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Tissue & Cell, 1999 31 (2) 223–232 © 1999 Harcourt Brace & Co. Ltd Article no. tice.1999.0021
Tissue&Cell
The ultrastructural effect and subcellular localization of mercuric chloride and methylmercuric chloride in insect cells (Aedes albopictus C6/36) B. Braeckman, H. Raes
Abstract. The ultrastructural effects of mercuric chloride (Hg) and methylmercuric chloride (MeHg) were studied in the Aedes albopictus C6/36 cell line. Both metal salts caused nuclear indentations, chromatin clumping and proliferation of the nucleolus. The mitochondria became pleomorphous. An increase of both free and membranebound ribosomes, swelling of the rough endoplasmic reticulum caused by accumulated protein and the appearance of well developed Golgi stacks all indicated the activation of protein synthesis. The activation was probably a cellular response to general stress, and the synthesized proteins may be members of the heat shock protein family. Apart from these common ultrastructural features, Hg-treated cells showed typical clusters of small electron-lucent vacuoles near the Golgi stacks. In cells exposed to MeHg, cytoplasmic tube-like structures were often observed and the disorganization of the organelles together with the appearance of blebs suggested disruption of the microtubules. Mercury accumulation was localized by an autometallographical silver staining technique both at the light and electron microscopic level; silver deposits were quantified by image analysis. For both Hg- and MeHg-treated cells, the degree of silver staining increased rapidly with increasing exposure time, but a considerable heterogeneity within the cell population was found. Lysosomes proved to be the major mercury storage sites in the Aedes cells and silver deposits could already be found after 30 min of Hg treatment. At sublethal concentrations, Hg inhibited the lysosomal marker enzyme acid phosphatase to some extent. For MeHg, no effect on this enzyme was found.
Keywords: Mercuric chloride, methylmercuric chloride, ultrastructure, insect cell line, autometallography, acid phosphatase
Introduction Mercury and cadmium (Cd) are some of the most toxic heavy metal contaminants. Environmental monitoring of these pollutants has become a major issue in the last decades. The use of insects as biomonitors for heavy metal contamination in terrestrial and aquatic ecosystems has been Department of Biology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
Received 2 December 1998 Accepted 2 February 1999 Correspondence to: Bart Braeckman. Tel: (+32) 9 264 52 22; Fax: (+32) 9 264 53 44; E-mail: Bart. Braeckman @rug.ac.be
reported to have a great potential (Seidman et al., 1986; Walton, 1989; Hare, 1992). We use the Aedes albopictus C6/36 cell line as a model to study heavy metal toxicity in insects at the cellular level. In previous research, we studied the effect of Cd on Aedes cells at the ultrastructural level (Braeckman et al., 1999a). Since mercury, like Cd, has a high affinity for sulfhydryls, we wanted to investigate the effect of this metal on subcellular morphology in a comparative way. In this study we examine the effects of mercuric chloride (Hg) and methylmercuric chloride (MeHg) as representatives of inorganic and organic mercury species. Both mercurials were shown to have a different effect on cell morphology at the light microscopical level (Braeckman et al., 1997a) and on 223
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the viability and proliferation of Aedes albopictus cells (Braeckman et al., 1997b). A study of the ultrastructural pathology may give important information on the subcellular targets of Hg and MeHg. We also study mercury accumulation histochemically using autometallography (Danscher et al., 1994). At the light microscopic level, the thereby formed silver deposits are quantified by image analysis. At the electron microscopical level the method enables us to localize the accumulation of mercury in the cells. As mercury is found to accumulate mainly in the lysosomes, the activity of acid phosphatase, which is a lysosomal marker enzyme, is also tested.
Materials and methods Materials HgCl2 and MeHgCl were purchased from Merck (Darmstadt, Germany). Lactalbumin hydrolysate, fetal calf serum and gentamicin were obtained from Gibco BRL (Paisley, UK). Yeast extract was a product of Oxoid (Unipath Ltd, Basingstoke, UK). Paraformaldehyde and 4-methylumbelliferyl phosphate were purchased from Sigma (St Louis, USA). Cacodylic acid, glutaraldehyde, OsO4, lead acetate and uranyl acetate were products from Fluka (Buchs, Switzerland). The epoxy resin kit (Araldite) was purchased from Agar Scientific Ltd (Stansted, UK). Propidium iodide was obtained from Molecular Probes (Eugene, Oregon, USA). DePeX was a product from BDH (Poole, UK). All other chemicals were purchased from Merck (Darmstadt, Germany). Culture recipients were obtained from Nunclon (Roskilde, Denmark). Cell culture In all our experiments we used Aedes albopictus cells (Diptera: Culicidae) belonging to the C6/36 clone, produced by lgarashi (1978) from a cell line of mixed embryonic origin (Singh, 1967). They were grown in modified Kitamura (MK) medium (132 mM NaCl, 8 mM KCl, 0.82 mM CaCl2, 0.86 mM KH2PO4, 24 mM D-glucose, 0.77% lactalbumin hydrolysate, 0.57% yeast extract, 5% fetal calf serum, 50 µg/ml gentamicin) initially described by Kitamura (1966). The pH of the medium was 6.8. The cultures were kept in the dark under ambient atmosphere at 27°C. The cells were routinely seeded at a density of 105 cells/ml. Under these conditions the cell population doubling time was about 30 h. For all experiments we used 7-day-old cultures. Metal exposure For each experiment, metal stock solutions were freshly prepared. HgCl2 and MeHgCl were dissolved directly in MK medium and, after filter sterilization, 5% fetal calf serum was added. These solutions were diluted to a final concentration of 40 µg/ml and used as a stock to prepare the appropriate treatment concentrations.
Evaluation of cell viability with the propidium iodide assay Cell viability was assessed by a dye exclusion test based on propidium iodide staining (Nieminen et al., 1992), adapted for Aedes cells. The method is basically a test for the membrane integrity and has been described in detail in Braeckman et al. (1997b). Cell cultures, at a final concentration of 1.0×106 cells/ml, were exposed for 24 h to the following HgCl2 concentrations: 0.5, 0.9, 1.9, 2.8, 3.7, 5.6, 7.4, 11.1, 14.8, and 22.2 µM. For MeHgCl, we used 0.4, 0.6, 0.8, 1.2, 1.6, 2.4, 3.2, 4.8, 6.4, and 9.6 µM. The viability curves show the average of two experiments, each containing eight replicates (Fig. 1). For the ultrastructural studies, metal concentrations in the LC10/24 h-LC50/24 h region were chosen in order to obtain the whole range of mercury provoked cellular injury (9.3 µM for Hg and 5.5 µM for MeHg). Electron microscopy and autometallographical procedure Cell suspensions (approx. 5×106 cells/ml) were treated with Hg (9.3 µM) or MeHg (5.5 µM) for a 6 h and 24 h period. For the autometallography (AMG), also shorter treatment times were applied (10 min, 30 min, 1 h, and 3 h). Prior to fixation, the MeHg samples were treated for 40 min with a 40 µg/ml Na2SeO3.5H2O solution to obtain Hg-Se complexes which can initiate the AMG reaction (Baatrup & Danscher, 1987). In preliminary experiments, this Se concentration and treatment time gave optimal staining results and caused no viability decrease (data not shown). The samples were fixed for 1 h at room temperature (2% glutaraldehyde and 2% paraformaldehyde in 0,1% cacodylate buffer, containing 60 mM of sucrose and 0.05% CaCl2, and adjusted to a pH of 7.4) and successively pelletted. In the samples that served for mercury localization, the fixative was replaced by the AMG developer. One hundred millilitres of this solution contained 60 ml of gum arabic, 10 ml citrate buffer (1.2 M citric acid monohydrate and 0.8 M trisodium citric acid dihydrate), and 30 ml of 0.5 M hydroquinone. Immediately before use, 0.5 ml of AgNO3 (0.75 M) was added to this mixture. After a 1 h developing time, the cells were washed twice with aq. dest. All samples were postfixed (1 h) in 1% OsO4 in cacodylate buffer. During dehydration the cells were stained ‘en bloc’ with 2% uranyl acetate in 50% ethanol and secondly in saturated lead acetate in ethanol-acetone (1:1), both for 1 h (Kushida, 1966). The pellets were embedded in Araldite. Thin and ultrathin sections were made with a Reichert-Jung Ultracut E. The sections were post-stained in a Reichert-Jung Ultrostainer programmed for 30 min uranyl acetate (40°C) and 3 min lead citrate (20°C) staining. The micrographs were made on a JEOL 1200 EX II transmission electron microscope. Light microscopy and image analysis Cells were seeded at a concentration of 6.105 cells/ml on plastic coverslips in four-well plates. Prior to mercury treatment, the cells were given a settling and adaptation
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Fig. 1 The effect of HgCl2 and MeHgCl on cell viability in Aedes albopictus C6/36 cultures. The viability, measured with a propidium iodide assay, is plotted against the micromolar concentration of the mercurials.
period of 24 h. Subsequently, they were treated with 3.0 µM HgCl2 or 2.4 µM MeHgCl. These mercury concentrations were chosen from a preliminary experiment in which sublethal Hg and MeHg concentrations were evaluated for optimal AMG staining (data not shown). The exposure times were: 1, 2, 4, 8, 16, and 24 h. MeHg-exposed cultures were treated with Se prior to fixation (see electron microscopy). The samples were fixed for 1 h at room temperature and the AMG developer was added for another hour (fixative and developer: see electron microscopy). After rinsing, the cells were counterstained with 1% toluidine blue, dehydrated and mounted with DePeX for subsequent light microscopical observation with a Reichert-Jung Polyvar. The percentage of the cell area that was covered with AMG grains was quantified with an IBAS KONTRON image analysis system. For each treatment, 200 cells from four separate preparations were measured. A special routine was programmed for this purpose. Acid phosphatase activity The acid phosphatase (AP) activity was determined in metal pre-treated cell cultures (this will be referred to as in vivo) and in metal-exposed crude cell extracts (in vitro). For both in vivo and in vitro experiments we used the following mercury concentrations: 0.9, 1.4, 1.9, 2.8, 3.7, 5.6, and 7.4 µM for Hg and 0.50, 0.75, 1.0, 1.5, 2.0, and 3.0 µM for MeHg. The experimental protocol for determination of AP activity is described in Braeckman et al. (1999a).
Results Effects of Hg and MeHg on the ultrastructure Aedes albopictus C6/36 cells had a similar morphology to insect granulocytes; for a detailed ultrastructural description
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we refer to Braeckman et al. (1999a). The nucleus had a nearly round shape and the chromatin was homogeneously distributed with few heterochromatin close to the nuclear envelope (Fig. 2a). Only few rough endoplasmic reticulum (RER) profiles could be found, which generally looked undilated (Fig. 3a). As a whole the Golgi system was poorly developed. The cytoplasm contained many lysosomes which, according to their contents, might be classified as heterolysosomes. The mitochondria had an electron-dense matrix with electron-lucent cristae; they often contained electron-dense granules (Fig. 2c). All the ultrastructural changes were already obvious after a 6 h Hg treatment; they were but slightly more pronounced after a 24 h treatment. The nucleus had become indented and irregular in form (Fig. 2b). The nucleolus had increased in volume and differentiation. Chromatin showed clumping and ‘empty spaces’ that gave the nucleoplasm a much less electron dense appearance. The mitochondria showed obvious changes: they had become pleomorphous. In some cells they were large and inflated with dense material, while in others there were accumulations of numerous dividing and small mitochondria. Mitochondria often showed matrix condensation (Fig. 2d) and sometimes the first signs of necrotic degeneration could be seen (Fig. 2e). As in the controls, the mitochondria contained large dense granules; after treatment these granules were sometimes replaced by flocculent densities. In most cells, the organelles involved in protein synthesis seemed activated: RER cisternae were inflated with proteinaceous material (Fig. 3b) and often a well developed Golgi stack could be found close to the dilated cisterna. The Golgi cisternae were surrounded by a cloud of transition vesicles and somewhat larger lucent vacuoles (Fig. 3c). The lysosomes were mostly heterophagous and contained a lot of membranous material. Some cells had become more electron dense due to an increased amount of free ribosomes. In these cells paraplasms could often be found: either accumulations of cytosol proteins or fat droplets. Also for MeHg the effects were already obvious after a 6 h treatment. Most of them were similar to those described for HgCl2. The nuclear deformation was more pronounced but the dilatations of the RER were less frequent, and the clouds of electron lucent small vacuoles surrounding the Golgi stacks were lacking. Moreover, in MeHg-treated cells some characteristic pathologies occurred. The cytoplasm regularly showed profiles of rolled-up RER-like membranes; these were filled with cytosol and were densely packed with ribosomes (Fig. 3e). They seemed randomly oriented. Also single-sheet RER-like membranes could be found that may represent precursors of the membranous tubes (Fig. 3f). After a 24 h treatment, there was a general disarrangement of the cytoplasmic organization that caused a disorderly appearance. Together with the occurrence of blebs (Fig. 3d) this seems to point at a disarrangement of the microtubules. Also, the tube-like structures had increased both in number and dimension.
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Fig. 2 Untreated Aedes albopictus cells (a, c) and Aedes cells exposed to 9.3 µM HgCl2 (d) and 5.5 µM MeHgCl (b, e) for 24 h. The nucleus of mercurytreated cells showed indentations (b), while mitochondria became condensed (d) or swollen (e). Magnifications: a: 17,400 x; b: 13,500 x; c: 65,300 x; d: 43,400 x; e: 22,100 x.
Light microscopy and AMG AMG staining was specific as the control cells showed almost no silver grains (Fig. 4a). In cells that were exposed to Hg- or MeHg, the AMG grains were found mainly over the cytoplasm. In well-spread cells almost no grains could be found over the nucleus (Fig. 4b). This was confirmed by ultrastructural localization (following section). To estimate the mercury accumulation in murine macrophages semiquantitatively, Christensen et al. (1991) used a 0–5 scale system. To diminish the subjectivity of this technique, we used an image analysis system and a doubleblind method to measure the surface ratio of AMG grains per cell (grains per cell ratio [GCR]). The mean GCR
increased with increasing exposure times for both Hg- and MeHg-treated cells (Fig. 5). In MeHg exposed cultures, a considerable background staining of 10% GCR was found. Despite the preliminary experiments to determine the optimal Se treatment, this background must originate from this treatment as no aspecific staining was found in Hg treated cells (which had no Se pre-treatment). The background probably originated from Zn-Se complexes that also catalyze the autometallographical reaction (Danscher & Møller-Madsen, 1985). The aspecific amount of 10% GCR was subtracted from the other values. The standard errors were large due to a considerable cell population heterogeneity (Fig. 6).
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Fig. 3 Untreated Aedes albopictus cell (a) and Aedes cells exposed to 9.3 µM HgCl2 (b, c) and 5.5 µM MeHgCl (d, e, f) for 24 h. Both mercury species caused severe dilatation of the RER cisternae (b). Clusters of small electron lucent vacuoles were observed close to Golgi-like structures in Hg-exposed cells (c). MeHg-treatment caused cytoplasmic disarrangement and cell blebs, which were devoid of organelles (d). In MeHg-exposed cells, tube-like structures (→, e) and their precursors (→, f) were found; they seem to consist of RER membranes. Magnifications: a: 52,900 x; b: 27,400 x; c: 38,900 x; d: 16,500 x; e: 20,500 x; f: 31,000 x.
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Fig. 4 Aedes cells stained by autometallography. Control cells showed almost no background staining (a). Cells treated with 3.0 µM HgCl2 for 24 h showed abundant silver deposits in the cytoplasm (b). Ultrastructurally, the silver grains were mainly found in the lysosomes (c) and to a lesser extent near the plasma membrane (d). a-b: 1,250 x; c: 15,000 x; d: 20,000 x.
Ultrastructural localization of AMG silver deposits Control cells, exposed to the AMG developer showed no silver deposits, which proved the absence of aspecific staining in the ultrastructural experiments. In Hg- and MeHg-treated cells, AMG grains were mainly localized in the lysosomes (Fig. 4c) and to a minor degree at the inner and outer side of the plasma membrane (Fig. 4d). Mercury accumulation was fast as silver deposits were found in the lysosomes after only 30 min of Hg treatment.
enzyme acid phosphatase. The in vitro study showed a significant inhibition (P<0.001) of AP activity at 2.8 µM HgCl2 (Fig. 7a). At higher concentrations, AP activity was similar compared to the control. In contrast, in the in vivo experiments, we found a significant inhibition (P<0.001) of the AP activity at the highest concentrations (3.7, 5.6 and 7.4 µM). MeHg had no effect on the AP activity, neither in vivo, nor in vitro (Fig. 7b).
AP activity The lysosomes seemed to be the major compartment for mercury storage. This prompted us to study the effect of the two mercurials on the activity of the lysosomal marker
Discussion We studied the effect of Hg and MeHg in insect cells at the ultrastructural level in order to find cellular pathologies or
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Fig. 5 The silver grains of autometallographically stained cells were measured by means of image analysis. The staining intensity (expressed as the percentage of cell area covered with silver grains, GCR) was plotted against the mercury treatment time and linear relationship was found for both mercury species. Cells were treated with 3.0 µM HgCl2 or 2.4 µM MeHgCl. Bars are SEM of 200 measurements.
Fig. 7 The effect of HgCl2 (A) and MeHgCl (B) on acid phosphatase activity. The effect of the metal on the enzyme activity was measured directly in crude cell extracts (in vitro) and after a 24 h mercury treatment of intact cells (in vivo).
Fig. 6 The population heterogeneity of silver stained cells is demonstrated in a histogram, which shows the number of cells (from a population of 200) in each 10% GCR interval. The cells were treated with 2.4 µM MeHg for 16 h.
target organelles for mercury toxicity. Untreated Aedes albopictus cells showed morphological features of phagocytic, non secretory cells. The general ultrastructural effects of Hg and MeHg were similar and clearly visible after a short incubation period of 6 h. The morphology of the mercury treated cells was characterized by indentation of the nucleus, chromatin clumping, the occurrence of pleomorphous mitochondria and indications of activated protein synthesis. Indented nuclei were also observed in mercury- and
Cd-treated hepatocytes (Hirano et al., 1991; Bucio et al., 1995). Chromatin clumping is a general prenecrotic effect. Also, mitochondrial pleomorphy is a prenecrotic event due to the disturbance of respiration and of mitochondrial growth and replication (Trump et al., 1978). In contrast to Cd (Braeckman et al., 1999a), mitochondrial swelling was less frequently found in mercury-treated Aedes cells. In our study, the activated protein synthesis was suggested by a series of changes in different organelles: the swelling of the nucleolus, increase of the free and membrane-bound ribosomes, the large and inflated RER cisternae filled with proteinaceous material and finally the appearance of well developed Golgi stacks. These morphological alterations were also found to some degree in Cd-treated cells (Braeckman et al., 1999a). Therefore they do not seem to be mercury-specific, instead they may reflect a general response to cell stress. The electron-dense content of the inflated RER differed clearly from the osmotically swollen electron-lucent cisternae that were described in other pathological situations (Miyakawa & Deshimaru, 1969; Ganote et al., 1974; Trump et al., 1978; Bucio et al., 1995). Although a pathological constipation of the RER, due to metalprovoked protein clustering, cannot be ruled out, it seems likely that the dilatation of the RER concerns an induction
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of certain stress proteins. In our previous experiments, data suggested that Cd is able to induce BiP, a RER-resident heat shock protein, in Aedes cells (Braeckman et al., 1999b). Probably, a similar induction takes place in mercury-treated Aedes cells. In contrast to our observations, Lachapelle et al. (1993) found a decrease of ER-bound ribosomes due to Hg treatment. Ganote et al. (1974) and Miura et al. (1979) described the Hg-induced dispersion of polysomes in the cytoplasm, which is also a prenecrotic event (Trump et al., 1978). Bucio et al. (1995) reported an increase of the free ribosomes due to Hg treatment. Apart from these general ultrastructural effects, we also noticed some alterations that were more specific for either Hg or MeHg. In the Hg-treated cells, clusters of small lucent vacuoles, close to the Golgi transition vesicles, were often seen. They might reflect an increased production of primary lysosomes, but a pathological effect (disturbance of vesicle transport or fusion) cannot be ruled out. In cells treated with MeHg, these clusters were almost lacking. On the other hand, here we observed tube-like structures that did not appear in Hg-treated cells. They are reminiscent of certain cytoplasmic inclusions found in oenocytes of the insect Calpodes ethlius (Locke, 1969), which seemed to be continuous with the smooth endoplasmic reticulum cisternae. We cannot explain the etiology of these remarkable structures. In MeHg-treated cells we found an obvious disorganization of the organelles together with blebbing. Both effects were probably due to the specific microtubule disrupting effect of this mercury species, which is in agreement with light microscopical observations (Braeckman et al., 1997a). MeHgcaused depolymerization of microtubules was described in detail by lmura et al. (1980) and Sager et al. (1983). AMG is a sensitive, histochemical silver amplification technique for the detection of mercury, gold and silver. This method has been used extensively for detection and localization of mercury in vertebrate tissues (Baatrup & Danscher, 1987; Nørgaard et al., 1991; Augier et al., 1993; Danscher et al., 1994; Christensen, 1996). Data on mercury staining is almost non-existent for invertebrates (Raes & De Coster, 1991; Marigómez et al., 1996) and to our knowledge, this is the first report on histochemical mercury staining in an invertebrate cell culture. In Hg and MeHg-treated cells, AMG grains were mainly located over the cytoplasm at the light microscopical level. The GCR, used as a measure for mercury accumulation, was quantified with an image analysis system. The mean GCR increased with increasing exposure times for both Hg- and MeHg-treated cells. The standard errors were large due to a considerable population heterogeneity. Because silver grains are quantified instead of mercury, this approach is a relative and indirect method. Mercury quantification by means of atomic fluorescence spectrometry is far more accurate and is described in Braeckman et al. (1998). However, in contrast to this analytical technique, the histochemical AMG method provides information about the population heterogeneity regarding mercury accumulation, which proved to be considerable in Aedes cell cultures. For both mercury species we found
widely differing accumulation capacities among the cells. An additional difference between the analytical and the histochemical technique concerns the type of mercury, which is quantified (Christensen, 1996). In atomic spectroscopy, all internalized mercury is quantified, whereas for histochemical staining, a cluster of sulfur- or seleniumbound mercury atoms is necessary to start the autokatalytical silver staining; one Hg-SH or Hg-Se center is not sufficient for histochemical detection (Danscher & Møller-Madsen, 1985; Danscher et al., 1994). This implies that, with the histochemical method, mainly mercury storage sites will be stained (and quantified). Ultrastructural localization of the AMG grains showed us the preferential accumulation of Hg and MeHg in the lysosomes. Lysosomal accumulation of mercury is thoroughly described in vertebrate literature (Baatrup & Danscher, 1987; Møller-Madsen, 1990; Danscher, 1991; Nørgaard et al., 1991; Christensen, 1996). In invertebrate literature, however, references are very scarce. Early publications on mercury detection in invertebrate cells came from BallanDufrançais et al. (1980) and Jeantet et al. (1980). They used electron probe microanalysis for localization of this heavy metal in the lysosomes of cockroach cells. Very few data exists on AMG demonstration of mercury in invertebrates (Raes & De Coster, 1991; Marigómez et al., 1996), and here also, mercury seemed to be mainly located in the lysosomes. Despite all these indications of lysosomes being the preferential accumulation sites for mercury, the uptake mechanism and transport routes of mercury to this organelle are still unclear. Hg has a high affinity for sulfhydryl groups. Proteins carrying Hg on their sulfhydryl groups may be broken down in the lysosomes. This detoxification process may lead to a concentration of this heavy metal in the lysosomes (Christensen, 1996). In 1980, Ballan-Dufrançais and co-workers reported that in lysosomes one atom of mercury is engaged in a molecule in which three atoms of sulfur coexist. This molecule must be of organic nature because in the mineral form, the mercury/sulfur ratio is always equal or higher than 1:1. The AMG-grains we found on the surface of mercury-treated Aedes cells probably represent mercury bound to free sulfhydryl groups of membrane proteins. The mercury sulfide concentration at this site seemed to be high enough to start the AMG reaction. This is not surprising as Endo et al. (1995) found that the non-internalized mercury fraction, bound to the cell membrane, is much greater than the internalized fraction. Mercury seemed to be concentrated in the lysosomes, possibly as a result of a detoxification process. However, it is not certain whether these high mercury concentrations, although presumed to be biologically inert, do not interfere with the normal lysosomal function. Therefore we tested the effect of Hg and MeHg on the lysosomal marker enzyme, AP. The activity was measured in vitro and in vivo to discriminate between direct and indirect effects of the mercury species on the enzyme. In the in vitro experiments we found a slight but highly significant inhibition of APactivity (6.3%) for only one Hg concentration (2.8 µM).
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This suggested that the Hg2+ concentration was very critical for this inhibitory effect. The indirect in vivo effect could be due to the interaction of mercury with free sulfhydryl groups during synthesis of the AP in the RER or the inhibition of the AP synthesis process itself. Enhanced AP turnover may also be envisaged. The effect of Hg on AP activity is well documented but ambiguous. In the fish Clarias, AP activity was found to be inhibited due to severe Hg treatment (the fish were injected with 2 ml of a 5% solution) (Saxena & Tyagi, 1979). A decrease of AP was found by Blasco et al. (1993) in the clam Ruditapes at very high Hg concentrations (1 mM). Naidu et al. (1984) found a significant decrease (30%) of AP-activity at the LC50/48 h (5.5 µM HgCl2). All these Hg concentrations are much higher than the sublethal concentrations we used and therefore the results might not be comparable. The effect of low Hg concentrations (0.37 µM) on AP-activity in marine lamellibranchs was shown to be species dependent. AP-activity was either enhanced, inhibited or unaffected depending on the species studied (Patel et al., 1988). Unlike Hg, MeHg had no effect on the AP-activity, either in vitro or in vivo. In contrast to our findings, Fowler et al. (1975) and Hossein & Dutta (1986) found an increase of AP activity at low MeHg levels in rat kidney and fish intestine, respectively. In conclusion we found that both mercury species caused mostly similar ultrastructural effects that were indicative of the induction of a general defense system, probably heat shock proteins. AMG showed us that the lysosomes were the main sites for mercury accumulation. The mercury accumulating capacity differed considerably among the Aedes cells. At sublethal concentrations, Hg inhibited AP-activity mostly in an indirect way; only for one concentration in vitro inhibition was found. MeHg had no effect on APactivity in the sublethal concentration range. ACKNOWLEDGEMENTS The authors thank Urszula Rzeznik for excellent technical assistance. We are also grateful to L. De Ridder (Laboratory for Histology, University of Ghent, Belgium) for the use of the transmission electron microscope. H. Raes is indebted to the FGWO (Belgian Fund for Medical Scientific Research) for the purchase of equipment without which this work would not have been possible (grant no. 3.9001.92). REFERENCES Augier, H., Benkoël, L., Brisse, J., Chamilian, A. and Park, W.K. 1993. Microscopic localization of mercury-selenium interaction products in liver, kidney, lung and brain of mediterranean striped dolphins (Stenella coeruleoalba) by silver enhancement kit. Cell. Mol. Biol., 39, 765–772. Baatrup, E. and Danscher, G. 1987. Cytochemical demonstration of mercury deposits in trout liver and kidney following methyl mercury intoxication: differentiation of two mercury pools by selenium. Ecotox. Environ. Safe., 14, 129–141. Ballan-Dufrançais, C., Ruste, J. and Jeantet, A.Y. 1980. Quantitative electron probe microanalysis on insects exposed to mercury. I Methods. An approach on the molecular form of the stored mercury. Possible occurrence of metallothionein-like proteins. Biol. Cell., 39, 317–324.
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Blasco, J., Puppo, J. and Sarasquete, M.C. 1993. Acid and alkaline phosphatase activities in the clam Ruditapes philippinarum. Mar. Biol., 115, 113–118. Braeckman, B., Simoens, C., Rzeznik, U. and Raes, H. 1997a. Sublethal dose effect of cadmium, inorganic mercury and methylmercury on the cell morphology of an insect cell line (Aedes albopictus, C6/36). Cell Biol. Int., 21, 823–832. Braeckman, B., Raes, H. and Van Hoye, D. 1997b. Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells. Cell Biol. Toxicol., 13, 389–397. Braeckman, B., Cornelis, R., Rzeznik, U. and Raes, H. 1998. Uptake of HgCl2 and MeHgCl in an insect cell line (Aedes albopictus, C6/36). Environmental Research, 79 (A), 33–40. Braeckman, B., Brys, K., Rzeznik, U. and Raes, H. 1999a. Cadmium pathology in an insect cell line: ultrastructural and biochemical effects. Tissue Cell, 31, 45–53. Braeckman, B., Smagghe, G., Brutsaert, N., Cornelis, R. and Raes, H. 1999b Cadmium uptake and defense mechanism in insect cells. Environmental Research, 80(A), 231–243. Bucio, L., Souza, V., Albores, A. et al. 1995. Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells). Toxicology, 102, 285–299. Christensen, M., Ellermann-Eriksen, S., Rungby, J., Mogensen, S.C. and Danscher, G. 1991. Histochemical and functional evaluation of mercuric chloride toxicity in cultured macrophages. Prog. Histochem. Cytoc., 23, 306–315. Christensen, M. 1996. Histochemical localization of autometallographically detectable mercury in tissues of the immune system from mice exposed to mercuric chloride. Histochem, J., 28, 217–225. Danscher, G. and Møller-Madsen, B. 1985. Silver amplification of mercury sulfide and selenide. A histochemical method for light and electron microscopic localization of mercury in tissue. J. Histochem. Cytochem., 33, 219–228. Danscher, G. 1991. Histochemical tracing of zinc, mercury, silver and gold. Prog. Histochem. Cytoc., 23, 273–285. Danscher, G., Stoltenberg, M. and Juhl, S. 1994. How to detect gold, silver and mercury in human brain and other tissues by autometallographic silver amplification. Neuropath. Appl. Neuro., 20, 454–467. Endo, T., Sakata, M. and Shaikh, Z.A. 1995. Mercury uptake by primary cultures of rat renal cortical epithelial cells. I. Effects of cell density, temperature and metabolic inhibitors. Toxicol. Appl. Pharm., 132, 36–43. Fowler, B.A., Brown, H.W., Lucier, G.W. and Krigman, M.R. 1975. The effects of chronic oral methyl mercury exposure on the lysosome system of rat kidney. Lab. Invest., 32, 313–322. Ganote, C.E., Reimer, K.A. and Jennings, R.B. 1974. Acute mercuric chloride nephrotoxicity. An electron microscopic and metabolic study. Lab. Invest., 31, 633–647. Hare, L. 1992. Aquatic insects and trace metals: bioavailability, bioaccumulation and toxicity. Crit. Rev. Toxicol., 22, 327–369. Hirano, T., Ueda, H., Kawahara, A. and Fujimoto, S. 1991. Cadmium toxicity on cultured neonatal rat hepatocytes: Biochemical and ultrastructural analyses. Histol. Histopathol., 6, 127–133. Hossein, A. and Dutta, H.M. 1986. Acid phosphatase activity in the intestine and caeca of bluegill, exposed to methyl mercuric chloride. B. Environ. Contam. Tox., 36, 460–467. Igarashi, A. 1978. Isolation of a Singh’s Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J. Gen. Virol., 40, 531–544. Imura, N., Miura, K., Inokawa, M. and Nakada, S. 1980. Mechanism of methyl-mercury cytotoxicity: by biochemical and morphological experiments using cultured cells. Toxicology, 17, 241–254. Jeantet, A.Y., Ballan-Dufrançais, C. and Ruste, J. 1980. Quantitative electron probe microanalysis on insects exposed to mercury. II Involvement of the lysosomal system in detoxification processes. Biol. Cell., 39, 325–334. Kitamura, S. 1966. The in vitro cultivation of tissues from mosquitoes. Kobe J. Med. Sci., 12, 63–70. Kushida, H. 1966. Staining of thin sections with lead acetate. J. Electron. Microsc., 15, 93. Lachapelle, M., Guertin, F., Marion, M., Fournier, M. and Denizeau, F. 1993. Mercuric chloride affects protein secretion in rat primary hepatocyte cultures: a biochemical ultrastructural, and gold imunnocytochemical study. J. Toxicol. Env. Health, 38, 343–354. Locke, M. 1969. The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect. Tissue Cell, 1, 103–154.
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Marigómez, I., Soto, M. and Kortabitarte, M. 1996. Tissue-level biomarkers and biological effect of mercury on sentinel slugs, Arion ater. Arch. Environ. Con. Tox., 31, 54–62. Miura, K., Nakada, S., Suzuki, K. and Imura, N. 1979. Ultrastrucutral studies on the cytotoxic effects of mercuric chloride on mouse glioma. Ecotox. Environ. Safe., 3, 352–361. Miyakawa, T. and Deshimaru, M. 1969. Electron microscopic study of experimentally induced poisoning due to organic mercury compounds. Acta Neuropathol., 14, 126–136. Møller-Madsen, B. 1990. Localization of mercury in CNS of the rat. II. Intraperitoneal injection of methylmercuric chloride (CH3HgCI) and mercuric chloride (HgCl2). Toxicol. Appl. Pharm., 103, 303–323. Morselt, A.F.W., Copius Peereboom-Stegeman, J.H.J., Puvion, E. and Maarschalkerweerd, V.J. 1993. Investigation of the mechanism of cadmium toxicity at cellular level. II. An electron microscopical study. Arch. Toxicol., 52, 99–108. Naidu, K.A., Naidu, K.A. and Ramamurthi, R. 1984. Acute effect of mercury toxicity on some enzymes in liver of teleost Sarotherodon mossambicus. Ecotox. Environ. Safe., 8, 215–218. Nieminen, A.-L., Gores, G.J., Bond, J.M., Imberti, R., Herman, B. and Lemasters, J. 1992. A novel cytotoxicity screening assay using a multiwell fluorescence scanner. Toxicol. Appl. Pharm., 115, 147–155. Nørgaard, J.O.R., Møller-Madsen, B. and Danscher, G. 1991. Autometallographic localization of mercury in rat kidney – interaction between mercury and selenium. Prog. Histochem. Cytoc., 23, 187–193.
Patel, B., Chandy, J.P. and Patel S. 1988. Do selenium and glutathione inhibit the toxic effects of mercury in marine lamellibranchs? Sci. Total Environ., 76, 147–165. Raes, H. and De Coster, W. 1991. Storage and detoxication of anorganic and organic mercury in honeybees: histochemical and ultrastructural evidence. Prog. Histochem. Cytoc., 23, 316–320. Sager, P.R., Doherty, R.A. and Olomsted, J.B. 1983. Interaction of methylmercury with microtubules in cultured cells and in vitro. Exp. Cell Res., 146, 127–137. Saxena, R. and Tyagi, A.P. 1979. Effect of mercuric chloride on the phosphatases in the accessory respiratory organs of Clarias batrachus. Hydrobiologia, 63, 209–211. Seidman, L.A., Bergtrom, G., Gingrich, D.J. and Remsen C.C. 1986. Accumulation of cadmium by the fourth instar larva of the fly Chironomus thummi. Tissue Cell, 18, 395–405. Singh, K.R.P. 1967. Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci., 19, 506–508. Trump, B.F., Jesudason, M.L. and Jones, R.T. 1978. Ultrastructural features of diseased cells. In: Trump B.F. and Jones R.T. (eds). Diagnostic electron microscopy, volume 1. John Wiley & Sons, New York, 1–88. Walton, B.T. 1989. Insects as indicators of toxicity, bioaccumulation and bioavailability of environmental contaminants. Environ. Toxicol. Chem., 8, 649–658.
Tissue&Cell
The ultrastructural effect and subcellular localization of mercuric chloride and methylmercuric chloride in insect cells (Aedes albopictus C6/36) B. Braeckman, H. Raes
Abstract. The ultrastructural effects of mercuric chloride (Hg) and methylmercuric chloride (MeHg) were studied in the Aedes albopictus C6/36 cell line. Both metal salts caused nuclear indentations, chromatin clumping and proliferation of the nucleolus. The mitochondria became pleomorphous. An increase of both free and membranebound ribosomes, swelling of the rough endoplasmic reticulum caused by accumulated protein and the appearance of well developed Golgi stacks all indicated the activation of protein synthesis. The activation was probably a cellular response to general stress, and the synthesized proteins may be members of the heat shock protein family. Apart from these common ultrastructural features, Hg-treated cells showed typical clusters of small electron-lucent vacuoles near the Golgi stacks. In cells exposed to MeHg, cytoplasmic tube-like structures were often observed and the disorganization of the organelles together with the appearance of blebs suggested disruption of the microtubules. Mercury accumulation was localized by an autometallographical silver staining technique both at the light and electron microscopic level; silver deposits were quantified by image analysis. For both Hg- and MeHg-treated cells, the degree of silver staining increased rapidly with increasing exposure time, but a considerable heterogeneity within the cell population was found. Lysosomes proved to be the major mercury storage sites in the Aedes cells and silver deposits could already be found after 30 min of Hg treatment. At sublethal concentrations, Hg inhibited the lysosomal marker enzyme acid phosphatase to some extent. For MeHg, no effect on this enzyme was found.
Keywords: Mercuric chloride, methylmercuric chloride, ultrastructure, insect cell line, autometallography, acid phosphatase
Introduction Mercury and cadmium (Cd) are some of the most toxic heavy metal contaminants. Environmental monitoring of these pollutants has become a major issue in the last decades. The use of insects as biomonitors for heavy metal contamination in terrestrial and aquatic ecosystems has been Department of Biology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
Received 2 December 1998 Accepted 2 February 1999 Correspondence to: Bart Braeckman. Tel: (+32) 9 264 52 22; Fax: (+32) 9 264 53 44; E-mail: Bart. Braeckman @rug.ac.be
reported to have a great potential (Seidman et al., 1986; Walton, 1989; Hare, 1992). We use the Aedes albopictus C6/36 cell line as a model to study heavy metal toxicity in insects at the cellular level. In previous research, we studied the effect of Cd on Aedes cells at the ultrastructural level (Braeckman et al., 1999a). Since mercury, like Cd, has a high affinity for sulfhydryls, we wanted to investigate the effect of this metal on subcellular morphology in a comparative way. In this study we examine the effects of mercuric chloride (Hg) and methylmercuric chloride (MeHg) as representatives of inorganic and organic mercury species. Both mercurials were shown to have a different effect on cell morphology at the light microscopical level (Braeckman et al., 1997a) and on 223
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the viability and proliferation of Aedes albopictus cells (Braeckman et al., 1997b). A study of the ultrastructural pathology may give important information on the subcellular targets of Hg and MeHg. We also study mercury accumulation histochemically using autometallography (Danscher et al., 1994). At the light microscopic level, the thereby formed silver deposits are quantified by image analysis. At the electron microscopical level the method enables us to localize the accumulation of mercury in the cells. As mercury is found to accumulate mainly in the lysosomes, the activity of acid phosphatase, which is a lysosomal marker enzyme, is also tested.
Materials and methods Materials HgCl2 and MeHgCl were purchased from Merck (Darmstadt, Germany). Lactalbumin hydrolysate, fetal calf serum and gentamicin were obtained from Gibco BRL (Paisley, UK). Yeast extract was a product of Oxoid (Unipath Ltd, Basingstoke, UK). Paraformaldehyde and 4-methylumbelliferyl phosphate were purchased from Sigma (St Louis, USA). Cacodylic acid, glutaraldehyde, OsO4, lead acetate and uranyl acetate were products from Fluka (Buchs, Switzerland). The epoxy resin kit (Araldite) was purchased from Agar Scientific Ltd (Stansted, UK). Propidium iodide was obtained from Molecular Probes (Eugene, Oregon, USA). DePeX was a product from BDH (Poole, UK). All other chemicals were purchased from Merck (Darmstadt, Germany). Culture recipients were obtained from Nunclon (Roskilde, Denmark). Cell culture In all our experiments we used Aedes albopictus cells (Diptera: Culicidae) belonging to the C6/36 clone, produced by lgarashi (1978) from a cell line of mixed embryonic origin (Singh, 1967). They were grown in modified Kitamura (MK) medium (132 mM NaCl, 8 mM KCl, 0.82 mM CaCl2, 0.86 mM KH2PO4, 24 mM D-glucose, 0.77% lactalbumin hydrolysate, 0.57% yeast extract, 5% fetal calf serum, 50 µg/ml gentamicin) initially described by Kitamura (1966). The pH of the medium was 6.8. The cultures were kept in the dark under ambient atmosphere at 27°C. The cells were routinely seeded at a density of 105 cells/ml. Under these conditions the cell population doubling time was about 30 h. For all experiments we used 7-day-old cultures. Metal exposure For each experiment, metal stock solutions were freshly prepared. HgCl2 and MeHgCl were dissolved directly in MK medium and, after filter sterilization, 5% fetal calf serum was added. These solutions were diluted to a final concentration of 40 µg/ml and used as a stock to prepare the appropriate treatment concentrations.
Evaluation of cell viability with the propidium iodide assay Cell viability was assessed by a dye exclusion test based on propidium iodide staining (Nieminen et al., 1992), adapted for Aedes cells. The method is basically a test for the membrane integrity and has been described in detail in Braeckman et al. (1997b). Cell cultures, at a final concentration of 1.0×106 cells/ml, were exposed for 24 h to the following HgCl2 concentrations: 0.5, 0.9, 1.9, 2.8, 3.7, 5.6, 7.4, 11.1, 14.8, and 22.2 µM. For MeHgCl, we used 0.4, 0.6, 0.8, 1.2, 1.6, 2.4, 3.2, 4.8, 6.4, and 9.6 µM. The viability curves show the average of two experiments, each containing eight replicates (Fig. 1). For the ultrastructural studies, metal concentrations in the LC10/24 h-LC50/24 h region were chosen in order to obtain the whole range of mercury provoked cellular injury (9.3 µM for Hg and 5.5 µM for MeHg). Electron microscopy and autometallographical procedure Cell suspensions (approx. 5×106 cells/ml) were treated with Hg (9.3 µM) or MeHg (5.5 µM) for a 6 h and 24 h period. For the autometallography (AMG), also shorter treatment times were applied (10 min, 30 min, 1 h, and 3 h). Prior to fixation, the MeHg samples were treated for 40 min with a 40 µg/ml Na2SeO3.5H2O solution to obtain Hg-Se complexes which can initiate the AMG reaction (Baatrup & Danscher, 1987). In preliminary experiments, this Se concentration and treatment time gave optimal staining results and caused no viability decrease (data not shown). The samples were fixed for 1 h at room temperature (2% glutaraldehyde and 2% paraformaldehyde in 0,1% cacodylate buffer, containing 60 mM of sucrose and 0.05% CaCl2, and adjusted to a pH of 7.4) and successively pelletted. In the samples that served for mercury localization, the fixative was replaced by the AMG developer. One hundred millilitres of this solution contained 60 ml of gum arabic, 10 ml citrate buffer (1.2 M citric acid monohydrate and 0.8 M trisodium citric acid dihydrate), and 30 ml of 0.5 M hydroquinone. Immediately before use, 0.5 ml of AgNO3 (0.75 M) was added to this mixture. After a 1 h developing time, the cells were washed twice with aq. dest. All samples were postfixed (1 h) in 1% OsO4 in cacodylate buffer. During dehydration the cells were stained ‘en bloc’ with 2% uranyl acetate in 50% ethanol and secondly in saturated lead acetate in ethanol-acetone (1:1), both for 1 h (Kushida, 1966). The pellets were embedded in Araldite. Thin and ultrathin sections were made with a Reichert-Jung Ultracut E. The sections were post-stained in a Reichert-Jung Ultrostainer programmed for 30 min uranyl acetate (40°C) and 3 min lead citrate (20°C) staining. The micrographs were made on a JEOL 1200 EX II transmission electron microscope. Light microscopy and image analysis Cells were seeded at a concentration of 6.105 cells/ml on plastic coverslips in four-well plates. Prior to mercury treatment, the cells were given a settling and adaptation
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Fig. 1 The effect of HgCl2 and MeHgCl on cell viability in Aedes albopictus C6/36 cultures. The viability, measured with a propidium iodide assay, is plotted against the micromolar concentration of the mercurials.
period of 24 h. Subsequently, they were treated with 3.0 µM HgCl2 or 2.4 µM MeHgCl. These mercury concentrations were chosen from a preliminary experiment in which sublethal Hg and MeHg concentrations were evaluated for optimal AMG staining (data not shown). The exposure times were: 1, 2, 4, 8, 16, and 24 h. MeHg-exposed cultures were treated with Se prior to fixation (see electron microscopy). The samples were fixed for 1 h at room temperature and the AMG developer was added for another hour (fixative and developer: see electron microscopy). After rinsing, the cells were counterstained with 1% toluidine blue, dehydrated and mounted with DePeX for subsequent light microscopical observation with a Reichert-Jung Polyvar. The percentage of the cell area that was covered with AMG grains was quantified with an IBAS KONTRON image analysis system. For each treatment, 200 cells from four separate preparations were measured. A special routine was programmed for this purpose. Acid phosphatase activity The acid phosphatase (AP) activity was determined in metal pre-treated cell cultures (this will be referred to as in vivo) and in metal-exposed crude cell extracts (in vitro). For both in vivo and in vitro experiments we used the following mercury concentrations: 0.9, 1.4, 1.9, 2.8, 3.7, 5.6, and 7.4 µM for Hg and 0.50, 0.75, 1.0, 1.5, 2.0, and 3.0 µM for MeHg. The experimental protocol for determination of AP activity is described in Braeckman et al. (1999a).
Results Effects of Hg and MeHg on the ultrastructure Aedes albopictus C6/36 cells had a similar morphology to insect granulocytes; for a detailed ultrastructural description
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we refer to Braeckman et al. (1999a). The nucleus had a nearly round shape and the chromatin was homogeneously distributed with few heterochromatin close to the nuclear envelope (Fig. 2a). Only few rough endoplasmic reticulum (RER) profiles could be found, which generally looked undilated (Fig. 3a). As a whole the Golgi system was poorly developed. The cytoplasm contained many lysosomes which, according to their contents, might be classified as heterolysosomes. The mitochondria had an electron-dense matrix with electron-lucent cristae; they often contained electron-dense granules (Fig. 2c). All the ultrastructural changes were already obvious after a 6 h Hg treatment; they were but slightly more pronounced after a 24 h treatment. The nucleus had become indented and irregular in form (Fig. 2b). The nucleolus had increased in volume and differentiation. Chromatin showed clumping and ‘empty spaces’ that gave the nucleoplasm a much less electron dense appearance. The mitochondria showed obvious changes: they had become pleomorphous. In some cells they were large and inflated with dense material, while in others there were accumulations of numerous dividing and small mitochondria. Mitochondria often showed matrix condensation (Fig. 2d) and sometimes the first signs of necrotic degeneration could be seen (Fig. 2e). As in the controls, the mitochondria contained large dense granules; after treatment these granules were sometimes replaced by flocculent densities. In most cells, the organelles involved in protein synthesis seemed activated: RER cisternae were inflated with proteinaceous material (Fig. 3b) and often a well developed Golgi stack could be found close to the dilated cisterna. The Golgi cisternae were surrounded by a cloud of transition vesicles and somewhat larger lucent vacuoles (Fig. 3c). The lysosomes were mostly heterophagous and contained a lot of membranous material. Some cells had become more electron dense due to an increased amount of free ribosomes. In these cells paraplasms could often be found: either accumulations of cytosol proteins or fat droplets. Also for MeHg the effects were already obvious after a 6 h treatment. Most of them were similar to those described for HgCl2. The nuclear deformation was more pronounced but the dilatations of the RER were less frequent, and the clouds of electron lucent small vacuoles surrounding the Golgi stacks were lacking. Moreover, in MeHg-treated cells some characteristic pathologies occurred. The cytoplasm regularly showed profiles of rolled-up RER-like membranes; these were filled with cytosol and were densely packed with ribosomes (Fig. 3e). They seemed randomly oriented. Also single-sheet RER-like membranes could be found that may represent precursors of the membranous tubes (Fig. 3f). After a 24 h treatment, there was a general disarrangement of the cytoplasmic organization that caused a disorderly appearance. Together with the occurrence of blebs (Fig. 3d) this seems to point at a disarrangement of the microtubules. Also, the tube-like structures had increased both in number and dimension.
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Fig. 2 Untreated Aedes albopictus cells (a, c) and Aedes cells exposed to 9.3 µM HgCl2 (d) and 5.5 µM MeHgCl (b, e) for 24 h. The nucleus of mercurytreated cells showed indentations (b), while mitochondria became condensed (d) or swollen (e). Magnifications: a: 17,400 x; b: 13,500 x; c: 65,300 x; d: 43,400 x; e: 22,100 x.
Light microscopy and AMG AMG staining was specific as the control cells showed almost no silver grains (Fig. 4a). In cells that were exposed to Hg- or MeHg, the AMG grains were found mainly over the cytoplasm. In well-spread cells almost no grains could be found over the nucleus (Fig. 4b). This was confirmed by ultrastructural localization (following section). To estimate the mercury accumulation in murine macrophages semiquantitatively, Christensen et al. (1991) used a 0–5 scale system. To diminish the subjectivity of this technique, we used an image analysis system and a doubleblind method to measure the surface ratio of AMG grains per cell (grains per cell ratio [GCR]). The mean GCR
increased with increasing exposure times for both Hg- and MeHg-treated cells (Fig. 5). In MeHg exposed cultures, a considerable background staining of 10% GCR was found. Despite the preliminary experiments to determine the optimal Se treatment, this background must originate from this treatment as no aspecific staining was found in Hg treated cells (which had no Se pre-treatment). The background probably originated from Zn-Se complexes that also catalyze the autometallographical reaction (Danscher & Møller-Madsen, 1985). The aspecific amount of 10% GCR was subtracted from the other values. The standard errors were large due to a considerable cell population heterogeneity (Fig. 6).
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Fig. 3 Untreated Aedes albopictus cell (a) and Aedes cells exposed to 9.3 µM HgCl2 (b, c) and 5.5 µM MeHgCl (d, e, f) for 24 h. Both mercury species caused severe dilatation of the RER cisternae (b). Clusters of small electron lucent vacuoles were observed close to Golgi-like structures in Hg-exposed cells (c). MeHg-treatment caused cytoplasmic disarrangement and cell blebs, which were devoid of organelles (d). In MeHg-exposed cells, tube-like structures (→, e) and their precursors (→, f) were found; they seem to consist of RER membranes. Magnifications: a: 52,900 x; b: 27,400 x; c: 38,900 x; d: 16,500 x; e: 20,500 x; f: 31,000 x.
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Fig. 4 Aedes cells stained by autometallography. Control cells showed almost no background staining (a). Cells treated with 3.0 µM HgCl2 for 24 h showed abundant silver deposits in the cytoplasm (b). Ultrastructurally, the silver grains were mainly found in the lysosomes (c) and to a lesser extent near the plasma membrane (d). a-b: 1,250 x; c: 15,000 x; d: 20,000 x.
Ultrastructural localization of AMG silver deposits Control cells, exposed to the AMG developer showed no silver deposits, which proved the absence of aspecific staining in the ultrastructural experiments. In Hg- and MeHg-treated cells, AMG grains were mainly localized in the lysosomes (Fig. 4c) and to a minor degree at the inner and outer side of the plasma membrane (Fig. 4d). Mercury accumulation was fast as silver deposits were found in the lysosomes after only 30 min of Hg treatment.
enzyme acid phosphatase. The in vitro study showed a significant inhibition (P<0.001) of AP activity at 2.8 µM HgCl2 (Fig. 7a). At higher concentrations, AP activity was similar compared to the control. In contrast, in the in vivo experiments, we found a significant inhibition (P<0.001) of the AP activity at the highest concentrations (3.7, 5.6 and 7.4 µM). MeHg had no effect on the AP activity, neither in vivo, nor in vitro (Fig. 7b).
AP activity The lysosomes seemed to be the major compartment for mercury storage. This prompted us to study the effect of the two mercurials on the activity of the lysosomal marker
Discussion We studied the effect of Hg and MeHg in insect cells at the ultrastructural level in order to find cellular pathologies or
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Fig. 5 The silver grains of autometallographically stained cells were measured by means of image analysis. The staining intensity (expressed as the percentage of cell area covered with silver grains, GCR) was plotted against the mercury treatment time and linear relationship was found for both mercury species. Cells were treated with 3.0 µM HgCl2 or 2.4 µM MeHgCl. Bars are SEM of 200 measurements.
Fig. 7 The effect of HgCl2 (A) and MeHgCl (B) on acid phosphatase activity. The effect of the metal on the enzyme activity was measured directly in crude cell extracts (in vitro) and after a 24 h mercury treatment of intact cells (in vivo).
Fig. 6 The population heterogeneity of silver stained cells is demonstrated in a histogram, which shows the number of cells (from a population of 200) in each 10% GCR interval. The cells were treated with 2.4 µM MeHg for 16 h.
target organelles for mercury toxicity. Untreated Aedes albopictus cells showed morphological features of phagocytic, non secretory cells. The general ultrastructural effects of Hg and MeHg were similar and clearly visible after a short incubation period of 6 h. The morphology of the mercury treated cells was characterized by indentation of the nucleus, chromatin clumping, the occurrence of pleomorphous mitochondria and indications of activated protein synthesis. Indented nuclei were also observed in mercury- and
Cd-treated hepatocytes (Hirano et al., 1991; Bucio et al., 1995). Chromatin clumping is a general prenecrotic effect. Also, mitochondrial pleomorphy is a prenecrotic event due to the disturbance of respiration and of mitochondrial growth and replication (Trump et al., 1978). In contrast to Cd (Braeckman et al., 1999a), mitochondrial swelling was less frequently found in mercury-treated Aedes cells. In our study, the activated protein synthesis was suggested by a series of changes in different organelles: the swelling of the nucleolus, increase of the free and membrane-bound ribosomes, the large and inflated RER cisternae filled with proteinaceous material and finally the appearance of well developed Golgi stacks. These morphological alterations were also found to some degree in Cd-treated cells (Braeckman et al., 1999a). Therefore they do not seem to be mercury-specific, instead they may reflect a general response to cell stress. The electron-dense content of the inflated RER differed clearly from the osmotically swollen electron-lucent cisternae that were described in other pathological situations (Miyakawa & Deshimaru, 1969; Ganote et al., 1974; Trump et al., 1978; Bucio et al., 1995). Although a pathological constipation of the RER, due to metalprovoked protein clustering, cannot be ruled out, it seems likely that the dilatation of the RER concerns an induction
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of certain stress proteins. In our previous experiments, data suggested that Cd is able to induce BiP, a RER-resident heat shock protein, in Aedes cells (Braeckman et al., 1999b). Probably, a similar induction takes place in mercury-treated Aedes cells. In contrast to our observations, Lachapelle et al. (1993) found a decrease of ER-bound ribosomes due to Hg treatment. Ganote et al. (1974) and Miura et al. (1979) described the Hg-induced dispersion of polysomes in the cytoplasm, which is also a prenecrotic event (Trump et al., 1978). Bucio et al. (1995) reported an increase of the free ribosomes due to Hg treatment. Apart from these general ultrastructural effects, we also noticed some alterations that were more specific for either Hg or MeHg. In the Hg-treated cells, clusters of small lucent vacuoles, close to the Golgi transition vesicles, were often seen. They might reflect an increased production of primary lysosomes, but a pathological effect (disturbance of vesicle transport or fusion) cannot be ruled out. In cells treated with MeHg, these clusters were almost lacking. On the other hand, here we observed tube-like structures that did not appear in Hg-treated cells. They are reminiscent of certain cytoplasmic inclusions found in oenocytes of the insect Calpodes ethlius (Locke, 1969), which seemed to be continuous with the smooth endoplasmic reticulum cisternae. We cannot explain the etiology of these remarkable structures. In MeHg-treated cells we found an obvious disorganization of the organelles together with blebbing. Both effects were probably due to the specific microtubule disrupting effect of this mercury species, which is in agreement with light microscopical observations (Braeckman et al., 1997a). MeHgcaused depolymerization of microtubules was described in detail by lmura et al. (1980) and Sager et al. (1983). AMG is a sensitive, histochemical silver amplification technique for the detection of mercury, gold and silver. This method has been used extensively for detection and localization of mercury in vertebrate tissues (Baatrup & Danscher, 1987; Nørgaard et al., 1991; Augier et al., 1993; Danscher et al., 1994; Christensen, 1996). Data on mercury staining is almost non-existent for invertebrates (Raes & De Coster, 1991; Marigómez et al., 1996) and to our knowledge, this is the first report on histochemical mercury staining in an invertebrate cell culture. In Hg and MeHg-treated cells, AMG grains were mainly located over the cytoplasm at the light microscopical level. The GCR, used as a measure for mercury accumulation, was quantified with an image analysis system. The mean GCR increased with increasing exposure times for both Hg- and MeHg-treated cells. The standard errors were large due to a considerable population heterogeneity. Because silver grains are quantified instead of mercury, this approach is a relative and indirect method. Mercury quantification by means of atomic fluorescence spectrometry is far more accurate and is described in Braeckman et al. (1998). However, in contrast to this analytical technique, the histochemical AMG method provides information about the population heterogeneity regarding mercury accumulation, which proved to be considerable in Aedes cell cultures. For both mercury species we found
widely differing accumulation capacities among the cells. An additional difference between the analytical and the histochemical technique concerns the type of mercury, which is quantified (Christensen, 1996). In atomic spectroscopy, all internalized mercury is quantified, whereas for histochemical staining, a cluster of sulfur- or seleniumbound mercury atoms is necessary to start the autokatalytical silver staining; one Hg-SH or Hg-Se center is not sufficient for histochemical detection (Danscher & Møller-Madsen, 1985; Danscher et al., 1994). This implies that, with the histochemical method, mainly mercury storage sites will be stained (and quantified). Ultrastructural localization of the AMG grains showed us the preferential accumulation of Hg and MeHg in the lysosomes. Lysosomal accumulation of mercury is thoroughly described in vertebrate literature (Baatrup & Danscher, 1987; Møller-Madsen, 1990; Danscher, 1991; Nørgaard et al., 1991; Christensen, 1996). In invertebrate literature, however, references are very scarce. Early publications on mercury detection in invertebrate cells came from BallanDufrançais et al. (1980) and Jeantet et al. (1980). They used electron probe microanalysis for localization of this heavy metal in the lysosomes of cockroach cells. Very few data exists on AMG demonstration of mercury in invertebrates (Raes & De Coster, 1991; Marigómez et al., 1996), and here also, mercury seemed to be mainly located in the lysosomes. Despite all these indications of lysosomes being the preferential accumulation sites for mercury, the uptake mechanism and transport routes of mercury to this organelle are still unclear. Hg has a high affinity for sulfhydryl groups. Proteins carrying Hg on their sulfhydryl groups may be broken down in the lysosomes. This detoxification process may lead to a concentration of this heavy metal in the lysosomes (Christensen, 1996). In 1980, Ballan-Dufrançais and co-workers reported that in lysosomes one atom of mercury is engaged in a molecule in which three atoms of sulfur coexist. This molecule must be of organic nature because in the mineral form, the mercury/sulfur ratio is always equal or higher than 1:1. The AMG-grains we found on the surface of mercury-treated Aedes cells probably represent mercury bound to free sulfhydryl groups of membrane proteins. The mercury sulfide concentration at this site seemed to be high enough to start the AMG reaction. This is not surprising as Endo et al. (1995) found that the non-internalized mercury fraction, bound to the cell membrane, is much greater than the internalized fraction. Mercury seemed to be concentrated in the lysosomes, possibly as a result of a detoxification process. However, it is not certain whether these high mercury concentrations, although presumed to be biologically inert, do not interfere with the normal lysosomal function. Therefore we tested the effect of Hg and MeHg on the lysosomal marker enzyme, AP. The activity was measured in vitro and in vivo to discriminate between direct and indirect effects of the mercury species on the enzyme. In the in vitro experiments we found a slight but highly significant inhibition of APactivity (6.3%) for only one Hg concentration (2.8 µM).
MERCURY AND METHYLMERCURY IN AN INSECT CELL LINE
This suggested that the Hg2+ concentration was very critical for this inhibitory effect. The indirect in vivo effect could be due to the interaction of mercury with free sulfhydryl groups during synthesis of the AP in the RER or the inhibition of the AP synthesis process itself. Enhanced AP turnover may also be envisaged. The effect of Hg on AP activity is well documented but ambiguous. In the fish Clarias, AP activity was found to be inhibited due to severe Hg treatment (the fish were injected with 2 ml of a 5% solution) (Saxena & Tyagi, 1979). A decrease of AP was found by Blasco et al. (1993) in the clam Ruditapes at very high Hg concentrations (1 mM). Naidu et al. (1984) found a significant decrease (30%) of AP-activity at the LC50/48 h (5.5 µM HgCl2). All these Hg concentrations are much higher than the sublethal concentrations we used and therefore the results might not be comparable. The effect of low Hg concentrations (0.37 µM) on AP-activity in marine lamellibranchs was shown to be species dependent. AP-activity was either enhanced, inhibited or unaffected depending on the species studied (Patel et al., 1988). Unlike Hg, MeHg had no effect on the AP-activity, either in vitro or in vivo. In contrast to our findings, Fowler et al. (1975) and Hossein & Dutta (1986) found an increase of AP activity at low MeHg levels in rat kidney and fish intestine, respectively. In conclusion we found that both mercury species caused mostly similar ultrastructural effects that were indicative of the induction of a general defense system, probably heat shock proteins. AMG showed us that the lysosomes were the main sites for mercury accumulation. The mercury accumulating capacity differed considerably among the Aedes cells. At sublethal concentrations, Hg inhibited AP-activity mostly in an indirect way; only for one concentration in vitro inhibition was found. MeHg had no effect on APactivity in the sublethal concentration range. ACKNOWLEDGEMENTS The authors thank Urszula Rzeznik for excellent technical assistance. We are also grateful to L. De Ridder (Laboratory for Histology, University of Ghent, Belgium) for the use of the transmission electron microscope. H. Raes is indebted to the FGWO (Belgian Fund for Medical Scientific Research) for the purchase of equipment without which this work would not have been possible (grant no. 3.9001.92). REFERENCES Augier, H., Benkoël, L., Brisse, J., Chamilian, A. and Park, W.K. 1993. Microscopic localization of mercury-selenium interaction products in liver, kidney, lung and brain of mediterranean striped dolphins (Stenella coeruleoalba) by silver enhancement kit. Cell. Mol. Biol., 39, 765–772. Baatrup, E. and Danscher, G. 1987. Cytochemical demonstration of mercury deposits in trout liver and kidney following methyl mercury intoxication: differentiation of two mercury pools by selenium. Ecotox. Environ. Safe., 14, 129–141. Ballan-Dufrançais, C., Ruste, J. and Jeantet, A.Y. 1980. Quantitative electron probe microanalysis on insects exposed to mercury. I Methods. An approach on the molecular form of the stored mercury. Possible occurrence of metallothionein-like proteins. Biol. Cell., 39, 317–324.
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