JOURNAL OF APPLIED TOXICOLOGY, VOL. 12(4), 285-290 (1992)
Effects of Nickel Hydroxycarbonate on Alveolar Macrophage Functions K. Arsalane,'*'.t C. Aerts,z B. Wallaert,' C. Voisin' and H. F. Hildebrand' 'Institut de Mkdecine du Travail, Laboratoire de Toxicologie Professionnelle, FacultC de Mkdecine, 1 place de Verdun, 59045 Lille cedex. France, *Laboratoire de Pathologie Respiratoire ExpCrinientale et de Pollution Atmospherique, and CJF INSERM 90-06. Institut Pasteur, 59019 Lille cedex, France
Key words: nickel hydroxycarbonate; guinea pig alveolar macrophages; ATP; superoxide anion; glutathione; catalase; superoxide dismutase.
The metabolic effects of different concentrations of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophages (GPAMs) were investigated. Exposure to high concentrations of NiHC (6.25 and 12.5 pg cells) led to cell vacuolization. Morphological changes were associated with a dramatic reduction in the steadystate level of cellular adenosine triphosphate (ATP), i.e. ATP levels were reduced by 35% ( P < 0.001) and 53% ( P < 0.01), respectively. Low concentrations of NiHC (0.0625 and 0.125 pg cells) did not induce morphological changes but increased cellular ATPcontent by 19% ( P < 0.01) and 12% ( P < 0.05), respectively. cells) on cell oxidative metabolism were studied. The chemiluminescence Effects of NiHC (0.125 and 6.25 pg was significantly increased ( P < 0.001) by the lower but not the higher concentration. A slight inhibition of total superoxide dismutase ( P < 0.05) and a decrease of catalase activity were demonstrated ( P < 0.05) for the high dose, while the low dose decreased the levels of reduced and total glutathione. In conclusion, the effects of NiHC on alveolar macrophages are characterized by an overproduction of free radicals for low concentrations and the depletion of cellular reserve energy, particularly ATP, for high concentrations.
INTRODUCTION The carcinogenic, mutagenic and allergenic potential of certain nickel compounds is well established.'.' However, the mechanisms of the carcinogenic and toxic effects of nickel are still unknown. Ciccarelli and co-workers"-s have shown that intraperitoneally injected nickel carbonate induced DNA damage and nickel-binding to chromatin, nucleic acids and nuclear proteins. Several investigations have shown that after various routes (inhalation, intravenous, intraperitoneal, subcutaneous) of administration the lung is the target of nickel incorporation,Gx with alveolar macrophages as major target cells.9 In rats and rabbits, the inhalation of NiClz at atmospheric concentration near the threshold limit values diminishes the phagocytic and bactericidal efficiency of alveolar macrophages by reducing the production and release of hydrolytic enzymes (e.g. lysozyme)."" In contrast, Sunderman" reported that some nickel compounds induce the release of free radicals and increase the lipid peroxidation. Indeed, increased lipid peroxidation may reflect either cell injury leading to cell destruction or cell activation leading to the release of various bioactive mediators, Several lines of evidence suggest that alveolar macrophages activated by environmental agents can release a battery of inflammatory and immune products within the alveolar structures: in v i m studies have demonstrated that inorganic dusts can activate alveolar macrophages; (ii) activated alveolar macrophages are able to release (i)
t Author to whom correspondence should be addressed 0260-137W92/040285-06$08.00 Wiley & Sons, Ltd.
0 1992 by John
oxidants, including superoxide anion, hydrogen peroxide and hydroxyl radical, and various factors that modulate fibroblast proliferation. l3-l9 The chemical reaction leading to the in vivo induction of lipid peroxidation by Ni2+ has not yet been established. It may be an indirect mechanism whereby Ni'+ induces either the depletion of free-radical scavengers (e.g. glutathione) or the inhibition of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase). I' With this in mind, we initiated this study to determine the in vitro effects of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophage activities. The cellular behaviour against the aggression of this compound was assessed by light microscopy and by the determination of intracellular adenosine triphosphate (ATP), the release of oxygen free radicals and antioxidant activities.
~
~~
MATERIALS AND METHODS Nickel hydroxycarbonate was ALFA product 89019 (NiC03. X H 2 0 ) provided by the INCO J. Roy Gordon Research Laboratory (Mississanga, Ontario, Canada), where Dr Zatka analysed it and determined its molecular formula to be exactly NiC03.Ni(OH),.2H20. The material therefore consists of hydrated basic (1+ 1) nickel carbonate rather than normal carbonate (NiCO,. x H 2 0 ) as specified on the ALFA label. The X-ray diffraction pattern of this compound was very weak and characterized the salt as essentially amorphous. Received I5 July 1991 Accepted (revised) 2 Junuurv I992
286
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Cell culture Guinea pig alveolar macrophages (GPAMs) were obtained from Hartley albino guinea pig lungs by bronchoalveolar lavage with Hanks balanced saline solution (HBSS).”’ The cell suspension was centrifuged at 800 g and the pellet was resuspended in Basic Eagle’s Medium (BEM)(Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum and cultured in glass flasks to obtain purified GPAMs. After 24 h non-adherent cells were eliminated, whereas GPAMs adhering to the glass were removed by 0.125% (w/v) ethylene diamine tetraacetic acid (EDTA) solution in Dulbecco’s phosphate buffered saline (PBS). Cell numbers were determined manually with a standard haemocytometer. Cells were then suspended in BEM with glutamine (2 mM) and 10% fetal calf serum to obtain 10 x loh cells m1-I. Cells were cultured according to the biphasic cell culture method of Voisin et al.,”~” the GPAMs being layered on porous cellulose triacetate membrane (Metricel GA 8, 0.2 mm; Gelman Sciences Inc., Ann Arbor. MI). This membrane was applied to the surface of a reservoir filled with nutrient medium so as to be saturated by capillarity. Under these conditions, cells were in direct air contact without a liquid medium interface. In all studies, cell preparations were incubated in a special chamber (IGR Caisson Lequeux SA, Paris. France) under air enriched with 5 % C 0 2 (Alphagaz SA, France). Prior to treatment with NiHC, a 3-h period was chosen to allow the good adherence of cells to the cellulose membrane. Nickel hydroxycarbonate exposure Nickel hydroxycarbonate was suspended in the culture medium. The suspension was calculated to expose cells to 0.0625, 0.125, 1.25, 6.25 and 12.5 bg NiHC l W h cells for ATP determination. According to the effects of various doses on cell morphology, all other experiments were performed with a ‘high dose’ (6.25) and a ‘low dose’ (0.125). The exposure time was 24 h for the chemiluminescence assay and 48 h for all other experiments.
A minimum of eight separate experiments was carried out for each concentration. Chernilurninescence assay
Superoxide anion generation by alveolar macrophages was investigated with a lucigenin-dependent chemiluminescence (CL) method adapted from Williams and Cole,30 in which lucigenin (bis-N-methylacridium nitrate, Sigma Chemical, St. Louis, MO) served as a chemiluminescence-generating probe. Lucigenin ( M) was dissolved in HBSS buffered with 18 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid). Phorbol-myristate-acetate (PMA; Sigma) was dissolved in dimethylsulphoxide and the resulting concentration was 0.5 mg ml-I. A 10-p.1 aliquot of PMA was dissolved just before use in 5 ml of Hanks’ HEPES (1 bg m1-I). Superoxide dismutase (SOD; Sigma) was dissolved in Hanks’ HEPES (2 mg ml-I). Chemiluminescence was measured at 37°C with a Luminometer (Lumac System AG, Basel, Switzerland). Four membranes with 0.5 x 10” alveolar macrophages were added to each of four vials containing 90O-pA aliquots of the lucigenin solution and 50-p.1 aliquots of a 3% gelatin solution. One vial was measured without and another after the addition of a 100-pl aliquot of 2 mg ml-’ SOD. A IOO-pl aliquot from a 1 pg mi-I PMA solution was added to a third vial and both PMA and SOD were added to the last vial. The total volume in each vial was brought to 1.650 ml by adding the appropriate amount of HBSS-HEPES. All vials were incubated simultaneously . The luminescence intensity was measured for 60 s after a 12-min incubation period at 37°C. The results are expressed in relative luminescent units per 0.5 x 10’ alveolar macrophages. A minimum of six separate experiments was performed for both concentrations and controls. Evaluation of antioxidant activities
Cell injury index. Adenosine triphosphate was used as an index of the energy status. We evaluated intracellular ATP through bioluminescence using luciferin-luciferase21.’”.23 on a Berthold Biolumat LB 9500T (C.L.V. Interbio, Villeurbanne, France). The analysis was carried out on a cellular extract (one membrane with 0.5 X lo6 cells) in dimethyl sulphoxide (DMSO). The cell injury index (CII) corresponds to the percentage ATP content of the exposed cells with respect to the control cells. The CII was evaluated as follows
Superoxide dismutase (SOD) assay. The cells of three membranes with 2 X loh GPAMs on each were pooled in 2 ml of phosphate buffer (pH 7.8). The cellular suspension was sonicated on ice (MSE sonifier at a power of 100 W up to 20 kHz for 30 s). After centrifugation at 800 g for 10 min at 4”C, supernatants were removed and treated with 0.2% (vh) Triton X100. After 30 min of incubation, solutions were centrifuged at 45,000 g for 15 min at 4°C and supernatants were assayed for SOD with the method of McCord and Fridovich,26 modified by Crapo and McCord?’ at pH 10. This method is based on the capacity of SOD to inhibit cytochrome c reduction mediated by O2 generated during the oxidation of xanthine catalysed by xanthine oxidase. Manganesedependent SOD (Mn SOD) activity was measured by adding M KCN. One unit of SOD activity was defined as the amount of enzyme that inhibited the rate of reduction of cytochrome c by 50%” in a final volume of 3 ml.
(ATP content of exposed cells cells) _ATP _ - content _ _ _of_control ____x 100 ATP content of control cells
Catalase assay. Cellular extraction was performed with the method used for SOD assay but cells were placed in phosphate buffer at ph 7 (one membrane
Cell injury evaluation Light microscopy. Cells on culture membrane were stained by capillarity with May-Grunwald-Giemsa and light microscopy studies.
NICKEL HYDROXYCARBONATE EFFECTS ON MACROPHAGE FUNCTIONS
with 2 x loh cells in 2 ml). Catalase was measured with the method of Beers and Sizer:” one unit of catalase activity decomposes 1 pmol of hydrogen peroxide per minute at 25°C; 0.1 ml of supernatant was added to 2.9 ml of the substrate solution (H,O,). For reproducible results, the absorbance at 240 nni (A,-ro)should be between 0.55 and 0.52. The time required for A z j oto decrease from 0.45 and 0.40 was noted and corresponded to the decomposition of 3.45 pmol of HzOz in 3 ml of solution.
287
samples. For the glutathione disulphide assay, 10 pl of triethanolamine and 10 pI of 2-vinyl-pyridine were added to 200 pl of supernatant of the same cellular sample. After 1 h of contact, the assay was the same as the assay for total glutathione. At least eight repeat experiments were performed for both concentrations and controls. Total protein concentration was determined by the method of Lowry er al.” Statistical analysis
Intracellular glutathione assay. The membranes with 1.4 X 10‘ GPAMs were placed in 1 rnl of 0.05% (wh) Triton X-100 in EDTA buffer (6.3 mM). Samples of lysates (760 pl) were acidified with 40 p1 of 50% (wh) sulphosalicylic acid and removed by centrifugation. Supernatants were assayed for total glutathione (GSH and GSSG) by an enzymatic recycling procedure: glutathione was sequentially oxidized by 55’-dithiobis2-nitrobenzoic acid (DTNB) and was reduced by NADPH in the presence of glutathione reductase at 30”C.25 2-Nitro-5-thiobenzoic acid formation was monitored at 412 nm between the 10th and 16th min by comparison of the results with the standard curve. GSH standards contained Triton X-100, sulphosalicyclic acid and HCI in quantities identical to the
Cell injury Light microscopy. Guinea pig alveolar macrophages exposed to low concentrations of NiHC (0.0625 and 0.125 kg lo-‘ cells) did not exhibit any morphological alteration and did not differ from control cells (Fig. 1).
Figure 1. Control guinea pig alveolar macrophages grown for 48 h in aerobiosis. (~800.)
Figure 2. Guinea pig alveolar macrophages incubated with 6.25 kg nickel hydroxycarbonate cells for 48 h in aerobiosis. Note the presence of large vacuoles. (~800.)
All results were expressed as mean values. Statistical significances were calculated with the Wilcoxon rank sum test. P < 0.05 was considered as significant.
RESULTS
288
K. ARSALANE ET A L
401 30
significantly decreased the chemiluminescent response of GPAMs (Table 1). The extent of inhibition of chemiluminescence by SOD demonstrates that the chemiluminescent response of GPAMs was related to the generation of superoxide anion by GPAMs.
*
h
$
20
Y
x w n
lo
o -10
g ?
z,
a w
0
-20 -30
-40 -50
1
-60
* *
-,v
0.0625
0.125
1.25
6.25
pg NiHC
12.5
cells
Figure 3. ATP content in guinea pig alveolar macrophages cultured for 48 h in aerobiosis in the presence of different concentrations of nickel hydroxycarbonate. Number of experiments = 8. * P < 0.05, + * P i 0.07 and *** P i 0.001. with respect to controls.
A slight vacuolization was observed at a concentration of 1.25 pg cells. This vacuolization became particularly obvious in cells exposed to 6.25 and cells, as demonstrated in Fig. 2. 12.5 pg Cell injury index. Figure 3 demonstrates a biphasic change in intracellular ATP concentration, which could indicate 'activation' of GPAMs at low concentrations and 12% after exposure to concentrations of 0.0625 and 0.125 pg lop6 cells, respectively) and inhibition of energy generation at higher concentrations (35% and 53% after exposure to concentrations of 6.25 and 12.5 pg cells, respectively). The ATP level remained unchanged after exposure to cells. 1.25 p,g Cell cherniluminescence
Chemiluminescence of the GPAMs exposed to the low dose (0.125 pg cells) of NiHC was significantly ( P < 0.001) higher than that of control cells. No significant difference was noted after exposure to the high dose (6.25 pg cells). After stimulation by PMA, no significant difference was observed after exposure to the low dose. Exposure to the high dose
Evaluation of antioxidant activities Antioxidant activities were evaluated after exposure to two different concentrations (0.125 and 6.25 pg cells). A slight inhibition of total SOD ( P < 0.05) and a significant decrease of catalase activity were demonstrated after exposure to a concentration of 6.25 pg cells (Table 2). Intracellular levels of total glutathione, reduced glutathione and oxidized glutathione were determined. The amount of total glutathione per unit protein was slightly reduced compared to control cells. This decrease was associated with the loss of reduced glutathione ( P < 0.05). No significant difference was noted for the oxidized glutathione (Table 2). DISCUSSION
Our results demonstrate that, depending on the concentration, NiHC has different effects on alveolar macrophage functions. High concentrations of NiHC induce a cytotoxic effect in GPAMs. Cells became vacuolized, probably as a result of phagocytosis of NiHC particles. Furthermore, there was a dose-related reduction in the amount of ATP. Such a decrease influences cellular behaviour, particularly membrane transport, the activity of several enzymes and energy-consuming reactions. As to the antioxidant activities of GPAMs, catalase and SOD activities were slightly reduced and the glutathione level diminished. Andersen and Andersen?' have also described the reduction of hepatic glutathione concentrations after i.p. injections of NiClz in mice. In contrast to the selective binding of heavy metals (e.g. Cd, Hg, Pb) to the SH groupof glutathione, nickel is predominantly bound to the y-glutamyl . also ~ ~ shown, by in centre.32 Rodriguez et ~ 1 have vitro and in vivo assays, the inhibition of catalase activity by nickel. Its in vitro action appears to be directly induced by the interaction of nickel ions with the protein, whereas the in vivo mechanism remains unknown.
Table 1. Chemiluminescence of alveolar macrophages before and after PMA stimulation"
Treatment
Number of experiments
Control
13
NiHC (0.125 kg NiHC (6.25 Fg a
13 cells)
6 cells)
After stimulation by PMA Before stimulation by PMA Mean Suppression byMean Suppression by (min-max) SOD (%I (min-max) SOD (%I
5124 (1544-26156) 12 176'" (2628-788221 1957 (423-6308)
76 86 67
84,940 (10509-233214) 94 185 (9851-403 709) 6603* (390-17405)
96 96 59
Results are expressed as relative luminescent units per 0.5 x lo6 alveolar macrophages.
** P < 0.001, when compared to control values.
* P < 0.05
and
289
NICKEL HYDROXYCARBONATE EFFECTS O N MACROPHAGE FUNCTIONS
Table 2. Effect of nickel hydroxycarbonate on antioxidant systems in guinea pig alveolar macrophages" Superoxide dismutase (SOD) (U m g - ' protein) ( min- max)
Glutathione (ng mg-' protein) (min-max)
Treatment
Total
Reduced
Oxidized
Catalase (U mg-' protein) (min-max)
Control
5170 (984-11 123)
4618 (531-10630)
423 (154-863)
82 (22-159)
11 (6.3-22)
5.4 (1.4-13.9)
5.8 (1.5-20)
NiHC (0.125 pg
4319* (1333-8304)
3720* (433-7747)
464 (240-978)
74 (31-133)
13 (5.8-30)
6 (0.2-19.4)
6.6 (1.1-28)
4279 (821-10524)
3689 (16C-9658)
414 (125-866)
65* (0-126)
1o* (5.4-22)
4.4 (0.8-1 1.1)
5.5 (1.2-21)
NiHC (6.25 p g a
cells)
Total
Cu-Zn dependent
Mn dependent
Results are means ( n = 8). * P < 0.05 when compared with control values.
Another phenomenon is increased ATP synthesis at low concentration. A similar activation by NiHC has been described in previous studies on cultured human epithelial lung cells (L132 cell line),34 where at low concentrations this compound stimulated cellular growth. This phenomenon seems to be specific for nickel hydroxycarbonates, since other compounds (NiCI2, aNi3S2 and PNiS) did not elicit the same e f f e ~ t . Because ~ ~ . ~ ~ SOD significantly inhibited the chemiluminescent response of GPAMs, the observed increase in both chemiluminescence and the generation of superoxide anion confirms the presence of cellular activation. Evans et ~ 1 . 'also ~ observed an increase in free-radical generation in polymorphonuclear leucocytes exposed in vitro to Ni3S2and NiO. These authors correlated the enhanced generation of free radicals with the carcinogenic activity of nickel compounds. These radicals may attack nucleic acids, proteins and membrane lipids, leading to genotoxicity, membrane damage and subsequent cell In this way, the genotoxic action of NiHC should be considered an indirect effect. The amount of reduced glutathione decreased with the generation of free radicals. This can be explained
by the intervention of glutathione in the elimination of free radicals and its subsequent transformation to oxidized glutathione, which is normally released from cells. The glutathione depletion could be due to the defective regeneration of reduced glutathione from oxidized glutathione by glutathione reductase and glucose-6-phosphate dehydrogenase. Indeed, some a ~ t h o r s 'have ~ reported an inhibitory effect of nickel on the activity of both enzymes. The results of the current study demonstrate a complex interaction between NiHC and cellular constituents. Apart from the cytotoxic effect shown by depletion of the cellular reserve energy, it also induces cell activation expressed by the generation of free radicals.
Acknowledgements The authors thank C. Fourneau and C. Merdy for skilful technical assistance. This work was supported by grants from the University of L i l l e 11. from INSERM (CJF YO-06), from the French Ministry of Research and Technology (Department: Homme, Travail et technologie. Contract no. 89 D 0039) and from the Conseil Regional Nord/Pas de Calais (GIs SANTE-TRAVAIL).
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Effects of Nickel Hydroxycarbonate on Alveolar Macrophage Functions K. Arsalane,'*'.t C. Aerts,z B. Wallaert,' C. Voisin' and H. F. Hildebrand' 'Institut de Mkdecine du Travail, Laboratoire de Toxicologie Professionnelle, FacultC de Mkdecine, 1 place de Verdun, 59045 Lille cedex. France, *Laboratoire de Pathologie Respiratoire ExpCrinientale et de Pollution Atmospherique, and CJF INSERM 90-06. Institut Pasteur, 59019 Lille cedex, France
Key words: nickel hydroxycarbonate; guinea pig alveolar macrophages; ATP; superoxide anion; glutathione; catalase; superoxide dismutase.
The metabolic effects of different concentrations of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophages (GPAMs) were investigated. Exposure to high concentrations of NiHC (6.25 and 12.5 pg cells) led to cell vacuolization. Morphological changes were associated with a dramatic reduction in the steadystate level of cellular adenosine triphosphate (ATP), i.e. ATP levels were reduced by 35% ( P < 0.001) and 53% ( P < 0.01), respectively. Low concentrations of NiHC (0.0625 and 0.125 pg cells) did not induce morphological changes but increased cellular ATPcontent by 19% ( P < 0.01) and 12% ( P < 0.05), respectively. cells) on cell oxidative metabolism were studied. The chemiluminescence Effects of NiHC (0.125 and 6.25 pg was significantly increased ( P < 0.001) by the lower but not the higher concentration. A slight inhibition of total superoxide dismutase ( P < 0.05) and a decrease of catalase activity were demonstrated ( P < 0.05) for the high dose, while the low dose decreased the levels of reduced and total glutathione. In conclusion, the effects of NiHC on alveolar macrophages are characterized by an overproduction of free radicals for low concentrations and the depletion of cellular reserve energy, particularly ATP, for high concentrations.
INTRODUCTION The carcinogenic, mutagenic and allergenic potential of certain nickel compounds is well established.'.' However, the mechanisms of the carcinogenic and toxic effects of nickel are still unknown. Ciccarelli and co-workers"-s have shown that intraperitoneally injected nickel carbonate induced DNA damage and nickel-binding to chromatin, nucleic acids and nuclear proteins. Several investigations have shown that after various routes (inhalation, intravenous, intraperitoneal, subcutaneous) of administration the lung is the target of nickel incorporation,Gx with alveolar macrophages as major target cells.9 In rats and rabbits, the inhalation of NiClz at atmospheric concentration near the threshold limit values diminishes the phagocytic and bactericidal efficiency of alveolar macrophages by reducing the production and release of hydrolytic enzymes (e.g. lysozyme)."" In contrast, Sunderman" reported that some nickel compounds induce the release of free radicals and increase the lipid peroxidation. Indeed, increased lipid peroxidation may reflect either cell injury leading to cell destruction or cell activation leading to the release of various bioactive mediators, Several lines of evidence suggest that alveolar macrophages activated by environmental agents can release a battery of inflammatory and immune products within the alveolar structures: in v i m studies have demonstrated that inorganic dusts can activate alveolar macrophages; (ii) activated alveolar macrophages are able to release (i)
t Author to whom correspondence should be addressed 0260-137W92/040285-06$08.00 Wiley & Sons, Ltd.
0 1992 by John
oxidants, including superoxide anion, hydrogen peroxide and hydroxyl radical, and various factors that modulate fibroblast proliferation. l3-l9 The chemical reaction leading to the in vivo induction of lipid peroxidation by Ni2+ has not yet been established. It may be an indirect mechanism whereby Ni'+ induces either the depletion of free-radical scavengers (e.g. glutathione) or the inhibition of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase). I' With this in mind, we initiated this study to determine the in vitro effects of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophage activities. The cellular behaviour against the aggression of this compound was assessed by light microscopy and by the determination of intracellular adenosine triphosphate (ATP), the release of oxygen free radicals and antioxidant activities.
~
~~
MATERIALS AND METHODS Nickel hydroxycarbonate was ALFA product 89019 (NiC03. X H 2 0 ) provided by the INCO J. Roy Gordon Research Laboratory (Mississanga, Ontario, Canada), where Dr Zatka analysed it and determined its molecular formula to be exactly NiC03.Ni(OH),.2H20. The material therefore consists of hydrated basic (1+ 1) nickel carbonate rather than normal carbonate (NiCO,. x H 2 0 ) as specified on the ALFA label. The X-ray diffraction pattern of this compound was very weak and characterized the salt as essentially amorphous. Received I5 July 1991 Accepted (revised) 2 Junuurv I992
286
K. ARSALANE ET A L .
Cell culture Guinea pig alveolar macrophages (GPAMs) were obtained from Hartley albino guinea pig lungs by bronchoalveolar lavage with Hanks balanced saline solution (HBSS).”’ The cell suspension was centrifuged at 800 g and the pellet was resuspended in Basic Eagle’s Medium (BEM)(Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum and cultured in glass flasks to obtain purified GPAMs. After 24 h non-adherent cells were eliminated, whereas GPAMs adhering to the glass were removed by 0.125% (w/v) ethylene diamine tetraacetic acid (EDTA) solution in Dulbecco’s phosphate buffered saline (PBS). Cell numbers were determined manually with a standard haemocytometer. Cells were then suspended in BEM with glutamine (2 mM) and 10% fetal calf serum to obtain 10 x loh cells m1-I. Cells were cultured according to the biphasic cell culture method of Voisin et al.,”~” the GPAMs being layered on porous cellulose triacetate membrane (Metricel GA 8, 0.2 mm; Gelman Sciences Inc., Ann Arbor. MI). This membrane was applied to the surface of a reservoir filled with nutrient medium so as to be saturated by capillarity. Under these conditions, cells were in direct air contact without a liquid medium interface. In all studies, cell preparations were incubated in a special chamber (IGR Caisson Lequeux SA, Paris. France) under air enriched with 5 % C 0 2 (Alphagaz SA, France). Prior to treatment with NiHC, a 3-h period was chosen to allow the good adherence of cells to the cellulose membrane. Nickel hydroxycarbonate exposure Nickel hydroxycarbonate was suspended in the culture medium. The suspension was calculated to expose cells to 0.0625, 0.125, 1.25, 6.25 and 12.5 bg NiHC l W h cells for ATP determination. According to the effects of various doses on cell morphology, all other experiments were performed with a ‘high dose’ (6.25) and a ‘low dose’ (0.125). The exposure time was 24 h for the chemiluminescence assay and 48 h for all other experiments.
A minimum of eight separate experiments was carried out for each concentration. Chernilurninescence assay
Superoxide anion generation by alveolar macrophages was investigated with a lucigenin-dependent chemiluminescence (CL) method adapted from Williams and Cole,30 in which lucigenin (bis-N-methylacridium nitrate, Sigma Chemical, St. Louis, MO) served as a chemiluminescence-generating probe. Lucigenin ( M) was dissolved in HBSS buffered with 18 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid). Phorbol-myristate-acetate (PMA; Sigma) was dissolved in dimethylsulphoxide and the resulting concentration was 0.5 mg ml-I. A 10-p.1 aliquot of PMA was dissolved just before use in 5 ml of Hanks’ HEPES (1 bg m1-I). Superoxide dismutase (SOD; Sigma) was dissolved in Hanks’ HEPES (2 mg ml-I). Chemiluminescence was measured at 37°C with a Luminometer (Lumac System AG, Basel, Switzerland). Four membranes with 0.5 x 10” alveolar macrophages were added to each of four vials containing 90O-pA aliquots of the lucigenin solution and 50-p.1 aliquots of a 3% gelatin solution. One vial was measured without and another after the addition of a 100-pl aliquot of 2 mg ml-’ SOD. A IOO-pl aliquot from a 1 pg mi-I PMA solution was added to a third vial and both PMA and SOD were added to the last vial. The total volume in each vial was brought to 1.650 ml by adding the appropriate amount of HBSS-HEPES. All vials were incubated simultaneously . The luminescence intensity was measured for 60 s after a 12-min incubation period at 37°C. The results are expressed in relative luminescent units per 0.5 x 10’ alveolar macrophages. A minimum of six separate experiments was performed for both concentrations and controls. Evaluation of antioxidant activities
Cell injury index. Adenosine triphosphate was used as an index of the energy status. We evaluated intracellular ATP through bioluminescence using luciferin-luciferase21.’”.23 on a Berthold Biolumat LB 9500T (C.L.V. Interbio, Villeurbanne, France). The analysis was carried out on a cellular extract (one membrane with 0.5 X lo6 cells) in dimethyl sulphoxide (DMSO). The cell injury index (CII) corresponds to the percentage ATP content of the exposed cells with respect to the control cells. The CII was evaluated as follows
Superoxide dismutase (SOD) assay. The cells of three membranes with 2 X loh GPAMs on each were pooled in 2 ml of phosphate buffer (pH 7.8). The cellular suspension was sonicated on ice (MSE sonifier at a power of 100 W up to 20 kHz for 30 s). After centrifugation at 800 g for 10 min at 4”C, supernatants were removed and treated with 0.2% (vh) Triton X100. After 30 min of incubation, solutions were centrifuged at 45,000 g for 15 min at 4°C and supernatants were assayed for SOD with the method of McCord and Fridovich,26 modified by Crapo and McCord?’ at pH 10. This method is based on the capacity of SOD to inhibit cytochrome c reduction mediated by O2 generated during the oxidation of xanthine catalysed by xanthine oxidase. Manganesedependent SOD (Mn SOD) activity was measured by adding M KCN. One unit of SOD activity was defined as the amount of enzyme that inhibited the rate of reduction of cytochrome c by 50%” in a final volume of 3 ml.
(ATP content of exposed cells cells) _ATP _ - content _ _ _of_control ____x 100 ATP content of control cells
Catalase assay. Cellular extraction was performed with the method used for SOD assay but cells were placed in phosphate buffer at ph 7 (one membrane
Cell injury evaluation Light microscopy. Cells on culture membrane were stained by capillarity with May-Grunwald-Giemsa and light microscopy studies.
NICKEL HYDROXYCARBONATE EFFECTS ON MACROPHAGE FUNCTIONS
with 2 x loh cells in 2 ml). Catalase was measured with the method of Beers and Sizer:” one unit of catalase activity decomposes 1 pmol of hydrogen peroxide per minute at 25°C; 0.1 ml of supernatant was added to 2.9 ml of the substrate solution (H,O,). For reproducible results, the absorbance at 240 nni (A,-ro)should be between 0.55 and 0.52. The time required for A z j oto decrease from 0.45 and 0.40 was noted and corresponded to the decomposition of 3.45 pmol of HzOz in 3 ml of solution.
287
samples. For the glutathione disulphide assay, 10 pl of triethanolamine and 10 pI of 2-vinyl-pyridine were added to 200 pl of supernatant of the same cellular sample. After 1 h of contact, the assay was the same as the assay for total glutathione. At least eight repeat experiments were performed for both concentrations and controls. Total protein concentration was determined by the method of Lowry er al.” Statistical analysis
Intracellular glutathione assay. The membranes with 1.4 X 10‘ GPAMs were placed in 1 rnl of 0.05% (wh) Triton X-100 in EDTA buffer (6.3 mM). Samples of lysates (760 pl) were acidified with 40 p1 of 50% (wh) sulphosalicylic acid and removed by centrifugation. Supernatants were assayed for total glutathione (GSH and GSSG) by an enzymatic recycling procedure: glutathione was sequentially oxidized by 55’-dithiobis2-nitrobenzoic acid (DTNB) and was reduced by NADPH in the presence of glutathione reductase at 30”C.25 2-Nitro-5-thiobenzoic acid formation was monitored at 412 nm between the 10th and 16th min by comparison of the results with the standard curve. GSH standards contained Triton X-100, sulphosalicyclic acid and HCI in quantities identical to the
Cell injury Light microscopy. Guinea pig alveolar macrophages exposed to low concentrations of NiHC (0.0625 and 0.125 kg lo-‘ cells) did not exhibit any morphological alteration and did not differ from control cells (Fig. 1).
Figure 1. Control guinea pig alveolar macrophages grown for 48 h in aerobiosis. (~800.)
Figure 2. Guinea pig alveolar macrophages incubated with 6.25 kg nickel hydroxycarbonate cells for 48 h in aerobiosis. Note the presence of large vacuoles. (~800.)
All results were expressed as mean values. Statistical significances were calculated with the Wilcoxon rank sum test. P < 0.05 was considered as significant.
RESULTS
288
K. ARSALANE ET A L
401 30
significantly decreased the chemiluminescent response of GPAMs (Table 1). The extent of inhibition of chemiluminescence by SOD demonstrates that the chemiluminescent response of GPAMs was related to the generation of superoxide anion by GPAMs.
*
h
$
20
Y
x w n
lo
o -10
g ?
z,
a w
0
-20 -30
-40 -50
1
-60
* *
-,v
0.0625
0.125
1.25
6.25
pg NiHC
12.5
cells
Figure 3. ATP content in guinea pig alveolar macrophages cultured for 48 h in aerobiosis in the presence of different concentrations of nickel hydroxycarbonate. Number of experiments = 8. * P < 0.05, + * P i 0.07 and *** P i 0.001. with respect to controls.
A slight vacuolization was observed at a concentration of 1.25 pg cells. This vacuolization became particularly obvious in cells exposed to 6.25 and cells, as demonstrated in Fig. 2. 12.5 pg Cell injury index. Figure 3 demonstrates a biphasic change in intracellular ATP concentration, which could indicate 'activation' of GPAMs at low concentrations and 12% after exposure to concentrations of 0.0625 and 0.125 pg lop6 cells, respectively) and inhibition of energy generation at higher concentrations (35% and 53% after exposure to concentrations of 6.25 and 12.5 pg cells, respectively). The ATP level remained unchanged after exposure to cells. 1.25 p,g Cell cherniluminescence
Chemiluminescence of the GPAMs exposed to the low dose (0.125 pg cells) of NiHC was significantly ( P < 0.001) higher than that of control cells. No significant difference was noted after exposure to the high dose (6.25 pg cells). After stimulation by PMA, no significant difference was observed after exposure to the low dose. Exposure to the high dose
Evaluation of antioxidant activities Antioxidant activities were evaluated after exposure to two different concentrations (0.125 and 6.25 pg cells). A slight inhibition of total SOD ( P < 0.05) and a significant decrease of catalase activity were demonstrated after exposure to a concentration of 6.25 pg cells (Table 2). Intracellular levels of total glutathione, reduced glutathione and oxidized glutathione were determined. The amount of total glutathione per unit protein was slightly reduced compared to control cells. This decrease was associated with the loss of reduced glutathione ( P < 0.05). No significant difference was noted for the oxidized glutathione (Table 2). DISCUSSION
Our results demonstrate that, depending on the concentration, NiHC has different effects on alveolar macrophage functions. High concentrations of NiHC induce a cytotoxic effect in GPAMs. Cells became vacuolized, probably as a result of phagocytosis of NiHC particles. Furthermore, there was a dose-related reduction in the amount of ATP. Such a decrease influences cellular behaviour, particularly membrane transport, the activity of several enzymes and energy-consuming reactions. As to the antioxidant activities of GPAMs, catalase and SOD activities were slightly reduced and the glutathione level diminished. Andersen and Andersen?' have also described the reduction of hepatic glutathione concentrations after i.p. injections of NiClz in mice. In contrast to the selective binding of heavy metals (e.g. Cd, Hg, Pb) to the SH groupof glutathione, nickel is predominantly bound to the y-glutamyl . also ~ ~ shown, by in centre.32 Rodriguez et ~ 1 have vitro and in vivo assays, the inhibition of catalase activity by nickel. Its in vitro action appears to be directly induced by the interaction of nickel ions with the protein, whereas the in vivo mechanism remains unknown.
Table 1. Chemiluminescence of alveolar macrophages before and after PMA stimulation"
Treatment
Number of experiments
Control
13
NiHC (0.125 kg NiHC (6.25 Fg a
13 cells)
6 cells)
After stimulation by PMA Before stimulation by PMA Mean Suppression byMean Suppression by (min-max) SOD (%I (min-max) SOD (%I
5124 (1544-26156) 12 176'" (2628-788221 1957 (423-6308)
76 86 67
84,940 (10509-233214) 94 185 (9851-403 709) 6603* (390-17405)
96 96 59
Results are expressed as relative luminescent units per 0.5 x lo6 alveolar macrophages.
** P < 0.001, when compared to control values.
* P < 0.05
and
289
NICKEL HYDROXYCARBONATE EFFECTS O N MACROPHAGE FUNCTIONS
Table 2. Effect of nickel hydroxycarbonate on antioxidant systems in guinea pig alveolar macrophages" Superoxide dismutase (SOD) (U m g - ' protein) ( min- max)
Glutathione (ng mg-' protein) (min-max)
Treatment
Total
Reduced
Oxidized
Catalase (U mg-' protein) (min-max)
Control
5170 (984-11 123)
4618 (531-10630)
423 (154-863)
82 (22-159)
11 (6.3-22)
5.4 (1.4-13.9)
5.8 (1.5-20)
NiHC (0.125 pg
4319* (1333-8304)
3720* (433-7747)
464 (240-978)
74 (31-133)
13 (5.8-30)
6 (0.2-19.4)
6.6 (1.1-28)
4279 (821-10524)
3689 (16C-9658)
414 (125-866)
65* (0-126)
1o* (5.4-22)
4.4 (0.8-1 1.1)
5.5 (1.2-21)
NiHC (6.25 p g a
cells)
Total
Cu-Zn dependent
Mn dependent
Results are means ( n = 8). * P < 0.05 when compared with control values.
Another phenomenon is increased ATP synthesis at low concentration. A similar activation by NiHC has been described in previous studies on cultured human epithelial lung cells (L132 cell line),34 where at low concentrations this compound stimulated cellular growth. This phenomenon seems to be specific for nickel hydroxycarbonates, since other compounds (NiCI2, aNi3S2 and PNiS) did not elicit the same e f f e ~ t . Because ~ ~ . ~ ~ SOD significantly inhibited the chemiluminescent response of GPAMs, the observed increase in both chemiluminescence and the generation of superoxide anion confirms the presence of cellular activation. Evans et ~ 1 . 'also ~ observed an increase in free-radical generation in polymorphonuclear leucocytes exposed in vitro to Ni3S2and NiO. These authors correlated the enhanced generation of free radicals with the carcinogenic activity of nickel compounds. These radicals may attack nucleic acids, proteins and membrane lipids, leading to genotoxicity, membrane damage and subsequent cell In this way, the genotoxic action of NiHC should be considered an indirect effect. The amount of reduced glutathione decreased with the generation of free radicals. This can be explained
by the intervention of glutathione in the elimination of free radicals and its subsequent transformation to oxidized glutathione, which is normally released from cells. The glutathione depletion could be due to the defective regeneration of reduced glutathione from oxidized glutathione by glutathione reductase and glucose-6-phosphate dehydrogenase. Indeed, some a ~ t h o r s 'have ~ reported an inhibitory effect of nickel on the activity of both enzymes. The results of the current study demonstrate a complex interaction between NiHC and cellular constituents. Apart from the cytotoxic effect shown by depletion of the cellular reserve energy, it also induces cell activation expressed by the generation of free radicals.
Acknowledgements The authors thank C. Fourneau and C. Merdy for skilful technical assistance. This work was supported by grants from the University of L i l l e 11. from INSERM (CJF YO-06), from the French Ministry of Research and Technology (Department: Homme, Travail et technologie. Contract no. 89 D 0039) and from the Conseil Regional Nord/Pas de Calais (GIs SANTE-TRAVAIL).
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