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The Pesticide Hexachlorobenzene Induces Alterations in the Human Erythrocyte Membrane
Pesticide Biochemistry and Physiology 65, 205–214 (1999) Article ID pest.1999.2443, available online at http://www.idealibrary.com on
The Pesticide Hexachlorobenzene Induces Alterations in the Human Erythrocyte Membrane Mario Suwalsky,*,1 Claudia Rodrı´guez,* Fernando Villena,† Felipe Aguilar,‡ and Carlos P. Sotomayor‡ *Faculty of Chemical Sciences and †Faculty of Biological Sciences, University of Concepcio´n, Casilla 160-C, Concepcio´n, Chile; and ‡Institute of Chemistry, Catholic University of Valparaı´so, Valparaı´so, Chile Received March 30, 1999; accepted August 5, 1999 Hexachlorobenzene (HCB) is one of the most widely distributed organochlorine residues in the biosphere. High concentrations have been found in developing and industrialized countries. Due to its persistence in the environment it has been detected in fish, birds, eggs, and human adipose tissue and serum, and chronic administration of this compound causes a number of toxic effects. Due to the lipophilic character of HCB, lipid-rich membranes are important targets for its interaction with living organisms. HCB was incubated with human erythrocytes and molecular models of biomembranes in order to better understand the molecular mechanism of its interaction with cell membranes. The models consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Electron microscopy showed that HCB interacted with the erythrocyte membrane, modifying its normal discoid morphology to cup-shaped stomatocytes. This result indicates that HCB was inserted in the inner layer of the red cell membrane, a conclusion supported by X-ray diffraction analyses of DMPC and DMPE bilayers. In fact, HCB incorporated into and perturbed the bilayer structures of DMPC and DMPE. However, the extent of the interaction was higher in DMPE. Therefore, the experimental results confirmed the important role played by the phospholipid bilayer in the molecular mechanism of HCB interaction with the red cell membrane. q1999 Academic Press Key Words: hexachlorobenzene; fungicide; erythrocyte membrane; phospholipid bilayer.
INTRODUCTION
Hexachlorobenzene (HCB)2 is one of the most widely distributed organochlorine residues in the biosphere (1). High concentrations have been found in developing and industrialized countries, which continue to be highly contaminated (2). Although its manufacture has been discontinued in most countries, it has been found as a by-product in the waste resulting from the manufacture of vinyl chloride, chlorine, and 1 To whom correspondence should be addressed. Fax: (56-41) 245974. E-mail: [email protected]. 2 Abbreviations used: HCB, hexachlorobenzene; DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; LUV, large unilamellar vesicles; Laurdan, 6-dodecanoyl-2-dimethylaminonaphthalene; DPH, 1,6-diphenyl-1,3,5-hexatriene; GP, general polarization; SEM, scanning electron microscopy.
chlorinated solvents (3). It has been used as a fungicide for control of smut infestation in barley, oat, and wheat seed (4). Due to its high persistence in the environment (5), HCB has been detected in tissues of freshwater and marine fish, birds, eggs (4), and human adipose tissue and serum (6). The human body burden, estimated at 0.7 mg, is derived mainly from dietary intake of fatty foods and inhalation, with diet contributing approximately 0.2 mg per day and inhalation estimated to contribute two orders of magnitude less (5). Chronic exposure of laboratory animals and humans to HCB damages several tissues and may induce hepatic porphyria (7), liver morphological lesions (8), liver plasma membrane damage (9), increased synthesis of liver microsomal enzymes, hypothyroxinemia, and thyroid adenomas (10). HCB is also known
205 0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
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to be carcinogenic (11, 12); it changes the red/ white blood cell profiles (13), induces alteration of microsomal membrane composition and monooxygenase activity (14), alters lipid metabolism (15), induces ultrastructural changes in ovarian follicles (16), affects neutrophil functions (17), and increases immunoglobulin levels (18). Despite these well-documented experimental results, there are no reports concerning the effects of HCB on the structure and function of cell membranes, particularly those of human erythrocytes. Based on its chemical similarity with lindane (hexachlorocyclohexane), which affects the morphology of the human red cell membrane (19), and its highly lipophilic character (log Ko/w 5.6) (20) that makes lipid-rich membranes probable targets for its interaction with living organisms, the present study was undertaken to determine whether HCB interacts with and alters the structure of cell membranes. During the development of in vitro systems for the toxicity screening of chemicals, different cellular models have been applied to examine the adverse effects of pesticides. This article describes the interaction of HCB with the human erythrocyte membrane and molecular models consisting of phospholipid multilayers and large unilamellar vesicles. These systems have been used to determine the interaction and perturbing effects on cell membranes by pesticides, such as pentachlorophenol (21, 22), 2,4-D (23), heptachlor (24), dieldrin (25), chloridazon (26), and lindane (19). A close relation between structural effects observed in these systems and functional effects in vivo has been found at physiologically relevant concentrations (21, 22). The multilayers consisted of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively (27). Considering the lipophilic nature of HCB and the amphiphilic character of phospholipids, the interactions were assayed in hydrophobic and aqueous media under a range of concentrations. The capacity of HCB to perturb the multilayer structure of DMPC and DMPE was determined
by X-ray diffraction. Fluorescent steady-state anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) and the fluorescence spectral shifts of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) were determined in DMPC large unilamellar vesicles (LUV). DPH is a useful probe for the hydrophobic regions of phospholipid bilayers, and its fluorescence steady-state anisotropy provides a measure of the rotational diffusion of the fluorophore according to the phospholipid acyl chain order. On the other hand, excitation and emission spectra of Laurdan are very sensitive to the physical state of membranes (28). With the fluorophore moiety located in a shallow position of the bilayer normal in the phospholipid polar headgroup environment, Laurdan provides information of dynamic properties in this zone of the bilayer (29, 30). Interaction of HCB with human erythrocytes was observed by scanning electron microscopy (SEM) to detect shape changes induced by the pesticide. MATERIALS AND METHODS
Scanning Electron Microscope Studies of Human Erythrocytes HCB was made to interact in vitro with red blood cells by incubating erythrocyte suspensions derived from healthy adult human male volunteers not currently receiving treatment with any pharmacological agent. Blood samples were obtained by puncture of the ear lobule and by aspiration into a tuberculin syringe (lacking the needle), without anticoagulant, and containing 1 ml of saline solution (0.9% NaCl) at room temperature. This blood stock suspension was used to prepare the following samples in tuberculin syringes: (a) control, prepared by mixing 0.1 ml of blood stock with 0.9 ml of saline and (b) 1 mM HCB suspension, prepared by mixing the blood with saline containing HCB. Samples were incubated at 378C for 1 h. They were then fixed overnight at 58C by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde. The fixed samples were washed twice with saline solution; drops of the suspensions were placed directly on Al stubs
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with the aid of tuberculin syringes without the needle, air dried at 378C for 30 min, and gold coated for 3 min at 1021 torr in a S 150 Edwards sputter device (Sussex, England). Resulting specimens were examined in an ETEC Autoscan scanning electron microscope (Etec Corp., Hayward, CA). These procedures were approved by the Ethics Committee of the University of Concepcion. X-Ray Diffraction Studies of Phospholipid Bilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma (St. Louis. MO) and HCB (lot LA 7676, MW 284.8) from Supelco (Bellefonte, PA) were used without further purification. About 1 mg of each phospholipid was mixed with the corresponding weight of HCB in order to attain DMPC:HCB and DMPE:HCB powder mixtures in the molar ratios of 10:1, 5:1, and 1:1. Each mixture was dissolved in chloroform:methanol 3:1 v/v and left to dry. The recrystallized samples were placed into special glass capillaries (Glas Technik & Konstruktion, Germany). They were diffracted in Debye– Scherrer cameras of 114.6 mm diameter (Philips, The Netherlands) and flat-plate cameras built by us with 0.25-mm diameter glass collimators provided with rotating devices. The same procedure was used for samples of each phospholipid and HCB. The aqueous specimens were prepared in glass capillaries, mixing each phospholipid and HCB in the proportions described above. Each capillary was then filled with about 200 mL of distilled water. These specimens were Xray diffracted 2 days after preparation in flatplate cameras. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuKa radiation from a PW1140 Philips X-ray generator was used. The relative reflection intensities on film were measured by peak-integration in a Bio-Rad GS-700 (Hercules, CA) densitometer using the Molecular Analyst/PC image software. No correction factors were applied. The experiments in water were performed at 17 6
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28C, which is below the main transition temperature of both DMPC and DMPE. Higher temperatures would have induced transitions to more fluid phases, making the detection of structural changes more difficult. The interplanar spacings and reflection intensities of each DMPC:HCB and DMPE:HCB molar mixture were compared with those of the corresponding pure phospholipid obtained under the same physicochemical conditions. At least two X-ray images of each specimen were taken. Fluorescence Measurements of Large Unilamellar Vesicles DMPC LUV suspended in water were prepared by extrusion of frozen and thawed multilamellar liposome suspension through two stacked polycarbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp., Cambridge, MA) employing nitrogen pressure at 108C over the lipid transition temperature, to a final concentration of 0.3 mM. DPH and Laurdan (Molecular Probes Inc., Eugene, OR) were incorporated into LUV by addition of small aliquots of concentrated solutions of the probe in tetrahydrofurane and ethanol, respectively, to LUV suspensions and gently shaken for ca. 30 min. Fluorescence spectra and anisotropy measurements were respectively performed in a Spex Fluorolog (Spex Industries Inc., Edison, NJ) and in a phase shift and modulation Greg-200 steadystate and time-resolved spectrofluorometer (I.S.S Inc., Champaign, IL), both interfaced to computers. Software from I.S.S. were used for data collection and analysis. Measurements of LUV suspensions were made at 188C using 10mm path-length square quartz cuvettes. Sample temperature was controlled by an external bath circulator (Cole Parmer, Chicago, IL) and measured prior and after each measurement using a digital thermometer (Omega Eng. Inc., Stanford, CT). Anisotropy measurements were made in the “L” configuration using Glan Thompson prism polarizers (I.S.S.) in both exciting and emitting beams. The emission was measured with a Schott WG-40 high-pass filter (Germany), which showed negligible fluorescence.
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Laurdan fluorescence spectral shifts were quantified through the General Polarization (GP) concept which was evaluated by GP 5 (Ib 2 Ir)/ (Ib 1 Ir), where Ib and Ir are, respectively, the intensities at the blue and red edges of the emission spectrum. These intensities have been measured at the emission wavelengths of 440 and 490 nm, which correspond respectively to the emission maxima of Laurdan in the gel and liquid crystalline phases (28). HCB was incorporated into LUV suspensions by addition of small aliquots of a concentrated ethanol solution and incubated at 408C for ca. 15 min. Samples with probes but without HCB showed no variation in the measured parameters during periods longer than those employed in the experiments. Blank subtraction was performed in all measurements using unlabeled samples without probes. HCB was added to sample as well as to blank cuvettes in order to take into account possible changes in light scattering due to the incorporation of the pesticide. Dilution and ethanol effects were considered in control experiments measuring both fluorescence parameters as function of water and ethanol addition, respectively, with no significant effects. RESULTS
Scanning Electron Microscopy Studies of Human Erythrocytes Human red blood cells were incubated with HCB suspensions equivalent to 1 mM. The SEM examination revealed that the erythrocytes lost their normal discoid biconcave aspect and underwent stomatocytic deformation, i.e., altered forms characterized by a deepening on one surface of their normal central concavity, taking up cup-shaped forms (Fig. 1a) in contrast to the forms of normal eythrocytes (Fig. 1b). In all the samples examined, the cell population showed similar intense shape changes. X-Ray Diffraction Studies of Phospholipid Bilayers The molecular interactions of HCB with multilayers of the phospholipids DMPC and
DMPE were studied in hydrophobic and aqueous media. Figure 2 shows a comparison of the diffraction patterns of DMPC, HCB, and their 10:1, 5:1, and 1:1 molar mixtures after interacting and recrystallizing from chloroform:methanol 3:1 v/v solutions. As may be observed, DMPC pattern was considerably affected by HCB when they were in a 10:1 molar ratio. Most reflections decreased their intensities by about two-thirds and the three strong reflections of 4.30, 4.07, ˚ fused, causing a weaker reflection and 3.83 A ˚ of 4.07 A. However, increasing ratios of HCB did not induce a further perturbation of the lipid structure. On the other hand, reflections arising from the fungicide were present practically only in the 1:1 mixture with DMPC. This result meant that lower concentrations of HCB completely incorporated into DMPC bilayers, causing its molecular disorder. Figure 3 presents the results obtained after DMPC, HCB, and their molar mixtures in the same ratio as above were immersed in distilled water. As expected (31), water changed the structure of DMPC, as its ˚ in its dry bilayer width increased from 54.7 A ˚ crystalline form to 64.4 A when immersed in water and its reflections were reduced to only the first three orders of the bilayer width. Fur˚ thermore, a new and strong reflection of 4.2 A appeared, indicative of the fluid state reached by DMPC, which corresponds to the average distance between the fully extended acyl chains organized with rotational disorder in hexagonal packing. As observed in dry specimens, the DMPC pattern was also considerably affected by HCB when they were in a 10:1 molar ratio. The fact that the reflection intensities decreased between 50 and 75% indicated that HCB, despite its low solubility in water, interacted with DMPC in an aqueous medium. Similarly, higher ratios of HCB did not significantly enhance the molecular perturbation of the DMPC structure. However, the strongest HCB reflections were present in the 5:1 and 1:1 mixtures with DMPC, a finding which led to the conclusion that only part of the fungicide was absorbed by the lipid. The results of the interaction of HCB with DMPE in the hydrophobic medium are shown in Fig. 4. As reported elsewhere (32), DMPE
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FIG. 1. Effect of hexachlorobenzene (HCB) on the morphology of human erythrocytes. Shown are scanning electron microscope (SEM) images of (a) untreated erythrocytes, 35003, and (b) erythrocytes incubated with 1 mM HCB, 32003.
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Fluorescence Measurements of Large Unilamellar Vesicles
FIG. 2. Microdensitograms from X-ray diffraction diagrams of specimens recrystallized from CHCl3:CH3OH 3:1 (v/v); flat-plate cameras; specimen-to-film distance: 8 cm.
The effect of HCB upon DMPC LUV was explored at two different depths of the lipid bilayer: at the hydrophilic/hydrophobic interface level and in the deep hydrophobic core. This was achieved by evaluating the Laurdan fluorescence spectral shift through the general polarization (GP) parameter and the DPH steady-state fluorescence anisotropy, respectively. As shown in Table 1, increasing concentrations of HCB did not change Laurdan GP but monotonously decreased DPH fluorescence anisotropy. The latter is related primarily to the restriction of the rotational motion due to the packing order of the lipid acyl chains. The decrease in this parameter can therefore be rationalized as a disruption of the structural order in the deep hydrophobic bilayer domain induced by incorporation of HCB. The possibility cannot be ruled out that DPH, assumed to be randomly distributed in the hydrophobic core of the bilayer, aggregates as
presents in two polymorphic forms: the first form (Lc1) has extended acyl chains parallel to ˚ bilayer width. In the bilayer normal and a 52 A the second form (Lc2) the acyl chains are tilted by about 308 and the bilayer width is nearly 44 ˚ . At the 10:1 molar ratio DMPE underwent a A Lc1 to Lc2 phase transition with a major reduc˚ reflection intensity. However, tion of the 4.04 A at the 5:1 ratio, DMPE exhibited the Lc1 form, while both phases were present in their 1:1 ratio. The strongest HCB reflections were observed under only the latter condition. Finally, the results of the interaction of the fungicide with DMPE in water are presented in Fig. 5. It has been shown (33) that water does not significantly affect the bilayer structure of DMPE. On the other hand, HCB did not induce phase transitions in DMPE, which remained throughout in the Lc1 form. However, increasing concentrations of the fungicide significantly decreased DMPE reflection intensities, becoming about 25% of HCBfree DMPE in their 1:1 ratio. HCB reflections were present in the 1:1 and 5:1 mixtures with DMPE.
FIG. 3. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPC and HCB; flatplate cameras; specimen-to-film distances: (A) 14 cm, (B) 8 cm.
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FIG. 4. Microdensitograms from X-ray diffraction diagrams of specimens recrystallized from CHCl3:CH3OH 3:1 (v/v); flat-plate cameras; specimen-to-film distance: 8 cm.
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nisms. It has been suggested that some effects directly related to their toxicity could be due to changes in membrane fluidity as a primary pesticide effect (34). However, despite the implications that at least part of their toxic effects could be due to the perturbation of the lipid phase of membranes (21–26), a clear relationship between changes in fluidity evoked by pesticides and their chronic toxicity still needs to be established. Results of the present study indicate that HCB interacted with the human erythrocyte membrane, inducing a gross alteration of its morphology from the normal discoid shape to a stomatocytic form. According to the bilayer couple hypothesis (35), shape changes induced in erythrocytes by foreign molecules are due to differential expansion of their two monolayers. Thus, spiculated shapes (equinocytes) arise when the added compound is inserted into the outer monolayer, whereas invaginated shapes (stomatocytes) are induced when the compound accumulates in the inner monolayer. The fact
a result of HCB incorporation, leading to higher local concentration. Consequently, radiationless energy transfer may occur between fluorophore molecules, thus lowering its fluorescence anisotropy. However, we have not considered this possibility because this effect should be concomitant with a concentration quenching, a result which was not observed in our experiments. The null effect of HCB on Laurdan GP indicates that this compound did not perturb the molecular dynamics of DMPC polar head groups. DISCUSSION
Organochlorine pesticides, including accompanying residues and metabolites, are ubiquitous in the environment because of their widespread use. Thus, the potential for human exposure and uptake is high. In general, the molecular mechanisms of pesticide action and toxicity are poorly understood. However, the lipophilicity of most pesticides makes lipid-rich membranes important targets of their interaction with living orga-
FIG. 5. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPE and HCB; flatplate cameras; specimen-to-film distances: (A) 14 cm, (B) 8 cm.
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TABLE 1 Effect of Hexachlorobenzene (HCB) on the Anisotropy (r) of DPH and the General Polarization (GP) of Laurdan Embedded in Large Unilamellar Dimyristoylphosphatidylcholine Vesicles HCB conc. (mM)
rDPH
GP Laurdan
0.00 0.01 0.10 1.00
0.298 0.240 0.223 0.208
0.411 0.412 0.411 0.414
Note. Each result represents the average of data in duplicate samples; between 6 and 12 determinations were performed in each sample; SEM 5 0.003. Probe:lipid ratio, 1:600.
that HCB induced the formation of stomatocytes indicates that this fungicide locates in the inner moiety of the red cell membrane. This conclusion is supported by the results obtained from the X-ray diffraction study of bilayers composed of DMPC and DMPE, which represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively (27). These phospholipids differ only in their terminal amino groups, being 1N(CH3)3 in DMPC and 1NH3 in DMPE. Moreover, both molecular conformations are very similar in their dry crystalline phases (31), with the acyl chains mostly parallel and extended and the polar groups lying perpendicularly to them. However, the gradual hydration of DMPC results in water filling the highly polar interbilayer spaces. Thus, its bilayer width increased ˚ when dry up to 64.4 A ˚ when it from 54.7 A was fully hydrated. This phenomenon led to its structural perturbation, by allowing the incorporation of HCB into DMPC bilayers, as shown by the X-ray and fluorescence experiments. DMPE molecules pack tighter due to their smaller polar groups and higher effective charge, resulting in a very stable bilayer system, which is not significantly affected by water (31) nor by a number of compounds (24, 26). Therefore, the observation that HCB incorporated into and perturbed the bilayer structure of DMPE to a larger extent than that of DMPC is rather peculiar. Given the chemical similarity of hexachlorobenzene with lindane (hexachlorocyclohexane)
it was not surprising to observe analogous effects on the erythrocyte membrane and on bilayers of DMPC and DMPE. In fact, both pesticides induced stomatocytosis in human erythrocytes; they interacted preferentially with DMPE multilayers and fluidized DMPC large unilamellar vesicles (19). Lindane, however, perturbed the polar head region of DMPC LUV to a larger extent than did HCB, whereas the latter had a greater effect on the acyl chain order. These findings may be explained by the higher lipophilicity of HCB (log Ko/w5.6) than that of lindane (log Ko/w 3.7). A recent report indicates that HCB affected rat hepatic microsomal membrane functions by alterating membrane-bound enzyme activities (10). HCB can certainly exert these actions through direct interactions with the proteins. However, there is a possibility that they are mediated by the membrane lipid bilayer through the “membrane bilayer pathway” (36). According to this possibility, HCB would first partition into the lipid bilayer, which would assist the pesticide by optimizing its location, orientation, and concentration with respect to the protein-binding site. Alternatively, enzyme functions may be altered by structural perturbations induced by HCB in the lipid bilayer surrounding the proteins (37). Although we still ignore the precise molecular mechanism of HCB toxicity, our experimental results confirm the important role played by the phospholipid bilayer of the erythrocyte membrane. Finally, it should be mentioned that the concentrations of HCB used in our studies are close to those found in the blood of the exposed population (18) and toxic to microorganisms (4). ACKNOWLEDGMENTS The authors thank Dr. Beryl Norris for her critical and thorough reading of the manuscript and Fernando Neira for his valuable technical assistance. This work was supported by grants from FONDECYT (1960680), DIUC (98.24.19-1) and DGIPUCV.
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The Pesticide Hexachlorobenzene Induces Alterations in the Human Erythrocyte Membrane Mario Suwalsky,*,1 Claudia Rodrı´guez,* Fernando Villena,† Felipe Aguilar,‡ and Carlos P. Sotomayor‡ *Faculty of Chemical Sciences and †Faculty of Biological Sciences, University of Concepcio´n, Casilla 160-C, Concepcio´n, Chile; and ‡Institute of Chemistry, Catholic University of Valparaı´so, Valparaı´so, Chile Received March 30, 1999; accepted August 5, 1999 Hexachlorobenzene (HCB) is one of the most widely distributed organochlorine residues in the biosphere. High concentrations have been found in developing and industrialized countries. Due to its persistence in the environment it has been detected in fish, birds, eggs, and human adipose tissue and serum, and chronic administration of this compound causes a number of toxic effects. Due to the lipophilic character of HCB, lipid-rich membranes are important targets for its interaction with living organisms. HCB was incubated with human erythrocytes and molecular models of biomembranes in order to better understand the molecular mechanism of its interaction with cell membranes. The models consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Electron microscopy showed that HCB interacted with the erythrocyte membrane, modifying its normal discoid morphology to cup-shaped stomatocytes. This result indicates that HCB was inserted in the inner layer of the red cell membrane, a conclusion supported by X-ray diffraction analyses of DMPC and DMPE bilayers. In fact, HCB incorporated into and perturbed the bilayer structures of DMPC and DMPE. However, the extent of the interaction was higher in DMPE. Therefore, the experimental results confirmed the important role played by the phospholipid bilayer in the molecular mechanism of HCB interaction with the red cell membrane. q1999 Academic Press Key Words: hexachlorobenzene; fungicide; erythrocyte membrane; phospholipid bilayer.
INTRODUCTION
Hexachlorobenzene (HCB)2 is one of the most widely distributed organochlorine residues in the biosphere (1). High concentrations have been found in developing and industrialized countries, which continue to be highly contaminated (2). Although its manufacture has been discontinued in most countries, it has been found as a by-product in the waste resulting from the manufacture of vinyl chloride, chlorine, and 1 To whom correspondence should be addressed. Fax: (56-41) 245974. E-mail: [email protected]. 2 Abbreviations used: HCB, hexachlorobenzene; DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; LUV, large unilamellar vesicles; Laurdan, 6-dodecanoyl-2-dimethylaminonaphthalene; DPH, 1,6-diphenyl-1,3,5-hexatriene; GP, general polarization; SEM, scanning electron microscopy.
chlorinated solvents (3). It has been used as a fungicide for control of smut infestation in barley, oat, and wheat seed (4). Due to its high persistence in the environment (5), HCB has been detected in tissues of freshwater and marine fish, birds, eggs (4), and human adipose tissue and serum (6). The human body burden, estimated at 0.7 mg, is derived mainly from dietary intake of fatty foods and inhalation, with diet contributing approximately 0.2 mg per day and inhalation estimated to contribute two orders of magnitude less (5). Chronic exposure of laboratory animals and humans to HCB damages several tissues and may induce hepatic porphyria (7), liver morphological lesions (8), liver plasma membrane damage (9), increased synthesis of liver microsomal enzymes, hypothyroxinemia, and thyroid adenomas (10). HCB is also known
205 0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
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to be carcinogenic (11, 12); it changes the red/ white blood cell profiles (13), induces alteration of microsomal membrane composition and monooxygenase activity (14), alters lipid metabolism (15), induces ultrastructural changes in ovarian follicles (16), affects neutrophil functions (17), and increases immunoglobulin levels (18). Despite these well-documented experimental results, there are no reports concerning the effects of HCB on the structure and function of cell membranes, particularly those of human erythrocytes. Based on its chemical similarity with lindane (hexachlorocyclohexane), which affects the morphology of the human red cell membrane (19), and its highly lipophilic character (log Ko/w 5.6) (20) that makes lipid-rich membranes probable targets for its interaction with living organisms, the present study was undertaken to determine whether HCB interacts with and alters the structure of cell membranes. During the development of in vitro systems for the toxicity screening of chemicals, different cellular models have been applied to examine the adverse effects of pesticides. This article describes the interaction of HCB with the human erythrocyte membrane and molecular models consisting of phospholipid multilayers and large unilamellar vesicles. These systems have been used to determine the interaction and perturbing effects on cell membranes by pesticides, such as pentachlorophenol (21, 22), 2,4-D (23), heptachlor (24), dieldrin (25), chloridazon (26), and lindane (19). A close relation between structural effects observed in these systems and functional effects in vivo has been found at physiologically relevant concentrations (21, 22). The multilayers consisted of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively (27). Considering the lipophilic nature of HCB and the amphiphilic character of phospholipids, the interactions were assayed in hydrophobic and aqueous media under a range of concentrations. The capacity of HCB to perturb the multilayer structure of DMPC and DMPE was determined
by X-ray diffraction. Fluorescent steady-state anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) and the fluorescence spectral shifts of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) were determined in DMPC large unilamellar vesicles (LUV). DPH is a useful probe for the hydrophobic regions of phospholipid bilayers, and its fluorescence steady-state anisotropy provides a measure of the rotational diffusion of the fluorophore according to the phospholipid acyl chain order. On the other hand, excitation and emission spectra of Laurdan are very sensitive to the physical state of membranes (28). With the fluorophore moiety located in a shallow position of the bilayer normal in the phospholipid polar headgroup environment, Laurdan provides information of dynamic properties in this zone of the bilayer (29, 30). Interaction of HCB with human erythrocytes was observed by scanning electron microscopy (SEM) to detect shape changes induced by the pesticide. MATERIALS AND METHODS
Scanning Electron Microscope Studies of Human Erythrocytes HCB was made to interact in vitro with red blood cells by incubating erythrocyte suspensions derived from healthy adult human male volunteers not currently receiving treatment with any pharmacological agent. Blood samples were obtained by puncture of the ear lobule and by aspiration into a tuberculin syringe (lacking the needle), without anticoagulant, and containing 1 ml of saline solution (0.9% NaCl) at room temperature. This blood stock suspension was used to prepare the following samples in tuberculin syringes: (a) control, prepared by mixing 0.1 ml of blood stock with 0.9 ml of saline and (b) 1 mM HCB suspension, prepared by mixing the blood with saline containing HCB. Samples were incubated at 378C for 1 h. They were then fixed overnight at 58C by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde. The fixed samples were washed twice with saline solution; drops of the suspensions were placed directly on Al stubs
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with the aid of tuberculin syringes without the needle, air dried at 378C for 30 min, and gold coated for 3 min at 1021 torr in a S 150 Edwards sputter device (Sussex, England). Resulting specimens were examined in an ETEC Autoscan scanning electron microscope (Etec Corp., Hayward, CA). These procedures were approved by the Ethics Committee of the University of Concepcion. X-Ray Diffraction Studies of Phospholipid Bilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma (St. Louis. MO) and HCB (lot LA 7676, MW 284.8) from Supelco (Bellefonte, PA) were used without further purification. About 1 mg of each phospholipid was mixed with the corresponding weight of HCB in order to attain DMPC:HCB and DMPE:HCB powder mixtures in the molar ratios of 10:1, 5:1, and 1:1. Each mixture was dissolved in chloroform:methanol 3:1 v/v and left to dry. The recrystallized samples were placed into special glass capillaries (Glas Technik & Konstruktion, Germany). They were diffracted in Debye– Scherrer cameras of 114.6 mm diameter (Philips, The Netherlands) and flat-plate cameras built by us with 0.25-mm diameter glass collimators provided with rotating devices. The same procedure was used for samples of each phospholipid and HCB. The aqueous specimens were prepared in glass capillaries, mixing each phospholipid and HCB in the proportions described above. Each capillary was then filled with about 200 mL of distilled water. These specimens were Xray diffracted 2 days after preparation in flatplate cameras. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuKa radiation from a PW1140 Philips X-ray generator was used. The relative reflection intensities on film were measured by peak-integration in a Bio-Rad GS-700 (Hercules, CA) densitometer using the Molecular Analyst/PC image software. No correction factors were applied. The experiments in water were performed at 17 6
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28C, which is below the main transition temperature of both DMPC and DMPE. Higher temperatures would have induced transitions to more fluid phases, making the detection of structural changes more difficult. The interplanar spacings and reflection intensities of each DMPC:HCB and DMPE:HCB molar mixture were compared with those of the corresponding pure phospholipid obtained under the same physicochemical conditions. At least two X-ray images of each specimen were taken. Fluorescence Measurements of Large Unilamellar Vesicles DMPC LUV suspended in water were prepared by extrusion of frozen and thawed multilamellar liposome suspension through two stacked polycarbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp., Cambridge, MA) employing nitrogen pressure at 108C over the lipid transition temperature, to a final concentration of 0.3 mM. DPH and Laurdan (Molecular Probes Inc., Eugene, OR) were incorporated into LUV by addition of small aliquots of concentrated solutions of the probe in tetrahydrofurane and ethanol, respectively, to LUV suspensions and gently shaken for ca. 30 min. Fluorescence spectra and anisotropy measurements were respectively performed in a Spex Fluorolog (Spex Industries Inc., Edison, NJ) and in a phase shift and modulation Greg-200 steadystate and time-resolved spectrofluorometer (I.S.S Inc., Champaign, IL), both interfaced to computers. Software from I.S.S. were used for data collection and analysis. Measurements of LUV suspensions were made at 188C using 10mm path-length square quartz cuvettes. Sample temperature was controlled by an external bath circulator (Cole Parmer, Chicago, IL) and measured prior and after each measurement using a digital thermometer (Omega Eng. Inc., Stanford, CT). Anisotropy measurements were made in the “L” configuration using Glan Thompson prism polarizers (I.S.S.) in both exciting and emitting beams. The emission was measured with a Schott WG-40 high-pass filter (Germany), which showed negligible fluorescence.
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Laurdan fluorescence spectral shifts were quantified through the General Polarization (GP) concept which was evaluated by GP 5 (Ib 2 Ir)/ (Ib 1 Ir), where Ib and Ir are, respectively, the intensities at the blue and red edges of the emission spectrum. These intensities have been measured at the emission wavelengths of 440 and 490 nm, which correspond respectively to the emission maxima of Laurdan in the gel and liquid crystalline phases (28). HCB was incorporated into LUV suspensions by addition of small aliquots of a concentrated ethanol solution and incubated at 408C for ca. 15 min. Samples with probes but without HCB showed no variation in the measured parameters during periods longer than those employed in the experiments. Blank subtraction was performed in all measurements using unlabeled samples without probes. HCB was added to sample as well as to blank cuvettes in order to take into account possible changes in light scattering due to the incorporation of the pesticide. Dilution and ethanol effects were considered in control experiments measuring both fluorescence parameters as function of water and ethanol addition, respectively, with no significant effects. RESULTS
Scanning Electron Microscopy Studies of Human Erythrocytes Human red blood cells were incubated with HCB suspensions equivalent to 1 mM. The SEM examination revealed that the erythrocytes lost their normal discoid biconcave aspect and underwent stomatocytic deformation, i.e., altered forms characterized by a deepening on one surface of their normal central concavity, taking up cup-shaped forms (Fig. 1a) in contrast to the forms of normal eythrocytes (Fig. 1b). In all the samples examined, the cell population showed similar intense shape changes. X-Ray Diffraction Studies of Phospholipid Bilayers The molecular interactions of HCB with multilayers of the phospholipids DMPC and
DMPE were studied in hydrophobic and aqueous media. Figure 2 shows a comparison of the diffraction patterns of DMPC, HCB, and their 10:1, 5:1, and 1:1 molar mixtures after interacting and recrystallizing from chloroform:methanol 3:1 v/v solutions. As may be observed, DMPC pattern was considerably affected by HCB when they were in a 10:1 molar ratio. Most reflections decreased their intensities by about two-thirds and the three strong reflections of 4.30, 4.07, ˚ fused, causing a weaker reflection and 3.83 A ˚ of 4.07 A. However, increasing ratios of HCB did not induce a further perturbation of the lipid structure. On the other hand, reflections arising from the fungicide were present practically only in the 1:1 mixture with DMPC. This result meant that lower concentrations of HCB completely incorporated into DMPC bilayers, causing its molecular disorder. Figure 3 presents the results obtained after DMPC, HCB, and their molar mixtures in the same ratio as above were immersed in distilled water. As expected (31), water changed the structure of DMPC, as its ˚ in its dry bilayer width increased from 54.7 A ˚ crystalline form to 64.4 A when immersed in water and its reflections were reduced to only the first three orders of the bilayer width. Fur˚ thermore, a new and strong reflection of 4.2 A appeared, indicative of the fluid state reached by DMPC, which corresponds to the average distance between the fully extended acyl chains organized with rotational disorder in hexagonal packing. As observed in dry specimens, the DMPC pattern was also considerably affected by HCB when they were in a 10:1 molar ratio. The fact that the reflection intensities decreased between 50 and 75% indicated that HCB, despite its low solubility in water, interacted with DMPC in an aqueous medium. Similarly, higher ratios of HCB did not significantly enhance the molecular perturbation of the DMPC structure. However, the strongest HCB reflections were present in the 5:1 and 1:1 mixtures with DMPC, a finding which led to the conclusion that only part of the fungicide was absorbed by the lipid. The results of the interaction of HCB with DMPE in the hydrophobic medium are shown in Fig. 4. As reported elsewhere (32), DMPE
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FIG. 1. Effect of hexachlorobenzene (HCB) on the morphology of human erythrocytes. Shown are scanning electron microscope (SEM) images of (a) untreated erythrocytes, 35003, and (b) erythrocytes incubated with 1 mM HCB, 32003.
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Fluorescence Measurements of Large Unilamellar Vesicles
FIG. 2. Microdensitograms from X-ray diffraction diagrams of specimens recrystallized from CHCl3:CH3OH 3:1 (v/v); flat-plate cameras; specimen-to-film distance: 8 cm.
The effect of HCB upon DMPC LUV was explored at two different depths of the lipid bilayer: at the hydrophilic/hydrophobic interface level and in the deep hydrophobic core. This was achieved by evaluating the Laurdan fluorescence spectral shift through the general polarization (GP) parameter and the DPH steady-state fluorescence anisotropy, respectively. As shown in Table 1, increasing concentrations of HCB did not change Laurdan GP but monotonously decreased DPH fluorescence anisotropy. The latter is related primarily to the restriction of the rotational motion due to the packing order of the lipid acyl chains. The decrease in this parameter can therefore be rationalized as a disruption of the structural order in the deep hydrophobic bilayer domain induced by incorporation of HCB. The possibility cannot be ruled out that DPH, assumed to be randomly distributed in the hydrophobic core of the bilayer, aggregates as
presents in two polymorphic forms: the first form (Lc1) has extended acyl chains parallel to ˚ bilayer width. In the bilayer normal and a 52 A the second form (Lc2) the acyl chains are tilted by about 308 and the bilayer width is nearly 44 ˚ . At the 10:1 molar ratio DMPE underwent a A Lc1 to Lc2 phase transition with a major reduc˚ reflection intensity. However, tion of the 4.04 A at the 5:1 ratio, DMPE exhibited the Lc1 form, while both phases were present in their 1:1 ratio. The strongest HCB reflections were observed under only the latter condition. Finally, the results of the interaction of the fungicide with DMPE in water are presented in Fig. 5. It has been shown (33) that water does not significantly affect the bilayer structure of DMPE. On the other hand, HCB did not induce phase transitions in DMPE, which remained throughout in the Lc1 form. However, increasing concentrations of the fungicide significantly decreased DMPE reflection intensities, becoming about 25% of HCBfree DMPE in their 1:1 ratio. HCB reflections were present in the 1:1 and 5:1 mixtures with DMPE.
FIG. 3. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPC and HCB; flatplate cameras; specimen-to-film distances: (A) 14 cm, (B) 8 cm.
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FIG. 4. Microdensitograms from X-ray diffraction diagrams of specimens recrystallized from CHCl3:CH3OH 3:1 (v/v); flat-plate cameras; specimen-to-film distance: 8 cm.
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nisms. It has been suggested that some effects directly related to their toxicity could be due to changes in membrane fluidity as a primary pesticide effect (34). However, despite the implications that at least part of their toxic effects could be due to the perturbation of the lipid phase of membranes (21–26), a clear relationship between changes in fluidity evoked by pesticides and their chronic toxicity still needs to be established. Results of the present study indicate that HCB interacted with the human erythrocyte membrane, inducing a gross alteration of its morphology from the normal discoid shape to a stomatocytic form. According to the bilayer couple hypothesis (35), shape changes induced in erythrocytes by foreign molecules are due to differential expansion of their two monolayers. Thus, spiculated shapes (equinocytes) arise when the added compound is inserted into the outer monolayer, whereas invaginated shapes (stomatocytes) are induced when the compound accumulates in the inner monolayer. The fact
a result of HCB incorporation, leading to higher local concentration. Consequently, radiationless energy transfer may occur between fluorophore molecules, thus lowering its fluorescence anisotropy. However, we have not considered this possibility because this effect should be concomitant with a concentration quenching, a result which was not observed in our experiments. The null effect of HCB on Laurdan GP indicates that this compound did not perturb the molecular dynamics of DMPC polar head groups. DISCUSSION
Organochlorine pesticides, including accompanying residues and metabolites, are ubiquitous in the environment because of their widespread use. Thus, the potential for human exposure and uptake is high. In general, the molecular mechanisms of pesticide action and toxicity are poorly understood. However, the lipophilicity of most pesticides makes lipid-rich membranes important targets of their interaction with living orga-
FIG. 5. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPE and HCB; flatplate cameras; specimen-to-film distances: (A) 14 cm, (B) 8 cm.
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TABLE 1 Effect of Hexachlorobenzene (HCB) on the Anisotropy (r) of DPH and the General Polarization (GP) of Laurdan Embedded in Large Unilamellar Dimyristoylphosphatidylcholine Vesicles HCB conc. (mM)
rDPH
GP Laurdan
0.00 0.01 0.10 1.00
0.298 0.240 0.223 0.208
0.411 0.412 0.411 0.414
Note. Each result represents the average of data in duplicate samples; between 6 and 12 determinations were performed in each sample; SEM 5 0.003. Probe:lipid ratio, 1:600.
that HCB induced the formation of stomatocytes indicates that this fungicide locates in the inner moiety of the red cell membrane. This conclusion is supported by the results obtained from the X-ray diffraction study of bilayers composed of DMPC and DMPE, which represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively (27). These phospholipids differ only in their terminal amino groups, being 1N(CH3)3 in DMPC and 1NH3 in DMPE. Moreover, both molecular conformations are very similar in their dry crystalline phases (31), with the acyl chains mostly parallel and extended and the polar groups lying perpendicularly to them. However, the gradual hydration of DMPC results in water filling the highly polar interbilayer spaces. Thus, its bilayer width increased ˚ when dry up to 64.4 A ˚ when it from 54.7 A was fully hydrated. This phenomenon led to its structural perturbation, by allowing the incorporation of HCB into DMPC bilayers, as shown by the X-ray and fluorescence experiments. DMPE molecules pack tighter due to their smaller polar groups and higher effective charge, resulting in a very stable bilayer system, which is not significantly affected by water (31) nor by a number of compounds (24, 26). Therefore, the observation that HCB incorporated into and perturbed the bilayer structure of DMPE to a larger extent than that of DMPC is rather peculiar. Given the chemical similarity of hexachlorobenzene with lindane (hexachlorocyclohexane)
it was not surprising to observe analogous effects on the erythrocyte membrane and on bilayers of DMPC and DMPE. In fact, both pesticides induced stomatocytosis in human erythrocytes; they interacted preferentially with DMPE multilayers and fluidized DMPC large unilamellar vesicles (19). Lindane, however, perturbed the polar head region of DMPC LUV to a larger extent than did HCB, whereas the latter had a greater effect on the acyl chain order. These findings may be explained by the higher lipophilicity of HCB (log Ko/w5.6) than that of lindane (log Ko/w 3.7). A recent report indicates that HCB affected rat hepatic microsomal membrane functions by alterating membrane-bound enzyme activities (10). HCB can certainly exert these actions through direct interactions with the proteins. However, there is a possibility that they are mediated by the membrane lipid bilayer through the “membrane bilayer pathway” (36). According to this possibility, HCB would first partition into the lipid bilayer, which would assist the pesticide by optimizing its location, orientation, and concentration with respect to the protein-binding site. Alternatively, enzyme functions may be altered by structural perturbations induced by HCB in the lipid bilayer surrounding the proteins (37). Although we still ignore the precise molecular mechanism of HCB toxicity, our experimental results confirm the important role played by the phospholipid bilayer of the erythrocyte membrane. Finally, it should be mentioned that the concentrations of HCB used in our studies are close to those found in the blood of the exposed population (18) and toxic to microorganisms (4). ACKNOWLEDGMENTS The authors thank Dr. Beryl Norris for her critical and thorough reading of the manuscript and Fernando Neira for his valuable technical assistance. This work was supported by grants from FONDECYT (1960680), DIUC (98.24.19-1) and DGIPUCV.
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