Toxicity and ultrastructural localization of mercuric chloride in cultured murine macrophages

Arch Toxicol (1988) 62:440-446 Archives of

Toxicology �9 Springer-Verlag 1988

Toxicity and ultrastructural localization of mercuric chloride in cultured murine macrophages

Margot Christensen 1, Soren C. Mogensen 2, and J~rgen Rungby 1

Department of Neurobiology, Institute of Anatomy 2 Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark

Abstract. The effects of mercuric chloride on cell survival, phagocytosis and cell migration were examined in cultured mouse peritoneal macrophages, and the accumulation of mercuric chloride in the cells was visualized by autometallography and evaluated by light and electron microscopy. Macrophages exposed to mercury concentrations from 1.25 ~tM to 10 I.tM mercuric chloride showed a concentration- and time-dependent increase in mercuric chloride accumulation, while cells exposed to 20 gM and 40 I.tM mercury showed an inverse relationship between mercury concentration and the accumulation of mercury. Mercury concentrations above these levels caused cell necrosis. Electron microscopy revealed that mercury was lo- cated primarily within lysosomes but also in the nucleus and cytoplasm. Mercury increased the death rate of macrophages in a concentration-dependent manner when cells were treated with mercury concentrations not causing cell necrosis. Further, we found that mercury clearly impaired macrophage random migration and possibly the capability for phagocytosis.

Key words: Macrophages - Mercuric chloride - Auto- metallography - Toxicity - Migration - Phagocytosis

Introduction

Cellular toxicity in response to mercury has been demonstrated in various cell types. Mercuric chloride induces necrosis of the renal tubular epithelium and hepatocytes in rats (Lindh and Johansson 1987), renal necrosis in rats (Parizek et al. 1967) and induces lipid peroxidation in rat hepatocytes (Stacey and Kappus 1982).

Because of their ability to migrate and phagocytize, macrophages play an important role in the defence against infections. A number of heavy metals affect macrophage function. Mercury has been shown to inhibit cellular re- sponses in alveolar macrophages such as increase in oxygen consumption and release of superoxide anions (Cas- tranova et al. 1980). Silver increases the death rate of murine peritoneal macrophages (Rungby et al. 1987) and shows autointerference of silver accumulation at higher concentrations of silver (Ellermann-Eriksen et al. 1987). However, macrophage functions like phagocytosis, ran-

Offprint requests to: M. Christensen

dom migration and interferon production were not affected by silver. Lead and cadmium were found by Koller and Roan (1977) to stimulate phagocytosis by peritoneal macrophages, whereas others have found cadmium to cause a reduced phagocytic activity of macrophages (Loose et al. 1978; Castranova et al. 1980). Macrophage mobility is impaired by zinc (Zukoski et al. 1974).

The present study was carried out to investigate the accumulation of mercury in cultured murine macrophages and the intracellular localization of mercury, and to pro- vide estimates of mercuric chloride toxicity to macrophages by examining cell survival, phagocytosis and random migration.

Materials and methods

Cell cultures. Mouse peritoneal macrophages (MPMs) were obtained by peritoneal lavage from 8-12-week-old specific pathogen-free BALB/c mice of both sexes. The cells were harvested by standard procedures (Ellermann-Erik- sen et al. 1987) in phosphate-buffered saline (PBS) supplemented with 10% foetal calf serum and 20 IU/ml heparin, collected in one pool and kept cool to prevent adhesion. After washing the cells were suspended in RPMI-1640 medium supplemented with 1% glutamine, 1% HEPES, 10% foetal calf serum, 200 IU/ml penicillin and 200 txg/ml streptomycin. Experiments were performed with resting macrophages, except the assay of random migration, in which macrophages from mice stimulated with light mineral oil (paraffin) were used.

Visualization of mercuric chloride by autometallography. The harvested macrophages were counted in a hemocy- tometer and 1 x t06 cells in 1 ml medium were seeded on round glass coverslips (diameter 13 mm; Chance Propper, England) in 24-well tissue culture multidish trays (NUNC, Roskilde, Denmark). MPMs were allowed to adhere to the coverslips at 37~ overnight, after which non-adherent cells were removed by washing coverslips in tissue culture medium-199 (TC-199). Experimental solutions were prepared by dissolving mercuric chloride in redistilled water and adding this to RPMI medium. The macrophages were transferred to trays containing freshly prepared medium with various concentrations of mercuric chloride (0, 1.25, 2.5, 5, 10, 20, 40, 80 t.tM), and cultured at 37 ~ C in a humidi- fied 5% CO2 atmosphere. After various exposure periods (2, 6, 12, 24, 48 h) the cells on coverslips were washed again

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and fixed in 3% glutaraldehyde in 0.15 M phosphate buffer. The fixative was changed after 10 min and the macrophages were postfixed for 12 h.

Light microscopy. The coverslips were dried and covered with protecting gelatine and after drying again for 1 h the cells were exposed to physical development in a dark box at 26~ for 70 min according to Danscher and Moller- Madsen (1985). The developer contained silver-lactate, hy- droquinone, citrate buffer and a protecting colloid based on gum arabic.

During development, intracellular accumulations of mercury sulphide or mercury selenide catalyze the hydro- quinone reduction of the silver ions by acting as an elec- trode that conducts electrons from the reducing molecules to the surface-bound silver ions. This results in the trans- formation of silver ions to metallic silver adherent to the surface of the catalyst, thus forming a shell of silver. This layer increases in size as long as the developer is present. Thus, mercury sulphide and mercury selenide in the macrophages are visualised by silver staining. After development coverslips were washed gently in running tap water at 40 ~ C for at least 40 min to remove the gelatine, dipped in Farmer's solution for 10 s and rinsed in redistilled water. They were then dipped in fixative and rinsed again. Fi- nally, the macrophages were counterstained with 1% toluidine blue, dehydrated in alcohol and xylol and embedded in permount on glass slides.

The percentage of cells containing silver grains was determined in the light microscope (magnification x 400), and the degree of intracellular silver staining in individual cells was evaluated semiquantitatively according to Eller- mann-Eriksen et al. (1987) on a 0 -5 scale: 0: no mercury visible; 1: a few just visible grains; 2: a few small but distinct grains; 3: many medium sized grains; 4: numerous medium to large sized grains; 5: diffuse staining covering the cytoplasm.

Electron microscopy. MPMs were exposed to mercuric chloride in various concentrations and with exposure periods as described above. After fixation in 3% glutaraldehyde cells were dehydrated in propylene oxide and embedded in Epon. Sections of the cells 3 ~tm thick were cut of the blocks and mounted on glass slides. After autometallography as described above and examination in light microscopy sections of interest were re-embedded in Epon. Ultra- thin sections were cut and counterstained with uranyl acetate and lead citrate. The cells were examined by trans- mission electron microscopy.

Long-term toxicity of mercuric chloride. MPMs in RPMI- 1640 medium were counted and 20 x 10 6 cells in 10 ml of medium were cultured in Falcon tissue culture flasks and allowed to adhere overnight at 37~ The cells were washed 4 times in TC-199 medium supplemented with 2% foetal calf serum. Medium containing 0, 2, 4, 8, 16 or 32 ~tM mercuric chloride was added. In each flask five areas with a medium density of macrophages were selected and marked. Adherent cells in these particular areas were counted in an inverted phase contrast microscope with a squared grid mounted in the eyepiece. Cell death was thus determined by the decrease in the number of adherent cells (dead cells detached from the culture substrate and disinte- grated in the medium). At the same time characteristics of

the macrophage morphology were noted. The cells were counted immediately after exposure to mercury and thereafter at 48-h intervals. The total cell numbers on days 2, 4, 6, 8, 10 were expressed as percentages of the cell count at day 0. Cell counts were performed blindly.

Assay of macrophage random migration. In these experiments a microdroplet method described previously by Mogensen (1982) was used. Stimulated peritoneal macrophages (SPMs) were obtained by peritoneal lavage of BALB/c mice inoculated intraperitoneally with 3 ml ster- ile light mineral oil 4 days before cell harvest. Agaros e microdroplets containing 5 x 105 peritoneal cells in 1111 were placed in the center of the wells of Falcon tissue culture plates (Microtest 11) and overlayed with 0.2 ml medium containing various concentrations of mercuric chloride (0, 2, 4, 8, 16, 32, 64, 128 pM HgCI2). The area of the cell migration from the microdroplet after overnight incubation was calculated by measuring two perpendicular diam- eters of cell migration in an inverted phase contrast microscope.

Assay of macrophage phagocytic activity. In this assay MPMs on coverslips were used. The cells were exposed to mercuric chloride (0, 2, 4, 8, 16, 32, 64 I.tM) in 2 ml medium and covered with latex particles (Difco, diameter 1.6 txm, approx. 10 7 particles/ml) in RPMI-1640 medium. The multidish trays were centrifuged to ensure contact between latex particles and the MPMs. After incubation for 30 min at 37 ~ C the cells were fixed in 3% glutaraldehyde, mounted in 50% glycerol-PBS and the amount of phagocytized latex particles was evaluated by phase contrast microscopy.

Statistics. All data were evaluated using Student's t-test (two tailed) for non-paired data.

Results

The localization of mercury in MPMs

Light microscopy. Macrophages cultured in mercury-free medium contained no autometallographic grains. The cells were fusiform or spherical and the nucleus was oval with condensed chromatin. Cells exposed to mercuric chloride showed autometallographic staining; with a low density of grains these were scattered in the cytoplasm and in the cy- toplasmatic extentions. In macrophages with a high density of grains they were situated primarily in the perinuclear re- gion. The morphology of cells exposed to concentrations of mercuric chloride up to 20 ~tM was normal, whereas cells exposed to 40 IxM mercuric chloride were affected by the mercury. In some cells the nucleus was pycnotic and the chromatin had lost its characteristic appearance and localization. Large vacuoles filled the cytoplasm, giving rise to swollen cells. Mercury staining was found scattered in the cytoplasm. In some cells the cell membrane had vanished and left the pycnotic nucleus surrounded by re- mains of the cytoplasm and extracellular mercury precipitates were observed (Fig. 1).

Electron microscopy. At the electron microscopical level autometallographic grains were situated mainly within lysosomes. However, precipitates were also found inside the nucleus and scattered in the cytoplasm (Fig. 2). A few

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Fig. 1. Light micrographs of toluidine blue stained MPMs exposed to medium containing a no mercuric chloride, b 1.25 ~tM, e 5 p.M, tl 10 I.tM, e 40 IxM or f 80 txM of mercuric chloride for 24 h and cells exposed to 10 I-tM mercuric chloride for g 12 h and h 48 h. In a, h, c, d, g and h the macrophages are normal and well-stretched, whereas cells in e have vacuolized cytoplasm or are pycnotic. In f the cell nucleus is pycnotic and the cell membrane and cytoplasm have vanished, b, e and d show concentrat ion-dependent mercury accumulation; cells in b have few a~d small grains (single arrow), cells in e have many medium sized grains and MPMs in d have numerous large sized grains. The double arrow in b shows MPM with engulfed mast cell granules, g and h show that mercury accumulation increases with exposure time; g showing few but distinct grains, h showing maximal staining. Magnification • 400

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Fig. 2. Electron micrographs showing macrophages exposed to 20 gM mercuric chloride for 2 h. In a autometallographic grains ate seen in lysosomes as well as in the nucleus and cytoplasm (x 7000). b shows larger magnification of macrophage (x 14000) with grains in lysosomes and nucleus. In general, most autometallographic grains were lysosomal

3 a 1 0 0

I I I I I 3b

; / I I I I I

2 6 12 24 48 TIME OF MERCURY EXPOSURE (HRS.)

Fig. 3. Kinetics of mercury accumulation in macrophages. MPMs were cultured in medium containing (O) 1.25 I.tM, (A) 2.5 p.M, (O) 5 ~tM, (O) 10 IxM, (&) 20 IxM or (11) 40 I.tM mercuric chloride. At each time indicated, cells were fixed and stained for mercury by autometallography. The percentage of cells containing grains a was determined by light microscopy. The degree of mercury staining b was graded semiquantitatively on a 0-5 scale

A 80

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Fig. 4. Percentage of adhering MPMs in culture at different time intervals after exposure to various concentrations of mercuric chloride: a control medium; b 2 ~tM; c 4 p.M; d 8 IxM; e 16 I.tM; f 32 IxM mercuric chloride. The values of each graph represents the mean percentage adhering cells and the vertical bars 1 s.d. in five culture areas

grains were found in mitochondria and some in the rough endoplasmic reticulum (RER). These findings were not re- lated to any specific mercury concentrat ion or time of exposure.

Influence of concentration and time of exposure to mercury on the amount of autometallographic grains

The results of the examinat ion in the light microscope of MPMs are illustrated in Fig. 1 and summarized in Fig. 3. Cells treated with 2.5 IxM, 5 txM and 10 IxM mercuric chloride showed mercury deposition after 2 h while cells exposed to higher or lower concentrations showed deposi- tions after 6 h. Examinat ion of cultures exposed to mercury for increasing time periods showed that for all concentrations of mercury the percentage of stained cells increased with exposure time. The same relation was found between the degree of mercury staining in individual cells

444

1 2 0 _

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I I I I I I I I c 2 4 8 16 32 64 128

MERCURY CONCENTRATION ( p M )

Fig. 5. Random migration of macrophages exposed to mercuric chloride. Mouse SPMs were placed in agarose microdroplets and overlayed with medium containing from 2 to 128 I-tM mercuric chloride. After overnight incubation the migration area was calculated and expressed as percentage of the mean area of migration from droplets overlayed with medium containing no mercuric chloride (controls). The dotted lines indicate the mean _+ SD of control cultures. Each point represents the mean of six cultures and the vertical bars l s.d.

and the exposure time. The maximal increase was found in cultures treated with l0 I.tM mercuric chloride and will be described in some detail. After exposure for 2 h, 6 h and 12 h these cells contained a few but distinct grains of mercury and the percentage of stained cells rose from 13% at 2 h to 30% at 12 h. Treatment for 24 h with l0 IxM mercury increased the percentage to 83 and the cells contained numerous medium to large sized grains. After 48 h exposure to this mercury concentrat ion all the cells were stained and the density of the staining was extensive. Comparing cell cultures exposed to medium containing 1.25 IxM, 2.5 IxM, 5 ~tM and l0 txM mercury revealed that the mercury staining increased with the concentrat ion of mercury. Cells exposed to medium containing 201.tM mercury contained just visible grains of mercury at 6 h and at 24 h 20% of the cells were stained and the grains were small and of low density. Maximal staining was found after 48 h of treatment with 20 IxM mercury. Mercury staining only reached 39% in cells treated with 40 txM mercury and the grains were medium to large in size but the density was lower than in cells treated With l0 p.M or 20 IxM mercury. Thus an inverse relationship between mercury concentration and mercury accumulation was present at concentrations above l0 p.M (autointerference). At 801.tM mercury all cells were dead and contained no intracellular mercury.

Fig. 6. Phase contrast micrographs showing phagocytic activity of macrophages pre-exposed to mercury chloride. MPMs were cultured in medium containing a no mercuric chloride, b 16 IxM, c 32 IxM or d 64 IxM of mercuric chloride for 24 h and thereafter allowed to phagocytize latex particles for 30 min. In a and b the macrophages are distended having phagocytized many particles, while cells in e show fewer engulfed particles. Macrophages in d are vacuolized and contain no particles. Magnification x 500

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Long-term toxicity of mercuric chloride

Preliminary experiments showed that mercury concentrations above approximately 40 p-M were highly toxic after 24 h exposure. For this reason mercury concentrations below 40 p-M were chosen for the experiment. Cell count data are shown in Fig. 4. No significant differences were found between the mean values on day 2. However, after 4 days of exposure cell counts in cultures treated with as little as 2 p-M mercury were dramatically lowered as com- pared with non-treated cultures (p < 0.001). Increasing the mercury concentration gradually from 2 to 16 p-M had a comparatively smaller effect on the macrophages, al- though values for 2 and 16 IxM were significantly different (p <0.001). A further increase in concentration to 32 p-M left only a few macrophages adhering beyond day 4.

The results showed that mercuric chloride significantly affects the viability of macrophages in a concentration dependent manner. The morphology of adherent cells treated with medium containing 2 I.tM or 4 p.M mercury was normal throughout the experiment (fusiform or spherical cells with no signs of pycnosis). Cells exposed to 8 I.tM or 16 p.M mercury maintained their spindle shape but seemed slimmer after day 6, while cells exposed to 32 I.tM were small and pycnotic already at day 2 and continued to have this appearance throughout the experiment.

Effects of mercuric chloride on random migration and phagocytic activity of macrophages

The capacity of stimulated peritoneal macrophages for random migration in medium con'caining mercuric chloride was examined (Fig. 5). The migration of cells exposed to 2 p.M, 4 p.M and 8 IxM mercury was normal when com- pared to cells migrating in medium only. Cells migrating in medium containing 16 p-M mercury showed a migration area 50% smaller than controls (p <0.001). Cells treated with mercury concentrations of 32 p.M, 64 IxM or 128 IzM did not migrate at all. This is in accordance with the result of the autometallography experiments which indicated that macrophages die when exposed to a mercury concentration of 40 p-M or above.

Figure 6 illustrates the phagocytic activity of macrophages pretreated with mercuric chloride for 24 h. Macro- phages exposed to medium containing mercury concentrations below 32 IxM were distended and filled with latex particles, while cells treated with 32 lxM mercury showed fewer engulfed particles and some had died. Medium containing 64 p.M mercury resulted in vacuolized necrotic macrophages containing no latex particles. Thus the phagocytic activity of macrophages was affected only at mercury concentrations shown by microscopy to be rapidly cyto- toxic.

Discussion

Autometallography demonstrates metals and molecules containing metals including mercury (Danscher and Moller-Madsen 1985; Danscher and Rungby 1986). By autometallography we found mercury mainly within the lysosomes, which is in accordance with previous studies, but also in the nucleus and scattered in the cytoplasm. Thus, mercury has been demonstrated in lysosomes of the renal tubule cell (Fowler et al. 1974; Madsen and Christen- sen 1978; Baatrup et al. 1986), in hepatocytes (Norseth and Brendeford 1971; Baatrup et al. 1986) and in neurones

(Danscher and Schroder 1979; Thorlacius-Ussing and Graaboek 1986).

It is well known that mercury has high affinity for sul- phydryl and selenium groups (Clarkson 1972; Magos et al. 1987) and forms complexes that can be visualized by autometallography (Danscher and Moller-Madsen 1985). His- tochemical demonstration of intracellular mercury by this method requires conglomerates of three to six or more molecules of mercury sulphide or mercury selenide (Danscher and Moller-Madsen 1985).

By using autometallography in fish exposed to mercuric chloride Baatrup et al. (1986) showed mercury in lysosomes only; however, by combined exposure to mercury and selenium, mercury was demonstrated in other organ- elles also. The presence of mercury in lysosomes might indicate a detoxifying process as indicated by Norseth et al. (1971), Fowler et al. (1974) and Sternlieb et al. (1976). However, if mercury binds to sulfhydryl groups of lysosomal enzymes, the presence of mercury within iysosomes may be of functional importance.

The mechanisms underlying mercury toxicity are wide- ly unknown. However, Stacey and Kappus (1982) found that mercuric chloride induced lipid peroxidation in iso- lated hepatocytes in a concentration- and time-dependent manner and that cellular toxicity of mercury was evi- denced by loss of cytoplasmic enzyme from the hepatocytes into the suspension medium. Other investigations have shown that mercury has a labilizing effect on lysosomal membranes (Lauwerys and Buchet 1972; Chvapil et al. 1972). Madsen and Christensen (1978) studied the effect of mercury on lysosomal protein digestion and found that the metal impaired lysosome capacity for protein degrada- tion. However, it was shown that the membranes of lysosomes were not disrupted during mercury intoxication. Our findings suggest that mercury may also impair cytoplasmic, mitochondrial and nuclear processes.

Defects in the functional capabilities of alveolar macrophages, which are part of specific and non-specific host defence mechanisms, have been studied in cells exposed to different metals and among these mercury (Castranova et al. 1980). The mechanisms most affected by mercury were oxygen consumption and production of reactive oxygen radicals. Additionally, this study showed that the phagocytic rate was inhibited by cadmium. Accordingly, other investigators (Loose et al. 1978) found that the phagocytic capacity of macrophages was severely affected by cadmium and that this metal had some effect on the microbicidal activity. These studies are not in accordance with an ear- lier study by Koller and Roan (1977) showing that the phagocytic activity was enhanced in macrophages treated with lead and cadmium. We found that the morphology and ultimate survival of macrophages as well as their migrating and possibly phagocytic capacities were affected by mercuric chloride, suggesting that an organism exposed to this substance may have decreased macrophage functions.

The autointerference of the autometallographic visuali- sation of mercury demonstrated in this study may also be regarded as an impairment of macrophage function, as- suming that the amount of lysosomal mercury is the result of a detoxifying process. A similar phenomenon has been described in silver toxicity (Ellerman-Eriksen et al. 1987).

Mercury altered the morphology of cells after long- term exposure while short-term exposure to mercury made

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no obvious structural changes in macrophages. Cell necrosis was found in macrophages exposed to mercury concentrations above 40 IxM.

The present investigations suggest that mercuric chloride is toxic to macrophages in vitro, affecting basic macrophage functions.

Acknowledgements. We thank Lone Podenphant and Bjarne Krunderup for excellent technical assistance and Gorm Danscher for advice and financial support. This study was supported by the Aarhus University Research Foundation.

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Received June 22, 1988/Received in revised form September 12, 1988/Accepted September 19, 1988

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Toxicity and ultrastructural localization of mercuric chloride in cultured murine macrophages