THE ACTION OF MERCURY ON CELL MEMBRANES

CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 6, (2001) pp 299 š 304 Received 13 May 2001 Accepted 23 July 2001

THE ACTION OF MERCURY ON CELL MEMBRANES MILAN SCHARA1, MARJANA NEMEC1, INGRID FALNOGA1, ALFRED BOGOMIR KOBAL2, MARINA KVEDER3 and JELKA SVETEK1 1 J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, 2Mercury Mine Idrija, Arkova ul. 43, 5280 Idrija, Slovenia, 3Rudjer Boskovic Institute, Bijeni“ ka 54, 10000 Zagreb, Croatia

Abstract: The action of mercuric chloride and methyl mercuric chloride on the membrane lateral domain organization of bovine, equine, and canine erythrocytes was studied. Electron paramagnetic resonance (EPR) spectra of spin-labeled erythrocytes were analyzed with respect to their lateral domain structure. Continuous alteration of the membrane domain populations revealed that mercuric compounds affect the membrane via the evolution of toxic events in the cells. Key words: Mercuric Compounds, Erythrocytes, Membrane Lateral Domains, EPR, ESR. INTRODUCTION Mercury is a pollutant in aquatic ecosystems. Its tendency to bio-accumulate in the food chain makes it significant for human health. The sources of mercury pollution have been identified, but the mechanism of the spread and local accumulation of this pollutant is still the focus of much research. Within the organism mercury can be found in the cell membranes at significantly higher concentrations than in the neighboring aqueous phase [1]. Human monocytes treated with mercury also exhibited changes in lipid organization within their plasma membranes. [2]. There are differences between the rate of penetration of the mercuric ions and their organic compounds [3]. The hemolytic action of mercuric ions on the erythrocyte membrane was studied. [4]. In our previous experiments, using electron paramagnetic resonance (EPR) and spin probes, the lateral domain structure of plasma membrane in non-destructed living cells was determined [5]. We also found that aluminum ions had an effect on the

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membrane domain structure [6]. Therefore, we expected similar response of the erythrocyte membrane to the action of mercuric compounds. MATERIALS AND METHODS Erythrocytes were prepared from freshly drawn cow, horse, or dog blood. The samples were treated with various concentrations of HgCl 2 or CH3HgCl for periods from 10 to 240 minutes, and subsequently labeled with the methyl ester of 5-doxyl palmitic acid (MeFASL(10,3)) a spin probe that can easily be dissolved homogeneously in the membrane lipids. The spectra were measured at 370C. A new sample was prepared for each spectrum. An X band Bruker ES 300 EPR spectrometer was used with a 100 Gauss molecular field scan, 10 mW microwave power, and 1 Gauss modulation amplitude. The experimental spectra were compared with theoretically calculated spectra using the fast motion approximation for the spin probe molecules and the lateral membrane domain model. The procedure for multi-parameter nonlinear optimization was used as described elsewhere [7].

1 mT

experiment fit

Domain 1 2 3

Fig. 1 A typical experimental EPR spectrum*, with the corresponding fit, and the evaluated spectra of the three domains. The parameters of the fit for each domain are given below. Si and τi are the order parameter and the rotational correlation time, ∆Bi the line width, pai and pgi the polarity correction factors, and Wi is the domain proportion. Domain i Si pai Pgi Wi τi,ns ∆Bi,gauss 1 0.16 1.8 0.4 0.94 1.00016 0.21 2 0.36 0.7 1.3 1.01 0.00012 0.34 3 0.67 0.3 3.1 1.05 0.99992 0.45 * Horse erythrocytes, after 200 min in contact with 8.9µM CH3HgCl, membrane labeled with MeFASL(10,3).


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The phenomenon of lateral domains was studied theoretically [8] as well as experimentally. EPR allows a relatively easy experimental approach. Cells and tissues can be studied. The usual assumption that the spin probe in the plasmalemma is relatively slowly degraded by reduction to EPR silent nonparamagnetic hydroxylamine has proven to be relevant since the nitroxide has to diffuse into the cytoplasm. If oxygen and iron ions were amply present in the cytosol, or even if a large amount of spin probe would be available, then the nitroxide spectra from the intracellular membrane structures might interfere with the spectra of the plasma membrane. This method does not give direct information about domain size but the overall relative abundance of the particular domain type in the membrane can be assessed. The domain class is selected according to the orientation ordering and the rotational dynamics of the nitroxide group of the spin probe. It should be mentioned that for cells of larger size the amount of the plasma membrane measured in a capillary would be too small to observe a detectable signal. For cells of diameters larger than 10 µm care must be taken to retain the molar ratio 1/100 between the membranedissolved spin probes and the constituent lipid molecules of the membrane. ” Tissue cells„ should be used to measure larger sample volumes, where the cell size exceeds 20 µm. With 9 GHz CW EPR spectrometers, samples containing cells close to 40 µm in size represent the limit of possible reasonable detection. RESULTS AND DISCUSSION The reduction rate of 0.0167 mM Tempone (2,2,6,6-tetramethyl-4-piperidone-1oxyl) with 0.167 mM ascorbate in PBS at pH 7.4 in the presence or absence of 1.7 mM HgCl2 shows pseudo first order kinetics with the same reaction rate constant of 0.08 min-1. Therefore, under the applied conditions, the mercuric ion would not directly interfere with nitroxide reduction. The erythrocyte membrane domain population, in the absence of mercuric compounds, is given in Fig.2. Domain i 1 2 3

0.9

Wi

0.6 0.3 0.0

bovine

horse

dog

Fig. 2. The weighing factors Wi of the lateral domains in the erythrocyte of the corresponding animals. Here, No.1 is the most fluid domain type. See text to Fig.1.


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The action of HgCl2 on the membrane lateral domain structure of bovine erythrocytes was measured for a shorter incubation time, 10 minutes. No differences could be obtained in the EPR line shape, therefore no alteration of domain structure could be expected. For dog erythrocytes larger periods of exposure to methyl mercury were applied (Fig.3).

Wi / Wi control

1.6

Domain i 1 2 3

1.4 1.2 1.0 0.8 0

40

80

t, min

120

160

Fig. 3. The normalized weighing factors for each domain in dog erythrocytes in the presence of 8.9 µM methyl mercury chloride in physiological solution at room temperature.

Wi / Wi control

1.6 Domain i 1 2 3

1.4 1.2 1.0 0.8 180

200

t, min 220

240

Fig. 4. The time dependence of the normalized weighing factors for each of the three lateral membrane domains of horse erythrocytes in by the action of 1mM HgCl2 solution.


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The population of the most fluid disordered domain decreased. On the other hand, no effect was observed for the horse erythrocytes with the same concentration of CH3HgCl, neither did the 0.1mM HgCl2 solution produces a significant effect. However, a 1 mM HgCl2 solution induced an increase of the most fluid domain population (Fig.4). The presented relative rates for the alterations in erythrocyte domain structure show that methyl mercury is more effective in the case of dog erythrocytes. Besides the concentration effect of HgCl2 on horse erythrocytes, the direction of alteration for horse erythrocytes is just the opposite of that observed for methyl mercury shown in Fig.3. The outcome of our experiments is somewhat surprising. Much less aggressive substances produced larger alterations in the lateral domain structure than the applied toxic mercury compounds. That would mean that, on a longer time scale, the toxic effect in vivo could trigger the processes, which lead to severe damage of the cell, either directly or via the apoptotic processes. It is assumed that the primary targets are the tiol groups. Several important supramolecular structures in the membrane, like the aquaporins, mediating the fast transport of water, are influenced by mercury compounds [9]. Intracellular antioxidants like glutathione or superoxide dismutase can be depleted or damaged, and thus the membrane domain structure could be influenced, producing the conditions which convene the observed slow rearrangements in the domain structure of the membranes. REFERENCES 1. Girault, L., Boudon, A. and Dufor, E.J. Methyl mercury interactions with phospholipid membranes as reported by fluorescence, P-31, and Hg-199 NMR. Biochim. Biophys. Acta - Biomembranes 1325 (1997) 250-262. 2. InSug, O., Datar, S., Koch, C.J., Shapiro, I.M. and Shenker, B.J. Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve. Toxicology 124 (1997) 211-224. 3. Repetto, G., Sanz, P. and Repetto, M. Invitro effects of mercuric chloride and methylmercury chloride on neuroblastoma cells. Toxicology in vitro 7 (1993) 353-357. 4. Zolla, L., Lupidi, G., Bellelli, A. and Amiconi, G. Effects of mercuric ions on human erythrocytes. Relationhip between hypotonic swelling and cell aggregation. Biochim. Biophys. Acta 1328 (1997) 273-280. 5. Svetek, J., Kirn, B., Vilhar, B. and Schara, M. Lateral domain diversity in membranes of callus and root cells of potato as revealed by EPR spectroscopy. Physiol. Plant. 105 (1999) 499-505.

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6. Zel, J., Schara, M., Svetek, J., Nemec, M. and Gogala, N. Influence of aluminum on the membranes of micorrhizal fungi. Water Air Soil Pollut. 71 (1993) 101-109. 7. S trancar, J., S entjurc, M. and Schara, M. Fast and accurate characterization of biological membranes by EPR spectral simulation of nitroxides. J. Magn. Reson. 142 (2000) 254-256. 8. Jorgensen, K., Ipsen, J.H. and Mouritsen, O.G. Lipid-bilayer heterogeneity. Principles of Medical Biology 7A, Membranes and Cell Signaling 1997 JAI Press, 19-38. 9. Lahajnar, G., Ma“ ek, P. and Zupan“ i“ , I. Suppression of red cell diffusional water permeability by lipophilic solutes. Bioelectrochemistry 52 (2000) 179-185.