al. (1977) e x p o s e d Syrian hamsters to 10 mg/m 3 o f C o O for their life span and found pneumoconiosis. K e r f o o t et al. (1975) found d e c r e a s e d lung compliance and increased content of collagen, elastic tissue, and fibroblasts in miniature swine e x p o s e d for 3 months to 0.1-1 mg/m 3 of cobalt metal powder. Tracheal injection in rats of c e m e n t e d tungsten carbide dust caused p a t c h y fibrosis after 6 months ( K i t a m u r a et al., 1980). The m o s t striking effect after e x p o s u r e of rabbits for 1--4 months to soluble cobalt as COC12, 0.4-2.0 mg/m 3, was a nodular growth pattern of the alveolar type II cells (Johansson et al., 1984, 1987a). T h e r e was also an increased n u m b e r of alveolar m a c r o p h a g e s in the lavage fluid (Johansson et al., 1983a, 1986a). A minor portion of these cells were large and engorged with suffactant-like laminated inclusions. This portion p r o b a b l y e m a n a t e d f r o m areas around the type I I cell nodules. Giant cells were found in lavage f r o m hard-metal w o r k e r s (Davidson et al., 1983; T a b a t o w s k i et al., 1988). Inhalation of metallic as well as soluble nickel caused a general increase in n u m b e r and size of alveolar type I I cells and an increase in surfactant in rabbits (Johansson and Camner, 1980; J o h a n s s o n et al., 1981, 1983b; Casarett-Bruce et aI., 1981; Curstedt et al., 1983, 1984). Secondary effects on the alveolar macro24 0013-9351/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
LUNG LESIONS AFTER COBALT AND NICKEL INHALATION
25
phages by the increase in surfactant were seen: increased number of surfactantlike inclusions, altered cell surfaces, and increased metabolic activity (Camner et al., 1978; johansson et al., 1980; Wiernik et al., 1983). The effect pattern is similar to that seen in rats following exposure to quartz dust (Corrin and King, 1969; Heppleston et al., 1970) and in the human disease pulmonary alveolar proteinosis (Rosen et al., 1958). The aim of the present study was to investigate alveolar tissue and alveolar macrophages in rabbits following combined exposure to cobalt, which causes a nodular growth of alveolar type II cells, and to nickel, which causes a general increase in type II cells with concomitant increase in surfactant. The exposure time of 4 months and the concentration 0.5 mg/m 3 of cobalt and nickel were chosen because these parameters had been used in earlier exposures. MATERIALS AND METHODS
Design and Exposure Twenty-four male rabbits, weighing 3-3.5 kg, were divided into three groups of eight rabbits each. The rabbits were exposed for 4 months, 5 days/week, and 6 hr/day to 0.5 +- 0.4 mg/m 3 (mean -+ SD) cobalt a s C o C I 2 (group Co), to 0.5 +- 0.4 mg/m 3 cobalt as CoC12 together with 0.5 -+ 0.4 mg/m 3 nickel as NiCI2 (group Co + Ni), or to filtered air (control group). Aerosols were produced with an ultrasonic nebulizer (DeVilbiss 35B). The mass median aerodynamic diameter was about 1 ~m (GSD 2.2) as measured with an impactor (Mitchell and Pilcher, 1959). Metal concentration was estimated by sucking air through a filter (Satorius, 100 N, pore size 0.8 p~m) and by analyzing the metal deposited on the filter with atomic absorption spectrophotometry (Varian AA6). After the exposure period the rabbits were killed by an overdose of sodium pentobarbital and their lungs excised. The upper left lung lobe was used for light microscopy. From the left lower lobe three pieces, 1-2 ~m 3 each, were taken for electron microscopic examination, and the remainder for phospholipid analysis. The right lung was lavaged and the cells were collected and studied with light and electron microscopy. To avoid biased evaluation all rabbits were coded prior to examination.
Light Microscopy Routine paraffin sections were prepared from the lung tissue, stained with hematoxylin and eosin, and studied with conventional light microscopy. Particular attention was paid to the growth pattern of type II cells, inflammatory lesions, and alveolar macrophages. Macrophage concentration in lavage fluid was measured in a B~irker chamber.
Transmission Electron Microscopy Pieces of lung tissue as well as alveolar macrophages were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, postfixed in 1% OsO4 in the same buffer, dehydrated through a series of graded alcohol, and embedded in Polarbed
26
JOHANSSON, CURSTEDT, AND CAMNER
812 (Polaron). Thin sections were examined in a Jeol I00 S electron microscope. Lung tissue was studied with reference to the growth pattern of alveolar epithelial type II ceils. The volume density of the type II cells was estimated from 21 randomly selected fields from each rabbit. Pictures were taken at a primary magnification of 1000 and morphometrical measurements were performed with a digitizer (Hipad) connected to a computer (Heathkit H 11). Areas occupied by type II cells (An) were compared with areas occupied with alveolar tissue (AT). The ratio An/A T (volume density) was estimated for each rabbit. The macrophages were investigated with specific attention paid to content of laminated inclusions (ingested surfactant) and cell surface. About 200 macrophages from each rabbit were scored.
Scanning Electron Microscopy Due to the limited number of cells obtained from lavages, scanning electron microscopy could be performed on alveolar macrophages from only three control rabbits, four rabbits from group Co, and seven rabbits from group Co + Ni. The cells were suspended in Hepes-buffered medium (Parker 199) with 15% fresh rabbit serum and allowed to settle on glass plates in Leighton tubes. The tubes were placed in an incubator, at 37°C, 5% CO2 in air, and 80% relative humidity, for 1 hr. Thereafter the cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, dehydrated in graded alcohol-acetone, and dried with the critical-point method (Anderson, 1951). The glass plates with macrophages were
FIG. 1. Histological findings in a rabbit exposed to C o 2+ and N i 2 + . Alveoli filled are with enlarged macrophages and groups of alveolar type II cells (arrowhead) associated with inflammatory reaction. H&E, x460.
LUNG LESIONS AFTER COBALT AND NICKEL INHALATION
27
FIG. 2. Interstitial as well as intraalveolar inflammatory reaction with accumulations of eosinophils
and neutrophils in a rabbit e x p o s e d to Co 2+ and Ni 2+ . H & E , x200.
mounted on carbon plates and coated with gold. The specimens were examined in a Jeol 1200 EX electron microscope equipped with an EM-ASID 10 scanning device. About 200 cells per rabbit were examined with reference to surface structures and contact with glass surface.
FIG. 3. N o r m a l histological appearance of lung p a r e n c h y m a from a control rabbit. H & E , x200.
28
JOHANSSON, CURSTEDT, AND CAMNER
Analysis o f Phospholipids The left lower lung lobes were homogenized in chloroform/methanol (2:1, v/v). After filtration 0.2 vol. of 0.58% NaC1 in water was added (Folch et al., 1957). The lower phase was evaporated to dryness and the phospholipids in the extract were isolated by liquid-gel chromatography on Lipidex-5000 (Packard Instruments Co, Downers Grove, IL) in the solvent system methanol:ethylene chloride 4:1 (v/v). The amount of phospholipids were determined according to Bartlett (1959). After addition of 1,2-dimyristoylphosphatidylcholine as internal standard, phosphatidylcholines were isolated by anionic exchange chromatography and thin-layer chromatography (Curstedt et al., 1983). The phosphatidylcholines were hydrolyzed with phospholipase C and the formed 1,2-diacylglycerols were determined to be trimethylsilyl ethers by capillary gas-liquid chromatography (Johansson et al., 1984). Statistical Methods Morphological data were evaluated with the Mann-Whitney U test and phospholipid data with the t test. Level of significance was 0.05 without prediction. RESULTS
Gross Findings All rabbits in group Co + Ni had red or black spots on the surface of their lungs
FIG. 4. Nodular aggregation of type II cells from a rabbit exposed to C o 2÷ and Ni 2+. Individual cells contain numerous lamellar bodies (LB) and dilated endoplasmic reticululm (arrowhead). Bar = 5 I~m.
L U N G LESIONS A F T E R COBALT A N D N I C K E L I N H A L A T I O N
29
and/or white subpleural nodules. The rabbits in group Co exhibited similar but less pronounced reactions. The lungs from control rabbits were unremarkable. The left lower lung lobe was significantly heavier in group Co + Ni, 3.7 -+ 0.6 g (mean -+ SD), then in group Co, 2.9 + 0.5 g (P < 0.01), or in the control group, 2.7 -+ 0.8 g (P < 0.02).
Lung Tissue Light microscopic findings. All animals in group Co + Ni showed areas of nodular aggregation of alveolar type II cells and accumulation of enlarged vacuolated macrophages. Several adjacent alveoli were filled with such cells (Fig. 1). These lesions were often combined with interstitial inflammatory changes including lymphocytic infiltration and intraalveolar accumulation of neutrophils and eosinophils (Fig. 2). Two of the animals in group Co showed a similar, but less prominent, reaction, while the other six rabbits and the controls had occasional focal inflammatory lesions but were essentially unremarkable (Fig. 3). Ultrastructural findings. As in previous experiments where rabbits were exposed to cobalt many of the alveolar type II cells were aggregated in noduli (Fig. 4). These aggregates were often larger in lungs from rabbits in group Co + Ni than in lungs from group Co. Some of these type II cells were large and engorged with lamellar bodies and had dilated endoplasmic reticula (Fig. 4). Also immature type II cells were found as well as type II cells with a moderate number of lamellar bodies, profiles of endoplasmic reticulum, and mitochondria, i.e., cells which appeared like most type II cells from control rabbits (Fig. 5).
FIG. 5. Alveolar type II cell from a control rabbit; LB, lamellar bodies. Bar = 2 ~tm.
30
JOHANSSON, CURSTEDT, AND CAMNER TABLE 1 VOLUME DENSITY OF ALVEOLAR EPITHELIAL TYPE II CELLS IN CONTROLS, GROUP Co, AND GROUP Co + Ni
Controls
Group Co
Group Co + Ni
0.040 0.045 0.040 0.055 0.050 0.037 0.048 0.046 0.046 0~045
0.164 0.039 0.046 0.040 0.053 0.034 0.035 0.098 0.064 0.054
0.072 0.088 0.056 0.084 0.109 0.083 0.075 0.084 0.081 0.080
Mean Geometric mean
The volume density of the type II cells was significantly higher in group Co + Ni than in the control group (P < 0.001), but not than in group Co (P > 0.05) (Table 1).
Alveolar Macrophages Light microscopicfindings. Significantly more macrophages were obtained by lavage from rabbits in group Co + Ni than from controls or rabbits in group Co (Table 2). The difference between group Co and the control group was not significant. Ultrastructuralfindings. In all three rabbit groups the vast majority of the cells in the lavage fluid were macrophages (Table 2). In the group exposed to cobalt and nickel there was a slight, but significant, increase in the percentage of neutrophils compared to controls. Cobalt alone caused a slight increase in macrophages containing surfactant-like TABLE 2 NUMBER AND DISTRIBUTION OF CELLS RECOVERED BY LAVAGE FROM LUNGS OF CONTROL RABBITS, GROUP Co, AND GROUP Co + Ni
Group
Number of cells (millions)
Macrophages
Monocytes
(%)
(%)
Lymphocytes
Neutrophils
Eosinophils
Controls 14 -+ 5
98.3 +-- 1.4
1.1 + 1.1
0.4 +- 0.5
0.3 +- 0.4
0
Co (n = 8)
(n = 8)
24 --+ 20
98.4 -+ 1.4
0.7 -+ 0.9
0.4 -+ 0.3
0.4 -+ 0.4
0.1 -+ 0.2
Co + Ni (n = 8)
70 -+ 22
97.4 -+ 1.5
0.5 +- 0.6
0.3 +- 0.3
1.6 z 1.2
0.3 +-- 0.4
(1) xx
(1) xx
(2) xx
(2) x
Note. Data are given as means -+ SD. (1) Compared to controls; (2) compared to group Co: x, P < 0.05; xx, P < 0.01.
LUNG
LESIONS
AFTER
COBALT
AND
NICKEL
INHALATION
31
TABLE 3 ULTRASTRUCTURAL DATA ON MACROPHAGES OBTAINED BY LAVAGE
Group Controls (n = 8) Co (n = 8) Co + Ni (n = 8)
Lamellar inclusions/cell profile
General impression abnormal (%)
0-3 (%)
4-10 (%)
>10 (%)
Active (%)
Normal (%)
Smooth (%)
Large Golgi complex (%)
13-+ 9
84-+ 10
15-+ 8
2-+ 2
6-+4
92-+ 4
2-+ 1
21 -+ 8
22-+ 17
76-+ 14
17+-8
7-+7 (1) x
7-6
85-+ 13
8-+9
28-+ 13
73-+ 14 (1) xxx (2) xxx
57-+ 14 (1) xxx (2) x
17-+ 5
29-+ 14
40-+ 15 (1) xxx (2) xxx
36-+7 (1) xx
26 + 16 (1) xxx (2) xx
Cell surface
31 -+ 16 (1) xxx (2) xxx
Note. Data are given as means -+ SD. (1) Compared to controls; (2) compared to group Co: x, P < 0.05; xx, P < 0.01; xxx, P < 0.001.
FIG. 6. A l v e o l a r m a c r o p h a g e f r o m a r a b b i t e x p o s e d t o C o 2+ a n d N i 2+ . T h e c y t o p l a s m is filled w i t h l a m i n a t e d , s u r f a c t a n t - l i k e i n c l u s i o n s (LI) a n d t h e s u r f a c e is s m o o t h . B a r = 2 ~ m .
32
JOHANSSON, CURSTEDT, AND CAMNER
laminated inclusions (Table 3). Exposure to cobalt and nickel resulted in a marked increase in macrophages with more than 10 laminated inclusion profiles per cell profile. The high amount of surfactant-like inclusion was often combined with a smooth cell surface (Fig. 6) or a surface covered by short microvilli. About 40% of the macrophages from rabbits in group Co + Ni had a smooth surface and about 30% had a surface rich in thin villi and protrusions (Fig. 7) (Table 3). These figures differed significantly from those of controls or rabbits in group Co. The percentage of macrophages with enlarged Golgi complexes was significantly higher in group Co + Ni than in the control group but not higher than in group Co (Table 3). In control rabbits and in rabbits in group Co most macrophages appeared unremarkable (Fig. 8) while most macrophages from rabbits in group Co + Ni showed one or several of the above-described alterations (Table 3). Scanning electron microscopy revealed that a higher percentage of the macrophages from rabbits in group Co + Ni had an active villous surface compared to macrophages from controls which expressed broad lamellipodia (Fig. 9) (Table 4). Macrophages from both exposed groups tended to be more ball-shaped than macrophages from controls; i.e., they did not spread out on the glass surface and often appeared in clusters (Fig. 10). Relatively few large and smooth cells were found. (A probable explanation is that these cells were loosely bound to the glass surface and fell off during the washing and fixation.)
Fro. 7. Alveolar m a c r o p h a g e from a rabbit e x p o s e d to C o 2+ and Ni a+ . The cell surface is covered with short, thin microvilli and the cell is phagocytizing surfactant (arrow). Bar = 2 ~m.
L U N G LESIONS A F T E R COBALT A N D N I C K E L I N H A L A T I O N
33
FIG. 8. Alveolar macrophage from a control rabbit. The cell has numerous vesiculated lysosomes (L). Bar = 2 p~m.
Lung Phospholipids Table 5 shows the concentrations of total phospholipids, phosphatidylcholines, and 1,2-dipalmitoylphosphatidylcholine in the unlavaged lung tissue. All concentrations were markedly and significantly higher for group Co + Ni than for group Co and the control group. Only the concentration of !,2-dipalmitoylphosphatidylcholine was significantly higher in group Co than in the controls. DISCUSSION The effects on lung macrophages, lung morphology, and lung phospholipids (present study) were compared in groups of rabbits exposed to a combination of Co 2+ and Ni 2+, to C o 2+ only, and to filtered air. Because of limited number of exposure chambers we were not able to include a group exposed to Ni 2+ only. However, we have recently exposed rabbits for 4 months to 0.6 mg/m3 Ni 2+ only (Johansson et al., 1989). The pulmonary toxicity of cobalt dust from the processing of hard metal is suggested to be related to its relatively high solubility in protein-containing fluids (Harding, 1950). The effects after exposure to soluble cobalt are therefore likely to be similar to the effects after exposure to dust from the processing of hard metal.
34
JOHANSSON,
CURSTEDT,
AND CAMNER
FIG. 9. Alveolar macrophage from a control rabbit. The cell expresses broad lamellipodia and is tightly bound to the glass surface. Bar = 5 Ixm.
Earlier studies in which rabbits inhaled soluble cobalt have shown that the first and most prominent effects were abnormal clustering of the alveolar epithelial type II cells and changed appearance of these cells (Johansson et al., 1984, 1987a). This reaction seems to be specific to cobalt as it was not seen after inhalation of nickel, cadmium, trivalent and hexavalent chromium, manganese, copper, or lithium (Johansson et al., 1981, 1983b, 1984, 1986b, 1987b, 1988a). Group Co + Ni showed more pronounced effects on most lung tissue and alveolar macrophage parameters than group Co. In Table 6 the data on lung weight, volume density of type II cells, and lung phospholipids in earlier studies with single exposures to nickel and cobalt of similar exposure periods and metal TABLE 4 SCANNING ELECTRON MICROSCOPIC DATA ON MACROPHAGES OBTAINED BY LAVAGE
Group
Villous cell surface
Cells with broad lamellipodia
Ball-shaped appearance
(%)
(%)
(%)
Controls (n = 3) Co (n = 4) Co + Ni (n = 7)
19 ± 4
74 ± lO
24±
25 ± 11
50 ± 33
40 ± 33
56 ± 18
35 ± 18
50±
Note. Data are given as means -+ SD.
24
18
L U N G LESIONS A F T E R C O B A L T A N D N I C K E L I N H A L A T I O N
35
FIG. 10. Alveolar m a c r o p h a g e s from a rabbit e x p o s e d to Co 2+ and Ni 2+. The cells stick to each other, are covered with short microvilli, and are not spread over the glass surface. Bar = 5 Ixm.
concentrations are compared with the data from the present study. The effects on lung weight and volume density of type II cells in group Co + Ni are apparently a combination of the effects in the single exposures. H o w e v e r , the formation of noduli of alveolar type II cells, which are not induced by nickel (Johansson et al., 1983b, 1989), appeared more pronounced in group Co + Ni than in group Co. Further, the increase in phospholipids in group Co + Ni seems more pronounced than just an additive combination. Co 2 + and Ni 2+ separately only increased the TABLE 5 TOTAL PHOSPHOLIPIDS, PHOSPHATIDYLCHOLINE, AND 1,2-DIPALMITOYLPHOSPHATIDYLCHOLINE IN UNLAVAGED LUNG TISSUE
Group Controls (n = 7) a Co (n = 8) Co + Ni (n = 8)
Phospholipids 0xmole/g lung)
Phosphatidylcholine 0xmole/g lung)
1,2-dipalmitoylphosphatidylcholine (ixmole/g lung)
14.6 -+ 1.0
6.9 -+ 0.8
2.4 --_ 0.4
16.9 - 3.4
9.1 -+ 2.5
3.6 +-- 1.4 (1) x
33.2 --_ 13.2
19.2 _+ 9.2
8.3 ± 4.8
(1) xx (2) xx
(1) xx (2) x
(1) xx (2) x
Note. (1) C o m p a r e d to controls; (2) C o m p a r e d to group Co: x, P < 0.05; xx, P < 0.01. a Sample from one rabbit lost due to technical error.
36
JOHANSSON, CURSTEDT, AND CAMNER TABLE 6
RATIOS BETWEEN METAL-ExPOSED AND CONTROL RABBITS CONCERNING LUNG WEIGHT, VOLUME DENSITY OF TYPE II CELLS, AND LUNG PHOSPHOLIPIDS IN THE PRESENT STUDY AND IN EARLIER STUDIES WITH SIMILAR EXPOSURE SITUATIONS
Metal conch. (mg/m 3)
Lung weight
Volume density of type II cells
Phospholipids/g wet lung
Phosphatidylcholines/g wet lung
Study
0.5 Co 2+ -~ 0.5 Ni 2+
1.4
1.8
2.3
2.8
Present
0.5 Co 2 +
1.2 1.0
1.2
1.3
0.4 Co 2+
1. l 1.0
2.0 Co 2+
1.3
1.5
0.6 Ni 2+
1.5
2.5
1.2
1.2
Present Johansson et al., 1987 Johansson et al., 1987 Johansson et al., 1989
phosphatidylcholines by about 30 and 20%, respectively, whereas the combination of Co ~+ and Ni 2+ caused an increase of 180% (Table 6). Nickel thus appears to potentiate the effect on type II cells induced by cobalt. A comparison of the data on alveolar macrophages in the present study with earlier data after inhalation of nickel alone (Johansson et al., 1983b) indicates that the effects in group Co + Ni are addititive combinations of single exposures of cobalt and nickel. The concentrations of cobalt and nickel in the present study were not far from the occupational threshold limit values, which are 0.05 mg/m 3 for cobalt and 0.1 mg/m 3 for nickel in Sweden. Nickel is, in addition to cobalt, sometimes added to hard metal (Balmes, 1987). As nickel potentiated the specific effect of cobalt, that is the formation of type II cell noduli and the increase in surfactant, combined exposure to cobalt and nickel seems to be more harmful than exposure to each of the metals separately.
ACKNOWLEDGMENTS We are grateful for the technical assistance from Ms. Elin Arvesen, Ms. Inger Granell, Ms. Kristina Nyberg, and Ms. Karin Widtski61d-Olsson. This study was supported by a grant from the Swedish Work and Environmental Foundation.
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Corrin, B., and King, E. (1969). Experimental endogenous lipid pneumonia and silicosis. J. Pathol. 97, 325-330. Curstedt, T. Casarett-Bruce, M., and Camner, P. (1984). Changes in glycerophosphatides and their ether analogs in lung lavage of rabbits exposed to nickel dust. Exp. Mol. Pathol. 41, 26-34. Curstedt, T., Hagman, M., Robertson, B., and Camner, P. (1983). Rabbit lungs after long-term exposure to low nickel dust concentration. I. Effects on phospholipid concentration and surfactant activity. Environ. Res. 30, 89-94. Davidson, A. G., Haslam, P. L., Coutts, I. I., Dewar, A., Ricling, W. D., Studdy, P. R., and Newman-Taylor, A. J. (1983). Interstitial lung disease and asthma in hard-metal workers: Bronchoalveolar lavage, ultrastructural, and analytical findings and results of bronchial provocation test. Thorax 38, 119-128. Folch, J., Lees, M., and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509. Harding, H. E. (1950). Notes on the toxicology of cobalt metal. Brit. J. Ind. Med. 7, 76--78. Hartung, M., Schaller, K. H., and Brand, E. (1982). On the question of the pathogenetic importance of cobalt for hard metal fibrosis of the lung. Int. Arch. Environ. Health 50, 53-57. Heppleston, A. G., Wright, N. A., and Steward, J. A. (1970). Experimental alveolar lipoproteinosis following the inhalation of silica. J. Pathol. 101, 293-307. Johansson, A., and Camner, P. (1980). Effects of nickel dust on rabbit alveolar epithelium. Environ. Res. 22, 510-516. Johansson, A., Camner, P., Jarstrand, C., and Wiernik, A. (1980). Morphology and function of alveolar macrophages after long-term nickel exposure. Environ. Res. 23, 170-180. Johansson, A., Camner, P., and Robertson, B. (1981). Effects of long-term nickel dust exposure on rabbit alveolar epithelium. Environ. Res. 25, 391--403. Johansson, A., Camner, P., Jarstrand, C., and Wiernik, A. (1983a). Rabbit alveolar macrophages after inhalation of soluble cadmium, copper, and cobalt: A comparison with the effect of soluble nickel. Environ. Res. 31, 340-354. Johansson, A., Curstedt, T., Robertson, B., and Camner, P. (1983b). Rabbit lung after inhalation of soluble nickel. II. Effects on lung tissue and phospholipids. Environ. Res. 31, 399-412. Johansson, A., Curstedt, T., Robertson, B., and Camner, P. (1984). Lung morphology and phospholipids after experimental inhalation of soluble cadmium, copper, and cobalt. Environ. Res. 34, 295-309. Johansson, A., Lundborg, M., Wiernik, A., Jarstrand, C., and Camner, P. (1986a). Rabbit alveolar macrophages after long-term inhalation of soluble cobalt. Environ. Res. 41, 488-496. Johansson, A., Robertsou, B., Curstedt, T., and Camner, P. (1986b). Rabbit lung after inhalation of hexa- and trivalent chromium. Environ. Res. 41, 110-119. Johansson, A., Robertson, B., and Camner, P. (1987a). Nodular accumulation of type II cells and inflammatory lesions caused by inhalation of low cobalt concentrations. Environ. Res. 43, 243277. Johansson, A., Robertson, B., Curstedt, T., and Camner, P. (1987b). Alveolar macrophage abnormalities in rabbits exposed to low concentrations of trivalent chromium. Environ. Res. 44, 279293. Johansson A., Camner, P., Curstedt, T., Jarstrand, C., Robertson, B., and Urban, T. (1988a). Rabbit lung after inhalation of lithium chloride. J. Appl. Toxicol. 8, 373-375. Johansson, A., Wiernik, A., Lundborg, M., Jarstrand, C., and Camner, P. (1988b). Alveolar macrophages in rabbits after combined exposure to nickel and trivalent chromium. Environ. Res. 46, 120-132. Johansson, A., Curstedt, T., Robertson, T., and Camner, P. (1989). Lung lesions after experimental combined exposure to nickel and trivalent chromium. Environ. Res. 50, 103-119. Kerfoot, E. J., Fredrick, W. G., and Domeier, E. (1975). Cobalt metal inhalation on miniature swine. Amer. Ind. Hyg. Assoc. J. 36, 17-25. Kitamura, H., Kitamura, H., Tozawa, T., and Kimula, Y. (1978). Cemented tungsten carbide pneumoconiosis. Acta Pathol. Japon. 28, 921-935. Kitamura, H., Yoshimuva, Y., Tozawa, T., and Koshi, K. (1980). Effects of cemented tungsten
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JOHANSSON, CURSTEDT, AND CAMNER
carbide dust on rat lungs following intratracheal injection of saline suspension. Acta Pathol. Japon. 30, 241-253. Mitchell, R. I., and Pilcher, J. M. (1959). Improved cascade impactor for measuring aerosol particle sizes. Ind. Eng. Chem. 51, 103%1042. Rosen, S. H., Castleman, B., and Liebow, A. A. (1958). Pulmonary alveolar proteinosis. N. Engl. J. Med. 258, 1123-1142. Tabatowski, U., Roggli, V. L., Fulkerson, W. J., Langley, R. L., Benning, T., and Johnston, W. W. (1988). Giant cell interstitial pneumonia in a hard-metal worker. Acta Cytol. 32, 240-246. Wehner, A. P., Busch, R. H., Olson, R. J., and Craig, D. K. (1977). Chronic inhalation of cobalt oxide and cigarette smoke by hamsters. Amer. Ind. Hyg. Assoc. J. 38, 338-346. Wiernik, A., Johansson, A., Jarstrand, C., and Camner P. (1983). Rabbit lung after inhalation of soluble nickel. I. Effects on alveolar macrophages. Environ. Res. 30, 12%141.
Lung Lesions after Combined Inhalation of Cobalt and Nickel ANNE JOHANSSON*'t'~ TORE CURSTEDT,§ AND PER CAMNER*'t *Section of Lung Medicine, Unit of Environmental Medicine, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden; ~Department of Environmental Hygiene, Karolinska Institute, Stockholm, Sweden; eThe Wenner-Gren Institute, Biology Building, University of Stockholm, Stockhohn, Sweden; and §Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden
Received December 14, 1989 Rabbits inhaled 0.5 mg/m3 Co2+ a s CoCl2, a combination of 0.5 mg/m3 Co2+ as CoC12 and 0.5 mg/m3 of Ni2+ as NiCI2, or filtered air (controls) for 4 months, 5 days/week, 6 hr/day. The pattern of morphological effects on lung tissue and alveolar macrophages after the simultaneous exposure to Co2+ and Ni2+ was a combination of the patterns seen after exposure to Co2+ and Niz+ alone. However, nickel seemed to potentiate the specific effect of cobalt, i.e., the formation of noduli of type II cells. Further, the increase in phospholipids, especially in 1,2-dipalmitoylphosphatidylcholine, appeared more pronounced after the combined exposure than after the additive combination. © 1991AcademicPress,Inc.
INTRODUCTION Cobalt is regarded as the main causative factor for hard-metal p n e u m o c o n i o s i s (Coates and Watson, 1971; K i t a m u r a et al., 1978; H a r t u n g et aI., 1982; Balmes, 1987). In the process of hard-metal production, c e m e n t e d tungsten carbide with cobalt as a binder is used. Also titanium, tantalum, chromium, and nickel are sometimes added. Fibrotic changes in the lung have b e e n d e m o n s t r a t e d in animals after cobalt exposure. W e h n e r et al. (1977) e x p o s e d Syrian hamsters to 10 mg/m 3 o f C o O for their life span and found pneumoconiosis. K e r f o o t et al. (1975) found d e c r e a s e d lung compliance and increased content of collagen, elastic tissue, and fibroblasts in miniature swine e x p o s e d for 3 months to 0.1-1 mg/m 3 of cobalt metal powder. Tracheal injection in rats of c e m e n t e d tungsten carbide dust caused p a t c h y fibrosis after 6 months ( K i t a m u r a et al., 1980). The m o s t striking effect after e x p o s u r e of rabbits for 1--4 months to soluble cobalt as COC12, 0.4-2.0 mg/m 3, was a nodular growth pattern of the alveolar type II cells (Johansson et al., 1984, 1987a). T h e r e was also an increased n u m b e r of alveolar m a c r o p h a g e s in the lavage fluid (Johansson et al., 1983a, 1986a). A minor portion of these cells were large and engorged with suffactant-like laminated inclusions. This portion p r o b a b l y e m a n a t e d f r o m areas around the type I I cell nodules. Giant cells were found in lavage f r o m hard-metal w o r k e r s (Davidson et al., 1983; T a b a t o w s k i et al., 1988). Inhalation of metallic as well as soluble nickel caused a general increase in n u m b e r and size of alveolar type I I cells and an increase in surfactant in rabbits (Johansson and Camner, 1980; J o h a n s s o n et al., 1981, 1983b; Casarett-Bruce et aI., 1981; Curstedt et al., 1983, 1984). Secondary effects on the alveolar macro24 0013-9351/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
LUNG LESIONS AFTER COBALT AND NICKEL INHALATION
25
phages by the increase in surfactant were seen: increased number of surfactantlike inclusions, altered cell surfaces, and increased metabolic activity (Camner et al., 1978; johansson et al., 1980; Wiernik et al., 1983). The effect pattern is similar to that seen in rats following exposure to quartz dust (Corrin and King, 1969; Heppleston et al., 1970) and in the human disease pulmonary alveolar proteinosis (Rosen et al., 1958). The aim of the present study was to investigate alveolar tissue and alveolar macrophages in rabbits following combined exposure to cobalt, which causes a nodular growth of alveolar type II cells, and to nickel, which causes a general increase in type II cells with concomitant increase in surfactant. The exposure time of 4 months and the concentration 0.5 mg/m 3 of cobalt and nickel were chosen because these parameters had been used in earlier exposures. MATERIALS AND METHODS
Design and Exposure Twenty-four male rabbits, weighing 3-3.5 kg, were divided into three groups of eight rabbits each. The rabbits were exposed for 4 months, 5 days/week, and 6 hr/day to 0.5 +- 0.4 mg/m 3 (mean -+ SD) cobalt a s C o C I 2 (group Co), to 0.5 +- 0.4 mg/m 3 cobalt as CoC12 together with 0.5 -+ 0.4 mg/m 3 nickel as NiCI2 (group Co + Ni), or to filtered air (control group). Aerosols were produced with an ultrasonic nebulizer (DeVilbiss 35B). The mass median aerodynamic diameter was about 1 ~m (GSD 2.2) as measured with an impactor (Mitchell and Pilcher, 1959). Metal concentration was estimated by sucking air through a filter (Satorius, 100 N, pore size 0.8 p~m) and by analyzing the metal deposited on the filter with atomic absorption spectrophotometry (Varian AA6). After the exposure period the rabbits were killed by an overdose of sodium pentobarbital and their lungs excised. The upper left lung lobe was used for light microscopy. From the left lower lobe three pieces, 1-2 ~m 3 each, were taken for electron microscopic examination, and the remainder for phospholipid analysis. The right lung was lavaged and the cells were collected and studied with light and electron microscopy. To avoid biased evaluation all rabbits were coded prior to examination.
Light Microscopy Routine paraffin sections were prepared from the lung tissue, stained with hematoxylin and eosin, and studied with conventional light microscopy. Particular attention was paid to the growth pattern of type II cells, inflammatory lesions, and alveolar macrophages. Macrophage concentration in lavage fluid was measured in a B~irker chamber.
Transmission Electron Microscopy Pieces of lung tissue as well as alveolar macrophages were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, postfixed in 1% OsO4 in the same buffer, dehydrated through a series of graded alcohol, and embedded in Polarbed
26
JOHANSSON, CURSTEDT, AND CAMNER
812 (Polaron). Thin sections were examined in a Jeol I00 S electron microscope. Lung tissue was studied with reference to the growth pattern of alveolar epithelial type II ceils. The volume density of the type II cells was estimated from 21 randomly selected fields from each rabbit. Pictures were taken at a primary magnification of 1000 and morphometrical measurements were performed with a digitizer (Hipad) connected to a computer (Heathkit H 11). Areas occupied by type II cells (An) were compared with areas occupied with alveolar tissue (AT). The ratio An/A T (volume density) was estimated for each rabbit. The macrophages were investigated with specific attention paid to content of laminated inclusions (ingested surfactant) and cell surface. About 200 macrophages from each rabbit were scored.
Scanning Electron Microscopy Due to the limited number of cells obtained from lavages, scanning electron microscopy could be performed on alveolar macrophages from only three control rabbits, four rabbits from group Co, and seven rabbits from group Co + Ni. The cells were suspended in Hepes-buffered medium (Parker 199) with 15% fresh rabbit serum and allowed to settle on glass plates in Leighton tubes. The tubes were placed in an incubator, at 37°C, 5% CO2 in air, and 80% relative humidity, for 1 hr. Thereafter the cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, dehydrated in graded alcohol-acetone, and dried with the critical-point method (Anderson, 1951). The glass plates with macrophages were
FIG. 1. Histological findings in a rabbit exposed to C o 2+ and N i 2 + . Alveoli filled are with enlarged macrophages and groups of alveolar type II cells (arrowhead) associated with inflammatory reaction. H&E, x460.
LUNG LESIONS AFTER COBALT AND NICKEL INHALATION
27
FIG. 2. Interstitial as well as intraalveolar inflammatory reaction with accumulations of eosinophils
and neutrophils in a rabbit e x p o s e d to Co 2+ and Ni 2+ . H & E , x200.
mounted on carbon plates and coated with gold. The specimens were examined in a Jeol 1200 EX electron microscope equipped with an EM-ASID 10 scanning device. About 200 cells per rabbit were examined with reference to surface structures and contact with glass surface.
FIG. 3. N o r m a l histological appearance of lung p a r e n c h y m a from a control rabbit. H & E , x200.
28
JOHANSSON, CURSTEDT, AND CAMNER
Analysis o f Phospholipids The left lower lung lobes were homogenized in chloroform/methanol (2:1, v/v). After filtration 0.2 vol. of 0.58% NaC1 in water was added (Folch et al., 1957). The lower phase was evaporated to dryness and the phospholipids in the extract were isolated by liquid-gel chromatography on Lipidex-5000 (Packard Instruments Co, Downers Grove, IL) in the solvent system methanol:ethylene chloride 4:1 (v/v). The amount of phospholipids were determined according to Bartlett (1959). After addition of 1,2-dimyristoylphosphatidylcholine as internal standard, phosphatidylcholines were isolated by anionic exchange chromatography and thin-layer chromatography (Curstedt et al., 1983). The phosphatidylcholines were hydrolyzed with phospholipase C and the formed 1,2-diacylglycerols were determined to be trimethylsilyl ethers by capillary gas-liquid chromatography (Johansson et al., 1984). Statistical Methods Morphological data were evaluated with the Mann-Whitney U test and phospholipid data with the t test. Level of significance was 0.05 without prediction. RESULTS
Gross Findings All rabbits in group Co + Ni had red or black spots on the surface of their lungs
FIG. 4. Nodular aggregation of type II cells from a rabbit exposed to C o 2÷ and Ni 2+. Individual cells contain numerous lamellar bodies (LB) and dilated endoplasmic reticululm (arrowhead). Bar = 5 I~m.
L U N G LESIONS A F T E R COBALT A N D N I C K E L I N H A L A T I O N
29
and/or white subpleural nodules. The rabbits in group Co exhibited similar but less pronounced reactions. The lungs from control rabbits were unremarkable. The left lower lung lobe was significantly heavier in group Co + Ni, 3.7 -+ 0.6 g (mean -+ SD), then in group Co, 2.9 + 0.5 g (P < 0.01), or in the control group, 2.7 -+ 0.8 g (P < 0.02).
Lung Tissue Light microscopic findings. All animals in group Co + Ni showed areas of nodular aggregation of alveolar type II cells and accumulation of enlarged vacuolated macrophages. Several adjacent alveoli were filled with such cells (Fig. 1). These lesions were often combined with interstitial inflammatory changes including lymphocytic infiltration and intraalveolar accumulation of neutrophils and eosinophils (Fig. 2). Two of the animals in group Co showed a similar, but less prominent, reaction, while the other six rabbits and the controls had occasional focal inflammatory lesions but were essentially unremarkable (Fig. 3). Ultrastructural findings. As in previous experiments where rabbits were exposed to cobalt many of the alveolar type II cells were aggregated in noduli (Fig. 4). These aggregates were often larger in lungs from rabbits in group Co + Ni than in lungs from group Co. Some of these type II cells were large and engorged with lamellar bodies and had dilated endoplasmic reticula (Fig. 4). Also immature type II cells were found as well as type II cells with a moderate number of lamellar bodies, profiles of endoplasmic reticulum, and mitochondria, i.e., cells which appeared like most type II cells from control rabbits (Fig. 5).
FIG. 5. Alveolar type II cell from a control rabbit; LB, lamellar bodies. Bar = 2 ~tm.
30
JOHANSSON, CURSTEDT, AND CAMNER TABLE 1 VOLUME DENSITY OF ALVEOLAR EPITHELIAL TYPE II CELLS IN CONTROLS, GROUP Co, AND GROUP Co + Ni
Controls
Group Co
Group Co + Ni
0.040 0.045 0.040 0.055 0.050 0.037 0.048 0.046 0.046 0~045
0.164 0.039 0.046 0.040 0.053 0.034 0.035 0.098 0.064 0.054
0.072 0.088 0.056 0.084 0.109 0.083 0.075 0.084 0.081 0.080
Mean Geometric mean
The volume density of the type II cells was significantly higher in group Co + Ni than in the control group (P < 0.001), but not than in group Co (P > 0.05) (Table 1).
Alveolar Macrophages Light microscopicfindings. Significantly more macrophages were obtained by lavage from rabbits in group Co + Ni than from controls or rabbits in group Co (Table 2). The difference between group Co and the control group was not significant. Ultrastructuralfindings. In all three rabbit groups the vast majority of the cells in the lavage fluid were macrophages (Table 2). In the group exposed to cobalt and nickel there was a slight, but significant, increase in the percentage of neutrophils compared to controls. Cobalt alone caused a slight increase in macrophages containing surfactant-like TABLE 2 NUMBER AND DISTRIBUTION OF CELLS RECOVERED BY LAVAGE FROM LUNGS OF CONTROL RABBITS, GROUP Co, AND GROUP Co + Ni
Group
Number of cells (millions)
Macrophages
Monocytes
(%)
(%)
Lymphocytes
Neutrophils
Eosinophils
Controls 14 -+ 5
98.3 +-- 1.4
1.1 + 1.1
0.4 +- 0.5
0.3 +- 0.4
0
Co (n = 8)
(n = 8)
24 --+ 20
98.4 -+ 1.4
0.7 -+ 0.9
0.4 -+ 0.3
0.4 -+ 0.4
0.1 -+ 0.2
Co + Ni (n = 8)
70 -+ 22
97.4 -+ 1.5
0.5 +- 0.6
0.3 +- 0.3
1.6 z 1.2
0.3 +-- 0.4
(1) xx
(1) xx
(2) xx
(2) x
Note. Data are given as means -+ SD. (1) Compared to controls; (2) compared to group Co: x, P < 0.05; xx, P < 0.01.
LUNG
LESIONS
AFTER
COBALT
AND
NICKEL
INHALATION
31
TABLE 3 ULTRASTRUCTURAL DATA ON MACROPHAGES OBTAINED BY LAVAGE
Group Controls (n = 8) Co (n = 8) Co + Ni (n = 8)
Lamellar inclusions/cell profile
General impression abnormal (%)
0-3 (%)
4-10 (%)
>10 (%)
Active (%)
Normal (%)
Smooth (%)
Large Golgi complex (%)
13-+ 9
84-+ 10
15-+ 8
2-+ 2
6-+4
92-+ 4
2-+ 1
21 -+ 8
22-+ 17
76-+ 14
17+-8
7-+7 (1) x
7-6
85-+ 13
8-+9
28-+ 13
73-+ 14 (1) xxx (2) xxx
57-+ 14 (1) xxx (2) x
17-+ 5
29-+ 14
40-+ 15 (1) xxx (2) xxx
36-+7 (1) xx
26 + 16 (1) xxx (2) xx
Cell surface
31 -+ 16 (1) xxx (2) xxx
Note. Data are given as means -+ SD. (1) Compared to controls; (2) compared to group Co: x, P < 0.05; xx, P < 0.01; xxx, P < 0.001.
FIG. 6. A l v e o l a r m a c r o p h a g e f r o m a r a b b i t e x p o s e d t o C o 2+ a n d N i 2+ . T h e c y t o p l a s m is filled w i t h l a m i n a t e d , s u r f a c t a n t - l i k e i n c l u s i o n s (LI) a n d t h e s u r f a c e is s m o o t h . B a r = 2 ~ m .
32
JOHANSSON, CURSTEDT, AND CAMNER
laminated inclusions (Table 3). Exposure to cobalt and nickel resulted in a marked increase in macrophages with more than 10 laminated inclusion profiles per cell profile. The high amount of surfactant-like inclusion was often combined with a smooth cell surface (Fig. 6) or a surface covered by short microvilli. About 40% of the macrophages from rabbits in group Co + Ni had a smooth surface and about 30% had a surface rich in thin villi and protrusions (Fig. 7) (Table 3). These figures differed significantly from those of controls or rabbits in group Co. The percentage of macrophages with enlarged Golgi complexes was significantly higher in group Co + Ni than in the control group but not higher than in group Co (Table 3). In control rabbits and in rabbits in group Co most macrophages appeared unremarkable (Fig. 8) while most macrophages from rabbits in group Co + Ni showed one or several of the above-described alterations (Table 3). Scanning electron microscopy revealed that a higher percentage of the macrophages from rabbits in group Co + Ni had an active villous surface compared to macrophages from controls which expressed broad lamellipodia (Fig. 9) (Table 4). Macrophages from both exposed groups tended to be more ball-shaped than macrophages from controls; i.e., they did not spread out on the glass surface and often appeared in clusters (Fig. 10). Relatively few large and smooth cells were found. (A probable explanation is that these cells were loosely bound to the glass surface and fell off during the washing and fixation.)
Fro. 7. Alveolar m a c r o p h a g e from a rabbit e x p o s e d to C o 2+ and Ni a+ . The cell surface is covered with short, thin microvilli and the cell is phagocytizing surfactant (arrow). Bar = 2 ~m.
L U N G LESIONS A F T E R COBALT A N D N I C K E L I N H A L A T I O N
33
FIG. 8. Alveolar macrophage from a control rabbit. The cell has numerous vesiculated lysosomes (L). Bar = 2 p~m.
Lung Phospholipids Table 5 shows the concentrations of total phospholipids, phosphatidylcholines, and 1,2-dipalmitoylphosphatidylcholine in the unlavaged lung tissue. All concentrations were markedly and significantly higher for group Co + Ni than for group Co and the control group. Only the concentration of !,2-dipalmitoylphosphatidylcholine was significantly higher in group Co than in the controls. DISCUSSION The effects on lung macrophages, lung morphology, and lung phospholipids (present study) were compared in groups of rabbits exposed to a combination of Co 2+ and Ni 2+, to C o 2+ only, and to filtered air. Because of limited number of exposure chambers we were not able to include a group exposed to Ni 2+ only. However, we have recently exposed rabbits for 4 months to 0.6 mg/m3 Ni 2+ only (Johansson et al., 1989). The pulmonary toxicity of cobalt dust from the processing of hard metal is suggested to be related to its relatively high solubility in protein-containing fluids (Harding, 1950). The effects after exposure to soluble cobalt are therefore likely to be similar to the effects after exposure to dust from the processing of hard metal.
34
JOHANSSON,
CURSTEDT,
AND CAMNER
FIG. 9. Alveolar macrophage from a control rabbit. The cell expresses broad lamellipodia and is tightly bound to the glass surface. Bar = 5 Ixm.
Earlier studies in which rabbits inhaled soluble cobalt have shown that the first and most prominent effects were abnormal clustering of the alveolar epithelial type II cells and changed appearance of these cells (Johansson et al., 1984, 1987a). This reaction seems to be specific to cobalt as it was not seen after inhalation of nickel, cadmium, trivalent and hexavalent chromium, manganese, copper, or lithium (Johansson et al., 1981, 1983b, 1984, 1986b, 1987b, 1988a). Group Co + Ni showed more pronounced effects on most lung tissue and alveolar macrophage parameters than group Co. In Table 6 the data on lung weight, volume density of type II cells, and lung phospholipids in earlier studies with single exposures to nickel and cobalt of similar exposure periods and metal TABLE 4 SCANNING ELECTRON MICROSCOPIC DATA ON MACROPHAGES OBTAINED BY LAVAGE
Group
Villous cell surface
Cells with broad lamellipodia
Ball-shaped appearance
(%)
(%)
(%)
Controls (n = 3) Co (n = 4) Co + Ni (n = 7)
19 ± 4
74 ± lO
24±
25 ± 11
50 ± 33
40 ± 33
56 ± 18
35 ± 18
50±
Note. Data are given as means -+ SD.
24
18
L U N G LESIONS A F T E R C O B A L T A N D N I C K E L I N H A L A T I O N
35
FIG. 10. Alveolar m a c r o p h a g e s from a rabbit e x p o s e d to Co 2+ and Ni 2+. The cells stick to each other, are covered with short microvilli, and are not spread over the glass surface. Bar = 5 Ixm.
concentrations are compared with the data from the present study. The effects on lung weight and volume density of type II cells in group Co + Ni are apparently a combination of the effects in the single exposures. H o w e v e r , the formation of noduli of alveolar type II cells, which are not induced by nickel (Johansson et al., 1983b, 1989), appeared more pronounced in group Co + Ni than in group Co. Further, the increase in phospholipids in group Co + Ni seems more pronounced than just an additive combination. Co 2 + and Ni 2+ separately only increased the TABLE 5 TOTAL PHOSPHOLIPIDS, PHOSPHATIDYLCHOLINE, AND 1,2-DIPALMITOYLPHOSPHATIDYLCHOLINE IN UNLAVAGED LUNG TISSUE
Group Controls (n = 7) a Co (n = 8) Co + Ni (n = 8)
Phospholipids 0xmole/g lung)
Phosphatidylcholine 0xmole/g lung)
1,2-dipalmitoylphosphatidylcholine (ixmole/g lung)
14.6 -+ 1.0
6.9 -+ 0.8
2.4 --_ 0.4
16.9 - 3.4
9.1 -+ 2.5
3.6 +-- 1.4 (1) x
33.2 --_ 13.2
19.2 _+ 9.2
8.3 ± 4.8
(1) xx (2) xx
(1) xx (2) x
(1) xx (2) x
Note. (1) C o m p a r e d to controls; (2) C o m p a r e d to group Co: x, P < 0.05; xx, P < 0.01. a Sample from one rabbit lost due to technical error.
36
JOHANSSON, CURSTEDT, AND CAMNER TABLE 6
RATIOS BETWEEN METAL-ExPOSED AND CONTROL RABBITS CONCERNING LUNG WEIGHT, VOLUME DENSITY OF TYPE II CELLS, AND LUNG PHOSPHOLIPIDS IN THE PRESENT STUDY AND IN EARLIER STUDIES WITH SIMILAR EXPOSURE SITUATIONS
Metal conch. (mg/m 3)
Lung weight
Volume density of type II cells
Phospholipids/g wet lung
Phosphatidylcholines/g wet lung
Study
0.5 Co 2+ -~ 0.5 Ni 2+
1.4
1.8
2.3
2.8
Present
0.5 Co 2 +
1.2 1.0
1.2
1.3
0.4 Co 2+
1. l 1.0
2.0 Co 2+
1.3
1.5
0.6 Ni 2+
1.5
2.5
1.2
1.2
Present Johansson et al., 1987 Johansson et al., 1987 Johansson et al., 1989
phosphatidylcholines by about 30 and 20%, respectively, whereas the combination of Co ~+ and Ni 2+ caused an increase of 180% (Table 6). Nickel thus appears to potentiate the effect on type II cells induced by cobalt. A comparison of the data on alveolar macrophages in the present study with earlier data after inhalation of nickel alone (Johansson et al., 1983b) indicates that the effects in group Co + Ni are addititive combinations of single exposures of cobalt and nickel. The concentrations of cobalt and nickel in the present study were not far from the occupational threshold limit values, which are 0.05 mg/m 3 for cobalt and 0.1 mg/m 3 for nickel in Sweden. Nickel is, in addition to cobalt, sometimes added to hard metal (Balmes, 1987). As nickel potentiated the specific effect of cobalt, that is the formation of type II cell noduli and the increase in surfactant, combined exposure to cobalt and nickel seems to be more harmful than exposure to each of the metals separately.
ACKNOWLEDGMENTS We are grateful for the technical assistance from Ms. Elin Arvesen, Ms. Inger Granell, Ms. Kristina Nyberg, and Ms. Karin Widtski61d-Olsson. This study was supported by a grant from the Swedish Work and Environmental Foundation.
REFERENCES Anderson, T. S. (1951). Techniques for the presentation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N . Y . Acad. Sci. 13, 130-134. Balmes, J. R. (1987). Respiratory effects of hard-metal dust exposure. State Art Rev. Occup. Med. 2, 327-344. Bartlett, G. R. (1959). Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466-468. Camner, P., Johansson, A., and Lundborg, M. (1978). Alveolar macrophages in rabbits exposed to nickel dust: Ultrastructural changes and effect on phagocytosis. Environ. Res., 16, 226-235. Casarett-Bruce, M., Camner, P., and Curstedt, T. (1981). Changes in pulmonary lipid composition of rabbits exposed to nickel dust. Environ. Res. 26, 353-362. Coates, E. O., and Watson, J. H. L. (1971). Diffuse interstitial lung disease in tungsten carbide workers. Ann. Intern. Med. 75, 70%716.
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