versión impresa ISSN 0366-1644
Bol. Soc. Chil. Quím. v.45 n.2 Concepción jun. 2000
A STRUCTURAL STUDY ON THE INTERACTION OF
MERCURIC CHLORIDE AND METHYLMERCURY WITH
MARIO SUWALSKY* AND BRIGITTE UNGERER
Faculty of Chemical Sciences, University of Concepción,
Casilla 160-C, Concepción, Chile.
(Received: April 12, 2000 - Accepted: May 5, 2000)
In memoriam of Doctor Guido S. Canessa C.
Biological membranes are the initial targets of pollutants from the surrounding environment. Understanding the mechanisms controlling their interactions at the molecular level is of primary importance to interpret the results of bio-accumulation and toxic effects. Mercury is a major environmental contaminant, which has been shown to cause increased morbidity and mortality in humans. In order to understand the molecular mechanism of mercury toxicity with cell membranes HgCl2 and CH3HgCl were made to interact with multilayers built up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylhosphatidylethanolamine (DMPE), which represent classes of phospholipids located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results presented in this paper indicated that both mercuric compounds interacted with and perturbed the molecular structures of DMPC and DMPE. These studies were performed by X-ray diffraction methods.
KEY WORDS: Mercuric chloride, methylmercury, cell membrane, phospholipid bilayer.
Las membranas biológicas constituyen el blanco inicial de la interacción de los contaminantes químicos con los seres vivos. Por tal razón es de una importancia fundamental entender los mecanismos moleculares que controlan sus interacciones, los que permiten interpretar los efectos de su bioacumulación y toxicidad. El mercurio es un contaminante químico ambiental de la mayor importancia ya que se ha demostrado que causa morbilidad y mortalidad en los seres humanos. Con el propósito de explicar los mecanismos moleculares de la toxicidad del mercurio sobre las membranas celulares, HgCl2 y CH3HgCl se hicieron interaccionar con multicapas de los fosfolípidos dimiristoilfosfatidilcolina (DMPC) y dimiristoilfosfatidiletanolamina (DMPE), que representan lípidos presentes respectivamente en la monocapa externa e interna de las membranas de los glóbulos rojos humanos. Los resultados obtenidos indican que ambos compuestos interaccionan y perturban las estructuras moleculares de la DMPC y DMPE. Estos estudios se efectuaron por métodos de difracción de rayos X.
PALABRAS CLAVES: Cloruro mercúrico, metilmercurio, membrana celular, bicapa lipídica.
The ubiquitous nature of mercury in the environment, its persistence and toxicity to humans are some of the current concerns associated with this pollulant1). These concerns have focused mostly on the presence of elevated mercury levels in commercial seafood2), particularly fish3), which is a major human dietary source of this element. The Environmental Protection Agency of U.S.A. has reported an average of 100-200 ng of mercury per gram of fish and shellfish4). It is toxic to microorganisms, plants, invertebrates and vertebrates. Mercury may also be ingested in tap water5). Mercury can exist in three oxidation states in natural waters: Hg0, Hg+, and Hg2+ 6) being their main species Hg2+, HgOH+, Hg(OH)2, HgCl+, HgCl2, HgCl3-, HgCl42-, and HgOHCl7). Their distribution depend upon the pH, redox potential and availability of anions which form stable complexes with mercury. In the environment, inorganic mercury can be transformed into organic mercury compounds, particularly in methylmercury6), which is considered the most toxic of all mercury chemical forms. It is also the main form accumulated along food webs accounting for up to 95% of total mercury into aquatic carnivorous species and leading to extremely high bio-accumulation factors (£106)8). Unfortunately, little is known about the molecular mechanism controlling the uptake and toxicity of mercury in its various forms8).
The cell membrane is a diffusion barrier which protects the cell interior. Therefore, its structure and functions are susceptible to alteration as a consequence of interactions with heavy metals9-10). In this paper, we report the interaction of mercuric chloride and methylmercury with bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids located in the outer and inner monolayers of the red cell membrane, respectively11). This study was performed by X-ray diffraction methods and its aim was to determine the capacity of both mercuric compounds to interact and perturb the structure of the lipid moiety of cell membranes.
MATERIALS AND METHODS
Synthetic DMPC (lot 80H-8371 A grade MW 677.9) and DMPE (lot 68F-8350 A grade MW 635.9) from Sigma (MO, USA), HgCl2 from Riedel-de Haën (Seelze, Germany, lot 21280, MW 271.5), and CH3HgCl from Pfalz & Bauer, Inc., Conn., USA, M 21890, MW 251.1) were used without further purification. About 1 mg of each phospholipid was introduced into 1 mm diameter special glass capillaries (Glas Technik & Konstruktion, Berlin, Germany), which were then filled with 200 ml of (a) distilled water, and (b) aqueous solutions of HgCl2 and CH3HgCl in a range of concentrations. The specimens were X-ray diffracted two days after preparation in flat plate cameras provided with rotating devices. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuKa radiation from a Philips PW 1140 X-ray generator (The Netherlands) was used. The relative reflection intensities on films were measured by peak-integration using a Bio-Rad GS-700 (CA, USA) microdensitometer and Bio-Rad Molecular Analyst/PC image software; no correction factors were applied. The experiments were performed at 17 ± 2°C, which is below the main transition temperature of both DMPC and DMPE.
Figure 1 illustrates the results obtained by incubating DMPC multilayers with water and HgCl2 in the 10-5 M to 10-1 M concentration range. As expected, water altered the crystalline structure of DMPC: its bilayer width increased from about 55 Å to 64.5 Å and its reflections were reduced to only the first three orders of the bilayer width12). On the other hand, a new and strong reflection of 4.2 Å showed up, which was indicative of the fluid state reached by DMPC as it corresponded to the average distance between the fully extended acyl chains organized with rotational disorder in hexagonal packing. Exposure to 10-5 M HgCl2 induced a marked decrease in the phospholipid reflection intensities: those of the first two reflections were about 75% less intense than those of Hg2+-free DMPC, whereas the intensity of the 4.2 Å reflection decreased by 50%. These results imply that this low concentration of Hg2+ ions induced molecular disorder of DMPC bilayers. However, 10-4 M HgCl2 increased the intensities of these reflections to values close to those of pure DMPC indicating that some sort of molecular reordering had occurred. Higher HgCl2 concentrations reversed the previous tendency once more, as the intensities again decreased, particularly those of the low angle reflections. Similar intensity fluctuations have also been observed in DMPC with CuCl2 9) and Al(acac)3 10). When the salt concentration was increased to 0.1 M no reflections were observed, indicating complete perturbation of the DMPC structure.
Fig. 1 Microdensitograms from X-ray diffraction diagrams of DMPC in water and aqueous solutions of HgCl2. Specimen-to- film distance (a) 14 cm, (b) 8 cm.
|Fig. 2 Microdensitograms from X-ray diffraction diagrams of DMPE in water and aqueous solutions of HgCl2. Specimen-to- film distance (a) 14 cm, (b) 8 cm|| |
Results from similar experiments with DMPE are exhibited in Figure 2. As reported elsewhere13), water did not affect significantly the bilayer structure of DMPE. However, increasing concentrations of HgCl2 progressively decreased DMPE reflection intensities; the maximal reduction was about 75% after addition of 0.1 M in both the low and high angle regions. These findings showed that HgCl2 perturbed with equal intensity both the polar and acyl chain regions of DMPE bilayer structure in a concentration-dependent manner. Despite this high degree of perturbation it was however somewhat lower than that induced in DMPC bilayers by equal concentrations of HgCl2.
The results obtained after DMPC and DMPE were made to interact with methylmercury are shown in Figures 3 and 4, respectively. As it can be appreciated, the effects of this compound upon the structure of DMPC were milder than those observed in the case of HgCl2 under the same physicochemical conditions. In fact, while up to 10-4 M CH3HgCl there was an increase of the reflection intensities, indicative of the reordering of DMPC molecules, higher concentrations showed only a slight decrease of this parameter, which at 10-2 M reached values similar to those observed in DMPC in the absence of methyl mercury. In the case of DMPE its structure was perturbed by 10-4 and 10-3 M CH3HgCl, whereas a moderate rearrangement was induced by 10-2 M CH3HgCl. In conclusion, both compounds interacted with DMPC and DMPE bilayers being this interaction somewhat higher with HgCl2.
Fig. 3 Microdensitograms from X-ray diffraction diagrams of DMPC in water and aqueous solutions of CH3HgCl. Specimen-to- film distance (a) 14 cm, (b) 8 cm.
|Fig. 4 Microdensitograms from X-ray diffraction diagrams of DMPE in water and aqueous solutions of CH3HgCl. Specimen-to- film distance (a) 14 cm, (b) 8 cm.|| |
Biological membranes are the initial targets of pollutants from the surrounding environment. Interactions of pollutants with bio-membranes also control their uptake process and subsequent toxicological effects at the cell and organ levels. The knowledge of the mechanisms controlling these interactions at the molecular level is therefore of primary importance. In order to understand the molecular mechanism of the toxicity of mercury the interaction of HgCl2 and CH3HgCl with phospholipid bilayers was explored in the concentration range of the reported toxic blood levels4). Results presented in this paper indicated that both mercuric compounds, at concentrations as low as 10-5 M, interacted with and perturbed the molecular structures of DMPC and DMPE, which represent classes of phospholipids located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Chemically both phospholipids only differ in their terminal amino groups, these being +N(CH3)3 in DMPC and +NH3 in DMPE. Moreover, both molecular conformations are very similar in their dry crystalline phases: their acyl chains are mostly parallel and extended with the polar groups lying perpendicularly to them14). However, DMPE molecules pack tighter than those of DMPC. This effect, due to DMPE smaller polar group and higher effective charge, makes for a very stable multilayer arrangement which is not significantly perturbed by water12,14) nor by a number of compounds9,15-16). This organization did not prevent low concentrations of HgCl2 and CH3HgCl from interacting with and perturbing DMPE structure. On the other hand, the gradual hydration of DMPC bilayers leads to water filling the highly polar interbilayer spaces14). Consequently, there is an increase in its bilayer width from about 55 Å when dry up to 64.5 Å when fully hydrated, and a decrease in the number of reflections due to the resulting fluidity. These conditions promoted the incorporation of both mercuric compounds into DMPC bilayers and the ensuing molecular perturbation of its bilayer structure, which was considerably greater with HgCl2 than that observed with CH3HgCl.
These findings do not seem to be in agreement with others reported in the literature. Studies with 199Hg-NMR7), 31P-NMR17), and fluorescence spectroscopy18) have found that HgCl2 decreased the fluidity of DMPE and phosphatidylethanolamine vesicles and micelles, an effect explained by Hg(II) interaction with the lipid primary amino group. These authors have also reported that HgCl2 did not interact significantly with phospholipids carrying a quaternary amino group such as dipalmitoyl- and egg-phosphatidylcholine . However, other investigators do not agree with this conclusion17). The explanation might lie in that the term "fluidity" has different interpretations. Thus, for instance, it has been used in the above technique as an operational term, which can be related but is not identical with the physical definition of fluidity19), whereas in X-ray diffraction this term means that there is a lack of periodical order, and consequently the reflections become weaker, broader and eventually can disappear.
The observed differences in the interactions of both mercuric compounds with the phospholipid bilayers can be due to their different lipophilicities. The methylmercury octanol/water partition coefficient is about four times higher than that of HgCl220). Therefore, this compound would tend to locate into the hydrophobic core of the lipid bilayers. In contrast, the permeation of a hydrophilic compound such as HgCl2 would be retarded in the water/lipid interfaces creating a high concentration in that region and a low one in the hydrophobic acyl chain environment.
The ordering-disordering effects induced by HgCl2 to DMPC and by CH3HgCl to DMPE might look surprising. However, similar effects have been observed in the interactions of ZnCl2 with DMPE21), CuCl2 with DMPC9) and Al(acac)3 with both DMPE and DMPC10). The structural fluctuations observed in the current work can be explained as follows. As previously described, the lipid polar head groups lie perpendicularly to the extended acyl chains; contacts between monolayers to form bilayers occur via the acyl chain terminal methyl groups. This arrangement is stabilized through hydrophobic interactions among the acyl chains and electrostatic interactions between the negatively charged phosphates and positively charged terminal amino groups of neighboring polar heads22). At low HgCl2 concentrations Hg2+ ions bind electrostatically to a few DMPC phosphates. As a consequence, inter-head group interactions that otherwise restrict the axial and lateral motions of the lipid molecules are disrupted; in addition, some head groups and acyl chains change their orientations. This type of structural perturbation would result in the fainting of DMPC reflection intensities. However, as the HgCl2 concentration increases, more Hg2+ ions link to more phosphate groups leading to a cooperative ordering of DMPC molecules. A further increase of HgCl2 concentration would induce repulsive interactions between the mercury-saturated DMPC head groups of neighboring bilayers with the consequent disruption of its structure. On the other hand, the interaction of increasing concentrations of CH3HgCl with DMPE would proceed according to the following steps:1) as explained above, the lipophilic nature of CH3HgCl promotes its incorporation among DMPE hydrophobic, long and flexible acyl chains thus stabilizing DMPE structure; 2) once this region is saturated a few additional Hg2+ ions link to head group phosphates breaking the weaker hydrogen bonds and electrostatic interactions that stabilize the lipid structure; 3) a further increase of Hg2+ ions again stabilize the DMPE structure through their interaction with the rest of the phosphates, thus bridging neighboring bilayers.
Most likely, the lethal toxicity of mercuric compounds arises from their interactions with proteins, either directly mainly through the formation of stable bonds with protein SH- groups, or indirectly. In the latter case, the interaction of mercuric compounds with membrane lipids would perturb their bilayer conformations. As a consequence, cell membrane structure and properties such as fluidity, permeability, receptor and channel function may be affected. Our results showed that both mercuric compounds interacted with and perturbed the lipid bilayer structures related to cell membranes. On the other hand, we have also found that both compounds irreversibly decreased the bio-electric parameters of isolated toad skin at concentrations as low as 8 mM23).
The authors thank Fernando Neira for technical assistance. This work was supported by grants from FONDECYT (1990289) and DIUC (98.24.19-1).
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