Toxicity of metallic ions and oxides to rabbit alveolar macrophages

ENVIRONMENTAL RESEARCH 48, 255--274

(1989)

Toxicity of Metallic Ions and Oxides to Rabbit Alveolar Macrophages MARIA LABEDZKA, HOLGER GULYAS, NORBERT SCHMIDT, AND GUENTHER GERCKEN 1

Institute of Biochemistry and Food Chemistry, Department of Biochemistry, University of Hamburg, D-2000 Hamburg 13, Federal Republic of Germany Received December 22, 1987 The effects of soluble compounds and oxides of As, Cd, Cu, Hg, Ni, Pb, Sb, Sn, V, and Zn on oxidative metabolism and membrane integrity of rabbit alveolar macrophages were studied by 24-hr in vitro exposure. Oxidative metabolism induced by phagocytosis of opsonized zymosan was measured by H202 and O2- release and by chemiluminescence in the presence of luminol. Membrane integrity was estimated by extracellular LDH activity. Metallic ions and oxides inhibited the release of active oxygen species. Cd(II), As(III), and V(V) were the most toxic elements as measured by all investigated parameters. Cu(II) decreased O2- release and chemiluminescence effectively but H202 release and membrane integrity less. Chemiluminescence was decreased strongly by Hg(II) while 02- and H202 release were depressed moderately. Zn(II) and Sb(III) compounds caused medium toxicity and the tested Sn, Ni, and Pb compounds showed only faint toxic effects. © 1989Academic Press, Inc.

INTRODUCTION Alveolar macrophages which come into close contact with inhaled metalcontaining particles play a crucial role in pulmonary defense against bacteria. They keep the respiratory surface of the alveoles clean by phagocytizing and killing infective organisms. Phagocytizing macrophages generate and secrete active oxygen species as superoxide anion radical 02- and hydrogen peroxide H202, which are required for efficient killing of microorganisms by phagocytic cells (Babior, 1984; Johnston, 1981a; Nathan, 1983). Thus, depression of zymosan-induced macrophage 02- and H202 secretion by toxic substances in vitro should be useful for predicting increased susceptibility to bacterial infections caused by inhalation of metal compounds. Chemiluminescence is a macrophage function very sensitive to toxic substances (Brennan and Kirchner, 1984) and closely related to production of active oxygen species (Miles et al., 1978). Membrane integrity of alveolar macrophages measured either by dye exclusion ("viability") or leakage of cytoplasmic enzymes, e.g., lactate dehydrogenase (LDH) 2 is an in vitro parameter related to immunotoxicity of inhaled mineral dusts. There is evidence that reducing viability of 1 To whom correspondence should be addressed. 2 Abbreviations used: DMEM, Dulbecco's minimum essential medium; HBSS, Hanks' balanced salts solution; Hepes, N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid; IDso, inhibition dose 50% of control level; LDH, lactate dehydrogenase; SOD, superoxide dismutase. 255 0013-9351/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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alveolar macrophages by mineral dusts in vitro corresponds to increased susceptibility to bacterial infections in mice following inhalation of the same dusts (Aranyi et al., 1981; Hatch et al., 1985). As, Cd, Cu, Hg, Ni, Pb, Sb, Sn, V, and Zn are constituents of rural and urban ~irborne dusts (Arbeitsgemeinschaft zur Foerderung der Radionuklidtechnik, 1983) as well as fly ashes from coal combustion (Biehusen, 1980; Muhle et al., 1984) or waste incinerators (Liu et al., 1984; Dannecker, 1983; Gulyas and Gercken, 1988). Inhalation or intratracheal injection of Cd, Cu, Pb, V, and Zn compounds (Hatch et al., 1981) and As203 (Aranyi et al., 1985) prior to inhalative burden with bacteria increase pulmonary susceptibility to bacterial infection in mice. Inhalation of PbC12 aerosols by mice induced a significant reduction in the elimination of bacteria from the lungs (Schlipkoeter and Bruch, 1980). Toxicological evaluation of metal compounds using alveolar macrophage in vitro tests contribute to the knowledge upon immune depressing effects of inhalable dusts in the lung. Several studies have dealt with the effects of toxic metal compounds on the zymosan-induced secretion of oxygen species by alveolar macrophages (Amoruso, 1980; Castranova et al., 1980, 1984; Galvin and Oberg, 1984; Loose et al., 1977, 1978; Wei and Misra, 1982). Unfortunately, only limited numbers of elements have been investigated in each work and different test systems concerning animal species, culture media, and state of macrophage activation have been employed. In this investigation oxides and soluble compounds of different elements were examined for their impairment of zymosan-induced secretion of H202 and 02and chemiluminescence by rabbit alveolar macrophages. Activated macrophages were used because of their availability in greater numbers. Effects on oxygen species secretion were compared to damage of the plasma membrane as measured by extracellular LDH activity. MATERIALS AND METHODS Harvesting macrophages. Activated alveolar macrophages from normal male rabbits, 3-3.5 kg, that had been injected iv Freund's complete adjuvant (Sigma Chemie, Deisenhofen, FRG) 4 and 3 weeks before cell harvest were collected by lung lavage according to the method of Myrvik et al. (1961) using a lavage fluid described by Castranova et al. (1980) containing 100 units/ml penicillin and 100 ixg/ml streptomycin (Serva Feinbiochemica, Heidelberg, FRG). The rabbits were killed by rapid iv injection of a 5-ml solution containing 200 mg sodium pentobarbital and 32.7 mg heparin. The thorax was opened and blood was removed by heart punctation. A cannula was tightened in the trachea and the lungs were filled with the lavage fluid by means of a 10-ml syringe with a fixed polyvinylchloride tube until all the air was removed from the airways. The tube of the lavage device consisting of a separation funnel as reservoir and a siliconized collecting vessel connected by a three-way stopcock was fitted to the cannula and lungs were filled with a further 40 ml lavage fluid by gravity from the reservoir at a height of 25 cm. After 10 rain and repeated lung massages the lavage fluid was allowed to flow into

TOXICITY OF METALLIC IONS AND OXIDES

257

the collecting vessel. This procedure was repeated until 420 ml of the suspension was obtained. Cell cultures. Freshly harvested cells were f'dtered over sixfold gauze and centrifuged for 5 min at 500g and the pellet was resuspended in HBSS (pH 7.4) containing 25 mM Hepes (Serva) resulting in a cell density of 2 × 106/ml. For incubation assays the cells were plated in polystyrene culture dishes (Costar, Cambridge, MA) and allowed to affix for I hr at 37°C. For the 0 2 - assay 1.25 × 106 cells in 23-mm-diameter dishes were used and for the H202 assay 5 x 106 cells were cultured in 50-mm-diameter dishes. The supernatant containing inviable and nonmacrophagic cells was sucked off with a Pasteur pipet and the cells were incubated for 24 hr with 0.6 ml of suspended or dissolved compounds per 10 6 macrophages in DMEM (Biochrom, Berlin) containing I00 units/ml penicillin, 100 ~g/ml streptomycin, and 50 mM Hepes (Serva) without phenol red (pH 7.4). For chemiluminescence assays the cell suspensions in HBSS were centrifuged for 5 min at 500g and the pellets were resuspended in DMEM. Cells (5 × 106/3 ml) were allowed to attach to 50-mm-diameter siliconized glass dishes (Brand, Wertheim, FRG) for 2-5 hr at 37°C. After withdrawing the medium 3 ml of DMEM containing the metallic compounds was added to the cells and incubated for 24 hr at 37°C. Preparation of suspensions and solutionsfor incubation. All compounds except vanadium (V) oxide (extra pure, Merck, Darmstadt, FRG) and cadmium oxide (CdO brown, Riedel de Haen, Seelze, FRG) were used in analytical grade. Cadmium chloride and copper(II) oxide were purchased from Riedel de Haen and nickel(III) oxide from F h k a (Neu-Ulm, FRG). All other compounds were provided by Merck. Ten to one hundred milligrams of the compound was sonicated 10 min twice in 5 ml bidistilled water by means of a supersonic bath (Sonorex RK 255, Bandelin, Berlin) and heat sterilized. To obtain soluble arsenic and antimony compounds, a 5 u sodium hydroxide solution was added to oxide suspensions. For incubation the stock solutions or suspensions were diluted with DMEM to concentrations between 0 and 1000 ~mole element medium. Preparation ofopsonized zymosan. Zymosan (Sigma) was suspended in 154 mM NaC1 solution at a concentration of 12.5 mg/ml and heated for 30 min in a boiling water bath. After cooling, the suspension was resuspended in NaC1 solution to a concentration of 50 mg/ml. Two milliliters of the zymosan suspension was diluted with 8 ml fresh bovine serum for opsonization, and the mixture was incubated for 30 min at 37°C. The opsonized zymosan was collected by centrifugation at 2500g for I0 min and washed with NaC1 solution (fivefold volume of pellet). After final centrifugation the pellet was resuspended in NaC1 solution to a concentration of 10 mg zymosan/ml. Assay for 02- anion release. The secretion of superoxide anion was determined by means of the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c, as described by Johnston (1981b). Cultured macrophages (1.25 × 106) were rinsed twice with 1 ml of HBSS. The cells were incubated for 90 min at 37°C with 0.75 ml of a reaction mixture containing 80 ~M ferricytochrome c (Type VI, Sigma) and 50 ~g zymosan in HBSS in the presence and absence of 30 Ixg SOD

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(from bovine blood, Sigma). The amount of reduced cytochrome c in the supernatants after centrifugation at 10,000g for 2 min was determined spectrophotometrically at 550 nm and the O2- generation calculated using the extinction coefficient of 21.0 × 103 M- 1 c m - 1. Assay for 11202 release. The oxidation of scopoletin by H202, catalyzed by horseradish peroxidase, was measured fluorometrically as described by Nathan (1981). The supernatants from the dishes with 5 × 106 adherent macrophages per dish were collected for determination of L D H activity. The cells were rinsed twice with HBSS and incubated for 90 rain at 37°C with 3 ml reaction mixture containing 250 nmole scopoletin (Fluka), 1 purpurogalin unit of peroxidase (Type II, Sigma), and 2 mg zymosan in HBSS. The supernatant was cleared by centrifugation at 2500g for 10 min and fuorescence was recorded in an Aminco SPF 500 fluorometer with excitation at 350 nm and emission at 462 nm. H202 standards titrated with KMnO4 were used for calibration. Preparation ofluminol solution. Luminol (10 rag) (Fluka) was dissolved in 1 ml DMSO and in HBSS without glucose to a stock concentration of 100 ~g/ml. Before use the stock solution was diluted 1:10 or 1:20 with HBSS. Measurement of chemiluminescence. At the end of the incubation time the cells were carefully rinsed with HBSS and harvested by washing the dishes three times with 1 ml of air-saturated HBSS. At time intervals, 0.75 ml of cell suspension was removed and mixed with 150 pJ of air saturated HBSS, 10 txl of luminol, and 50 pJ of zymosan. Luminescence assays were conducted at 37°C using a luminescence analyzer Biolumat LB (Berthold, Wildbad, FRG). Light intensity was monitored continuously by a recorder and expressed in counts/2 sec. At the beginning of the measurements (time 0) the average background of samples was less than 35 counts/2 sec and was subtracted from the maximum of chemiluminescence. Determination of extracellular LDH activity. Membrane leakage was estimated by extracellular L D H activity according to the method of Bergmeyer and Bernt (1974). Centrifuged (2.5 rain, 10,000g) supernatants (100 Ixl) from 24-hr macrophage cultures were added to a mixture of 867 Ixl of a 0.67 mM pyruvate solution in 50 mM phosphate buffer (pH 7.5) and 33 pJ of a 6.37 mM N A D H solution in 57.6 mM sodium bicarbonate solution. The decrease of NADH concentration was measured photometrically at 334 nm. RESULTS The results of the measurements plotted as percentages of control level against element concentrations are shown in Figs. 1-10. In general, metallic ions and oxides inhibit the release of active oxygen species. Dose-response relationships for the effects on H202 and O2- release and chemiluminescence were similar for the same compound and they correlated mutually for CdO, A s 2 0 3 , N a V O 3, HgO, Sb203, ZnO, and Cu 2+ (coefficient of first-order correlation r >i 0.818). Extracel-

Firs. 1-10. H202 release, 0 2 - release, maximal chemiluminescence, and extracellular LDH activity of rabbit alveolar macrophages incubated with metallic ions and oxides. Values are means standard deviation of three to nine experiments. CL, chemiluminescence.

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lular L D H activity increased with increasing concentrations of metals and correlated with release of active oxygen species and chemiluminescence for CdO, A s 2 0 3 (without chemiluminescence), Sb203, and ZnO (r ~< -0.882). Because Hg inhibited LDH in the measuring system this parameter could not be calculated. The investigated compounds could be divided into three different groups: 1. CdO, C d 2+ , A s 2 0 3 , and NaVO 3 caused significant differences (P < 0.001) from control values concerning the effects on active oxygen species at element concentrations t>10 ~M and on L D H release at element concentrations I>100 IXM. 2. HgO, Sb203, Cu 2+, Zn 2+, and ZnO caused significant differences (P < 0.001) from control values for all measured parameters at element concentrations I>100 ~M. 3. PbO2, Ni203, and SnO2 did not cause significant effects at element concentrations ~<500 IxM. In general the effects of metallic ions were similar to the effects of oxides for the same element. Lead ions decreased chemiluminescence more effectively (P < 0.001) than lead oxides did. Lead ions were also more toxic toward alveolar macrophages than PbO and PbO2 as evaluated by 0 2 - and H202 release (P < 0.05). Hg 2+ ions inhibited active oxygen species more than HgO. On the other hand the effects on O2- and H202 release were significantly higher by SbzO 3 than by SbO2-. IDs0 values, i.e., the element concentration which results in 50% inhibition compared to control levels, were calculated by intrapolation. The IDso values of H202 release for different compounds are summarized in Table 1. CdO caused the greatest inhibition, while Ni203, PbO2, and SnO2 had no significant effect up to a dose of 500 ~M. The order of potency for inhibition of hydrogen peroxide release TABLE 1 INHIBITORY EFFECTS OF METALLIC IONS AND OXIDES ON H202 RELEASE OF CULTIVATED RABBIT ALVEOLAR MACROPHAGESa Compound CdO Cd 2+

NaVOa AS203

HgO Zn2+ ZnO Sb203 Cu 2+ Ni203 Pb02 Sn02

ID5o (~M element) 5.5 (+1.8) 6.6 (+-1.1) 20.0 (-+20.0) 22.6 ( - 10.5) 70.8 (-+19.4) 76.3 (+-9.5) 82.6 (-+9.4) 100.0 (± 18.4) 181.4 (-+20.3) >500 >500 >500

IDso = concentration required for 50% inhibition -+ standard deviation (calculated by intrapolation), n = 3 to 9. a

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was CdO -~ Cd 2+ > NaVO3 ~ AS203 ~ HgO ~ Z n 2 + ~ ZnO > Sb203 > C u E ÷ > Ni203 ~ PbO 2 ~ SnO2. IDso values for the effects on superoxide anion release are shown in Table 2. The order of potency for inhibition of superoxide anion release was Cd > AszO3 > Cu 2+ = NaVO3 > Zn 2+ > Sb203 > HgO > PbO 2 ~ Ni203 ~ SnO 2. The effects of metals on chemiluminescence are shown in Table 3. The order of potency of inhibition determined from IDso values w a s A s 2 0 3 ~ C d O ~ NaVO3 HgO > Cu 2+ -~ Sb203 > ZnO > PbO2 ~ SnO2. The effects on extracellular LDH activity of various compounds at element concentration of 100 ~M are summarized in Table 4. The potency of the metals in increasing membrane leakage was CdO ~ As203 ~ V205 -~ NaVO 3 > Sb203 > CHO = Sn 2+ ~ Ni203 > PbO ~ SnOz.

DISCUSSION In this study, we improved the method for a long time incubation of alveolar macrophages for chemiluminescence assay of cells in suspensions, Activated rabbit alveolar macrophages were cultured in siliconized glass dishes. The cells formed weakly affixed monolayers on the dishes and after incubation they could readily be harvested and resuspended. Maximal chemiluminescence of 24-hr incubated macrophages as well as of freshly harvested cells occurred about 15 min after exposure to zymosan. The time course of chemiluminescence was similar to that observed by Miles et al. (1978) with rabbit alveolar macrophages, but it was different from that observed with rat alveolar macrophages which were harvested by trypsin treatment after a 9-hr incubation in glass dishes yielding the maximal chemiluminescence immediately (time 0) after exposure to zymosan (Galvin and Oberg, 1984). Obviously, the chemiluminescence-triggering excitation in those experiments was the treatment with trypsin or mechanical perturbation during the removal of the intensely affixed cells. TABLE 2 INHIBITORY EFFECTS OF METALLIC IONS AND OXIDES ON 0 2 RELEASE OF CULTIVATED RABBIT ALVEOLAR MACROPHAGESa

Compound

IDso (~M element)

CdO AszO3 Cu 2+ NaVO 3 Zn 2+ ZnO SbzO3 HgO PbOz Ni203 SnO2

6.6 (-+0.5) 15.1 (-+6.8) 40.6 (-+21.6) 41.2 (-+7.2) 88.3 (-+6.5) 101.8 (-+23.8) 125.7 (+ 167.1) 145.0 (-+52.8) >500 >500 >500

a IDso = concentration required for 50% inhibition -+ standard deviation (calculated by intrapolation), n = 3 to 6.

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TABLE 3 INHIBITORY EFFECTS OF METALLIC IONS AND OXIDES ON CHEMILUMINESCENCE OF CULTIVATED RABBIT ALVEOLAR MACROPHAGESa IDso

Compound

(~M element)

As203 CdO NaVO3 HgO Cu 2+ Sb203

0.7 (-+0.3) 3.8 (-+2.4) 6.1 (+1.4) 9.7 (-+2.8) 42.7 (+24.6) 57.0 (-+60.0) 125.0 (+-75.0) >500 >500

ZnO PbO 2 SnO 2

a IDso = concentration required for 50% inhibition -+ standard deviation (calculated by intrapolation), n = 3 to 5.

Cd(II), As(III), and V(V) were the most toxic of the elements tested in this study while Sn, Ni, and Pb compounds exhibited no toxicity up to an element concentration of 500 ~M. This result agrees with studies by Waters et al. (1975) which demonstrated that Cd 2 ÷ and VO3- are unique macrophage toxicants. An IDso value of VO3- for chemiluminescence of bovine alveolar macrophages similar to the results of this study was described by Wei and Misra (1982). The ID5o values for reduction of active oxygen species release by Cu(II), Hg(II), Zn(II) and Sb(III) were approximately 10-fold higher than for Cd(II), As(III), and V(V). The same order of toxicity was observed on other cell parameters for Cd 2 +, Cu E+, and Zn2+ by Hatch et al. (1985) and for V(V), As(III), and Zn(II) by Fisher et al. TABLE 4 EFFECTS OF METALLIC IONS AND OXIDES AT AN ELEMENT CONCENTRATION OF 100 I.LMON EXTRACELLULAR L D H ACTIVITY OF CULTIVATED RABBIT ALVEOLAR MACROPHAGESa 100 bI,M -- effect

Compound CdO As203 V205 NaVO3 Sb203

ZnO PbO2 CuO Sn 2+ Ni203 PbO SnO2 Ni 2÷ " Values are means - standard deviation of 3 to 9 experiments.

(% of control) 1071.1 953.5 899.0 888.1 348.8 164.0 155.2 145.3 141.3 140.0 114.2 102.3 99.2

(_+64.3) ( - 191.9) (-+45.4) (-+ 12.4) (-+95.3) (-+37.3) (-+47.0) (-+18.9) (+-4.6) (-+68.0) (-+ 1.6) (---7.0) (-+2.0)

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(1986). The order of potency of inhibition determined from IDso values of chemiluminescence for the investigated compounds are generally in agreement with results reported by Castranova et al. (1980, 1984) using rat alveolar macrophages. However, they found in their short-time exposure experiments Cd 2+ to be less toxic than Cu 2+ , Hg 2+ , Zn 2+ , and Ni 2+ . The difference between their results and those presented in this report may reflect different conditions concerning incubation modalities or species sensitivities. The effects on H202 and 0 2 - release, chemiluminescence, and extracellular LDH activity can be reviewed to evaluate which parameter is the most sensitive indicator of cellular damage. Evaluation of element concentrations yielding toxicity parameters significantly different from control values (P < 0.001) indicates that assays of active oxygen species, including chemiluminescence, were more sensitive than extracellular LDH activity especially for the most toxic compounds. The orders of potency for inhibition of hydrogen peroxide and superoxide anion release and chemiluminescence based on IDs0 values were only slightly different. An exception was Cu(II) which decreased O 2- release and chemiluminescence fivefold more than H202 release. This result may reflect that Cu 2+ ions bind to target biomolecules and these protein-Cu(II) complexes can be reduced by 0 2 radicals yielding protein-Cu(I) complexes (Samuni et al., 1981). In this way superoxide anion could possibly react faster with Cu(II) complexes than, e.g., with ferricytochrome c in the in vitro assay or with bacterial constituents in vivo. In addition, the chemiluminescence reported in this study is inhibitable by superoxide dismutase, i.e., is closely related to O2- concentrations. Comparison of in vitro depression of the determined parameters by oxides and soluble compounds showed that the effects of metal compounds on alveolar macrophages generally were not dependent on the solubility of the compound with the exception of lead and mercury compounds being more toxic in soluble form and antimony which was less toxic if employed as oxide. These results contrast with investigations done by Waters et al. (1974) who found different toxicities of soluble and insoluble vanadium compounds toward alveolar macrophages. It is interesting that only the effects on active oxygen species exhibited significant differences between oxides and metallic ions of the three elements Pb, Sb, and Hg. The in vitro results of the present study are consistent with increased susceptibility to bacterial infections of mice which were treated with HgO, CdO, ZnO, and NaAsO 2 by intratracheal injection (Hatch et al., 1985) or As203 by inhalation (Aranyi et al., 1985). Ni compounds were neither immunotoxic in vitro as investigated in this work nor/n vivo (Hatch et al., 1981). It is very interesting that the rank order of in vitro toxicity of three metals (Cd 2+ > Cu 2+ > Zn 2+) measured by O2- release and chemiluminescence depression in this work corresponds to in vivo pulmonary immunodepression data of Ehrlich (1980) and Gardner et al. (1977). These data are summarized in the study of Hatch et al. (1981) who also found that intratracheal injection of CuSO 4 [but not CuO (Hatch et al., 1985)] increased the susceptibility to pulmonary bacterial infection to a degree similar to that of intratracheal injection of CdSO4 or ZnSO4. Because Cu 2+ has a far less dramatic effect on depression of H202 release or membrane

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integrity, 02- release by alveolar macrophages seems to be a potent mechanism in pulmonary defense against microbes. A general conclusion of this study is that ions and oxides of Cd(II), As(III), V(V), Cu(II), Hg(II), Zn(II), and Sb(III) in the micromolar range can affect the viability of alveolar macrophages and decrease their ability to release antibacterial substances. Therefore, inhalation of particles containing these elements may impair the pulmonary defense system and increase susceptibility to bacterial infections. ACKNOWLEDGMENTS The authors thank Mrs. Andrea Puschmann, Mrs. Helga Kahlert, and Ms. Sabine Birgfeld for technical assistance and Mr. Janusz Izykowski for assistance in graphic representations. This study was supported by the Forschungsbereich Umweltschutz und Umweltgestaltung of the University of Hamburg.

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