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The organochlorine herbicide chloridazon interacts with cell membranes
Comparative Biochemistry and Physiology Part C 120 (1998) 29 – 35
The organochlorine herbicide chloridazon interacts with cell membranes Mario Suwalsky a,*, Maritza Benites a, Fernando Villena b, Beryl Norris b, Ladislao Quevedo b a b
Faculty of Chemical Sciences, Uni6ersity of Concepcion, Casilla 3 -C, Concepcion, Chile Faculty of Biological Sciences, Uni6ersity of Concepcion, Casilla 3 -C, Concepcion, Chile Received 7 May 1997; accepted 14 November 1997
Abstract Chloridazon is a widely used organochlorine herbicide. In order to evaluate its perturbing effect on cell membranes it was made to interact with human erythrocytes, frog adrenergic neuroepithelial synapse and molecular models. These consisted in multilayers of dimyristoylphosphatidylethanolamine (DMPE) and of dimyristoylphosphatidyltidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that chloridazon interacted preferentially with DMPC multilayers. Scanning electron microscopy revealed that 0.1 mM chloridazon induced erythrocyte crenation. According to the bilayer couple hypothesis, this is due to the preferential insertion of chloridazon in the phosphatidylcholine-rich external moiety of the red cell membrane. Electrophysiological measurements showed that nerve stimulation was followed immediately by a transient increase in short-circuit current (SCC) and in the potential difference (PD) of the neuroepithelial synapse. Increasing concentrations of chloridazon caused a dose-dependent and reversible decrease of the responses of both parameters to 76% of their control values. The pesticide induced a similar (28%) significant time-dependent decrease in the basal values of the SCC and of PD. These results are in accordance with a perturbing effect of chloridazon on the phospholipid moiety of the nerve fibre membrane leading to interference with total ion transport across the nerve skin junction. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Chloridazon; Herbicide; Phospholipid bilayer; Erythrocyte membrane; Neuroepithelial synapse; X-ray diffraction; Scanning electron microscopy
1. Introduction Organochlorine pesticides, residues and metabolites, are ubiquitous in the environment because of their widespread use. Thus, the risk for human exposure and contamination is considerable [14]. Chloridazon (5 - amino - 4 - chloro - 2 - phenyl - 3(2H) - pyridazinone), also
Abbre6iations: DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; PD, potential difference; SCC, short-circuit current; SEM, scanning electron microscopy. * Corresponding author. Tel.: + 56 41 234985; fax: + 56 41 240280; e-mail: [email protected]. 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)00002-4
known as pyrazon [4], is an organochlorine herbicide used to control broad-level weeds, particularly in sugar beet crops [12]. In general, the molecular mechanisms of pesticide toxicity are poorly understood. However, the lipophilicity of most compounds makes lipid-rich membranes a plausible target of their interaction with living organisms [1]. It has been suggested that some effects related to their toxicity could be mediated by changes in membrane fluidity [23]. This is consistent with the hypothesis that alterations in the organization of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein functions [7]. Thus, the effect of dieldrin upon the activities of phospholipase C has been explained as a perturbation
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of the lipidic environment of the enzyme by the pesticide [3]. However, in spite of the putative toxic effects of pesticides in the perturbation of the membrane lipid phase, a clear relationship between changes in fluidity elicited by pesticides and their toxicity requires further support. In the course of in vitro systems search for the toxicity screening of chemicals, different cellular models have been applied to examine the adverse effects of pesticides in isolated organs [8]. This article describes the interaction of chloridazon with cell membranes of human erythrocytes and frog synapse as well as with molecular models of phospholipid multilayers. These systems have been used to determine the interaction and perturbing effects on membranes by the pesticides DDT [15], pentachlorophenol [16,17], 2,4-D [18], heptachlor [19] and dieldrin [20]. The multilayers consisted of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the human erythrocyte membrane, respectively [2]. Considering the lipophilic nature of chloridazon and the amphiphilic character of phospholipids, the interactions were assayed in hydrophobic and aqueous media in a wide range of concentrations. The capacity of chloridazon to perturb the multilayer structure of DMPC and DMPE was determined by X-ray diffraction. A simple model for ion movements across epithelia is the frog nerve-skin preparation. Electric stimulation of the synapse between sympathetic nerve endings and skin mucous glands has long been known to induce a transient rise in the bioelectric parameters. These are the transmembrane potential difference (PD) and the short-circuit current (SCC) across the skin [10], whose rise is due to an increase in active Cl − transport by the mucous gland [22]. The effects of chloridazon on the response of the frog neuroepithelial synapse to electrical stimulation and on the basal values of the bioelectric parameters of the frog skin were examined. The interaction of chloridazon with human erythrocytes was observed by scanning electron microscopy (SEM) to detect shape changes induced by the herbicide.
2. Materials and methods
2.1. X-ray diffraction analysis of phospholipid multilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma and chloridazon (99%, MW 221.6) from Riedel de Haen were used without further
purification. About 3 mg of each phospholipid was mixed with the corresponding weight of chloridazon in order to attain DMPC:chloridazon and DMPE:chloridazon powder mixtures in the molar ratios of 10:1, 5:1, 2:1 and 1:1. Each mixture was dissolved in chloroform:methanol 3:1 v/v and left to dry. The recrystallized samples were placed in special glass capillaries. They were diffracted in Debye-Scherrer cameras of 114.6 mm diameter and flat-plate cameras with 0.25 mm diameter glass collimators provided with rotating devices. The same procedure was followed with samples of each phospholipid and chloridazon. The aqueous specimens were prepared in glass capillaries mixing each phospholipid and chloridazon in the same proportions as described above. Each capillary was then filled with 200 ml of distilled water. These specimens were X-ray diffracted 2 days after preparation in flat-plate cameras. Specimen-tofilm distances were 8 or 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuKa radiation from a Philips PW1140 X-ray generator was used. The relative reflection intensities were obtained from films by peak-integration in a Joyce-Loebl MKIIICS microdensitometer interfaced to a PC. No correction factors were applied. The experiments in water were performed at 179 2°C, which is below the main transition temperatures of both DMPC and DMPE.
2.2. Scanning electron microscope (SEM) studies on human erythrocytes Chloridazon was made to interact in vitro with erythrocytes by incubating blood samples of five human healthy male adult donors. For this purpose, the samples were obtained by puncture of the ear lobule. One drop was aspirated with a plastic tuberculin syringe without the needle, 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) 0.1 ml of blood stock mixed with 0.9 ml of saline solution (control); and (b) 0.1 ml of blood stock mixed with saline chloridazon suspension to obtain concentrations equivalent to 0.1 and 1 mM. All the samples were incubated for 1 h at 37°C. Then, they were fixed with glutaraldehyde by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde in saline solution, reaching a final fixation concentration of 2.4%. After resting overnight at 5°C, the fixed samples were washed twice with saline solution, placed directly on Al stubs, air dried at 37°C for 30 min to 1 h and finally gold coated for 3 min at 10 − 1 torr in a S 150 Edwards sputter device. The observations and photographic records were performed in an ETEC Autoscan scanning electron microscope.
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2.3. Electrophysiological measurements on frog ner6e-skin preparation The experiments were performed on frogs of the species Caudi6erbera Caudi6erbera (180 – 350 g) which were kept in tap water at room temperature (18 –22°C) for at least 24 h before use. The frogs were pithed and the cutaneous branch of the tibial nerve was isolated together with the attached piece of skin and mounted in Ussing chambers as previously described [11]. An area of 1.33 cm2 was exposed to 3.5 ml of phosphatebuffered (pH 7.5) Ringer’s solution on both surfaces and gassed with a stream of air. The SCC was monitored with non-polarizable Ag/AgCl electrodes placed at 15 mm from the epithelium and connected to a voltage-clamp circuit (C. Me´traux Electronique) set to keep the PD across the skin at 0 mV. The PD was measured with calomel-agar electrodes at intervals of 2 min for 4 s. Both parameters were displayed on a two-channel Cole-Parmer recorder. Experiments were started 30 min after the bioelectric parameters of the preparation had reached a steady level. For electric stimulation, the nerve was placed on a pair of Ag electrodes connected to the isolation unit of a Grass S44 stimulator. Square wave pulses of 4 ms duration at a rate of 10 Hz and 10 V for 30 s were used. Preparations were stimulated at regular intervals (30 min). For each preparation the control responses were stable. Since each experiment lasted 8 – 10 h, usually only one set of readings was made for each neuroepithelial synapse. The effect of the pesticide on the steady basal values of the bioelectric parameters (non-stimulated preparations) was also assessed. The pesticide in aqueous suspensions was added to the solution bathing the inner (serosal) surface of the skin in the final equivalent concentrations indicated in the text. Statistical analysis was performed using Student’s paired ttest. Values throughout the work refer to means 9 SEM for each neuroepithelial synapse and for each skin.
3. Results
3.1. X-ray studies on phospholipid multilayers The molecular interaction of chloridazon with multilayers of the phospholipids DMPC and DMPE was studied in aqueous and hydrophobic media. Fig. 1 shows a comparison of the diffraction patterns of DMPC, chloridazon and of their 1:1 molar mixture after interacting and being recrystallized from chloroform:methanol 3:1 v/v solutions. The pattern of DMPC was affected by the herbicide: the lipid reflection intensities decreased, followed by the consequent disappearance of the weakest. However, several new reflections appeared, all due to chloridazon strongest reflections.
Fig. 1. Microdensitograms from X-ray diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v). Flat-plate cameras. Specimen-to-film distance 8 cm.
˚ DMPC bilayer width On the other hand, the 54.5 A remained practically constant (Table 1). These results indicated that part of the herbicide interacted with DMPC, penetrating into the phospholipid bilayer core and perturbing its structure. Fig. 2 illustrates the diffractograms obtained after DMPC, chloridazon and their 1:1 molar mixture were immersed in an excess of distilled water. Water produced an expansion of ˚ when dry to 63.1 A ˚. DMPC bilayer width from 54.5 A The observed reflections were reduced to only the first three orders of the bilayer width and a relatively intense ˚ . The latter arose from the stiff reflection of 4.2 A and fully extended hydrocarbon chains organized with rotational disorder in an hexagonal lattice [5]. Chloridazon produced a marked decrease in the phospholipid reflection intensities. On the other hand, the herbicide strongest reflections again showed up in the X-ray diagram of DMPC. These results meant that chloridaTable 1 DMPC and DMPE bilayer widths Phospholipid
Physical state
˚) Bilayer width (A
DMPC Dry DMPC:chloridazon 1:1 Dry DMPC Immersed in water DMPC:chloridazon 1:1 Immersed in water
54.5 54.5 63.1 63.1
DMPE Dry DMPE:chloridazon 1:1 Dry DMPE Immersed in water
51.4 51.4 51.4
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Fig. 2. Microdensitograms from X-ray diagrams of DMPC, chloridazon and of their 1:1 molar mixture in water. Flat-plate cameras. Specimen-to-film distance 8 cm.(The DMPC low-angle reflection of ˚ was observed in diagrams obtained at 14 cm specimen-to-film 63.1 A distance).
zon produced a perturbation in the DMPC bilayer structure, despite the fact that it is not very soluble in water. Fig. 3 exhibits the X-ray patterns obtained after DMPE was made to interact with chloridazon in the
Fig. 4. Microdensitograms from X-ray diagrams of DMPE, chloridazon and of their 1:1 molar mixture in water. Flat-plate cameras. Specimen-to-film distance 8 cm.
same way as described for DMPC in a hydrophobic medium. The perturbing effect of the pesticide upon the structure of DMPE multilayers was similar to that observed in DMPC. In fact, at their 1:1 molar ratio, a decrease of the lipid reflection intensities and the presence of several reflections from the herbicide were observed. Finally, the results of the interaction of DMPE and chloridazon in the presence of water are presented in Fig. 4. As may be observed, the X-ray pattern of DMPE, including its bilayer width (Table 1), is similar to the dry pattern shown in Fig. 3, although its reflections are weaker. On the other hand, at the 1:1 molar ratio, chloridazon produced a moderate decrease of DMPE reflection intensities. As in the previous experiments, many chloridazon reflections also showed up. These results indicated that the pesticide was also able to interact with DMPE bilayers, although the extent of the structural perturbation was less than that induced in DMPC under the same physicochemical conditions.
3.2. Scanning electron microscopy (SEM) studies on human erythrocytes
Fig. 3. Microdensitograms from X-ray diagrams of samples recrystallized from chloroform methanol 3:1 (v/v). Flat-plate cameras. Specimen-to-film distance 8 cm.
Blood samples were incubated with chloridazon suspensions equivalent to 0.1 and 1 mM. The SEM examinations indicated that chloridazon in both concentrations induced erythrocyte crenation, i.e. blebs or protuberances developed on their surface (Fig. 5b).
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In all cases, the cell population showed extensive crenation; only a few red cells were unchanged.
Table 2 Inhibitory effect of increasing concentrations of chloridazon (inner solution) on frog neuroepithelial synapse response to electric stimulation
3.3. Electrophysiological measurements on the frog ner6e-skin preparation
Chloridazon concentration % Decrease in (Mm) PD
Stimulation of the nerve was followed by a transient increase in the bioelectric parameters of the skin. The increase in SCC usually consisted of two main components. The first was a rapid rise in current from 36.49
0.01 0.10 1.00 1.00
2.6 90.5 16.7 92.1 24.5 93.6* (30.3 94.1) 24.5 93.6* (30.3 94.1)
% Decrease in SCC 1.5 90.5 13.6 92.1 23.6 9 3.2* (41.2 9 6.0) 23.6 9 3.2* (41.2 9 6.0)
Results are expressed as % decrease (means 9 SEM) in the skin response of the PD and of the SCC over the basal values of the non-stimulated skin; n =9. Figures in parentheses refer to basal bioelectric values in non stimulated synapses. * Significantly different from the response in the absence of pesticide: PB0.05 (Student’s paired t-test).
Fig. 5. Scanning electron microscope (SEM) images of human erythrocytes; × 2500. (a) control; (b) incubated with 1 mM chloridazon.
4.4 to 45.8 94.3 mA/cm2. The peak was reached in 0.399 0.03 min and the duration of the rapid rise was 1.029 0.07 min (n=22). The second component consisted of a slow rise when the rapid component was declining; the peak was variable, usually smaller than that of the first rise. The profile of the rise in PD was similar, although always smaller in magnitude than that of the rise in SCC: a rapid initial component rose from 33.69 3.1 to 36.2 9 3.2 mV (n= 22). Since the slow component was nearly continuous with the rapid component and very difficult to measure, it was not further analysed. The values throughout the work refer to the initial rapid rise in SCC and in PD. In 22 neuroepithelial synapses, stimulation of the nerve every 30 min for a period of 8–10 h induced repetitive responses which did not decline significantly in magnitude. The addition of aliquots of Ringer’s solution to the inner surface of the skin had no effect on the response to nerve stimulation in 12 preparations. Increasing concentrations of the pesticide, equivalent to 0.01 and 1.0 mM, produced a concentration-dependent reduction in the nerve-skin response to electric stimulation. Smaller concentrations had no effect. Table 2 shows that the decrease of the response to nerve stimulation at maximal concentration was 23.49 3.0 % in 140.09 20.6 min (n=9). By this time, the peak of the rapid rise in SCC was reached in 0.45 9 0.06 min and the duration of the rapid component was 1.489 0.11 min, values which were significantly different from control (PB 0.05 and B 0.01, respectively). The magnitude of the second component was not significantly reduced. All these effects were usually reversible after removal of chloridazon by a 3-fold washout. During the time course of the experimental runs, a gradual and significant (PB0.02) decline in the basal values of the PD and SCC was observed, both in synapses untreated
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and treated with the pesticide. The observed decrease was 27.59 3.1%; the slope of the line was significantly different from that of untreated synapses (r =0.89, P B 0.001, n= 8).
4. Discussion The widespread use of the herbicide chloridazon has stimulated interest in the study of its toxic effects on cell membranes. As described in the Section 1, the lipophilic character of most pesticides makes the phospholipid moiety of cell membranes a very plausible target for their interaction. The resulting changes in their fluidity should affect vital functions of membrane proteins. However, due to the comparatively low lipophilicity of chloridazon (log Ko/w 1.14) [9] as compared to other pesticides, slight or no interaction with phospholipid bilayers was expected. In fact, it has been reported that it is not significantly hazardous to fish and no symptoms of poisoning have been noted in humans [4]. The results indicate that chloridazon interacted with phospholipid bilayers present in the membranes of human erythrocytes, frog neuroepithelial synapses and molecular models. In fact, human erythrocytes exposed to chloridazon at concentrations as low as 0.1 mM developed spiculated shapes (echinocytes). According to the bilayer couple hypothesis [13], the shape changes induced in red cells by foreign molecules are due to differential expansion of the two monolayers. Thus, echinocytes are observed when the added molecules insert into the outer monolayer, whereas cup shapes (stomatocytes) are induced when they locate into the inner monolayer [6]. It can therefore be concluded that chloridazon preferentially interacted with phospholipids in the outer monolayer of the erythrocytes. Our studies on the membrane models support this conclusion. In fact, the X-ray studies on DMPC and DMPE multilayers showed that chloridazon preferentially interacted with the former. As previously described, DMPC and DMPE are representative of phospholipid classes located in the outer and inner monolayers of the red cell membrane, respectively [2]. The different type and degree of perturbation induced by chloridazon in DMPC and DMPE multilayers can be explained by differences in their packing arrangements. Chemically they only differ in their terminal amino groups, being + NH3 in DMPE and + N(CH3)3 in DMPC. 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 them. However, DMPE molecules pack tighter than those of DMPC. This effect, due to the DMPE smaller polar group and higher effective charge, makes for a very stable multi-
layer arrangement which is not significantly perturbed by the presence of water [21]. On the other hand, the gradual hydration of DMPC bilayers leads to water filling the highly polar interbilayer spaces. Consequently, there is an increase in its bilayer width from ˚ when dry up to nearly 63 A ˚ when fully hydrated 54.5 A at a temperature below that of its main transition. This condition promoted the incorporation of chloridazon into DMPC bilayers, its penetration into the acyl chain region and the ensuing molecular perturbation of the phospholipid bilayer structure. Electrophysiological measurements of an adrenergic synapse of the frog C. Caudi6erbera showed significant inhibition of the bioelectric response to nerve stimulation and a decrease of the basal bioelectric parameters. Since adrenergic synapse transmission is dependent on Ca2 + entry through the presynaptic nerve terminal membrane, and on active ion transport, principally Cl − , it may be concluded that the effect of the pesticide might be due to a change in noradrenaline activity and interference with total ion transport across the neuroepithelial membrane. The time-dependent reduction in the values of the basal bioelectric parameters of the neuroepithelial synapse induced by chloridazon might be ascribed to interference with active Na + transport across the skin. These results confirmed the fact, as previously reported [7], that the structural perturbation of membrane lipid bilayers affects the functioning of ion channels. A comparison of the effects produced by chloridazon with those induced by other organochlorine pesticides studied by the same methods indicates a low toxicity for chloridazon. In fact, heptachlor [19], 2,4-D [18] and dieldrin [20] affected the structure of DMPC multilayers at considerably lower concentrations than those of chloridazon. On the other hand, the first two pesticides produced greater perturbation in human erythrocytes than that induced by chloridazon. These differences might be due to dissimilar lipophilicities; the partition coefficients of heptachlor, 2,4-D, dieldrin and chloridazon expressed as log Ko/w, are 5.38, 4.32, 2.81 and 1.14, respectively [9].
Acknowledgements This work was supported by grants from FONDECYT (1960680) and DIUC (95.24.09-1 and 94.33.75-1).
References [1] Blasiak J. Changes in membrane fluidity evoked by organophosphorous insecticide bromfenvinfos and its methylated analogue. Comp Biochem Physiol 1995;110c:15 – 21.
M. Suwalsky et al. / Comparati6e Biochemistry and Physiology, Part C 120 (1998) 29–35 [2] Devaux PF, Zachowsky A. Maintenance and consequences of membrane phospholipid asymmetry. Chem Phys Lipids 1994;73:107 – 20. [3] Fonovich TM, Pechen AM. The effect of dieldrin on the Clostridium perfringens phosphatidylcholine phospholipase C activity. Pest Biochem Physiol 1995;51:170–7. [4] Humburg NE, editor. Herbicide Handbook. 6th edn. USA: Weed Society of America, 1989. [5] Janiak MJ, Small DM, Shipley GG. Nature of the thermal pretransition of synthetic phospholipids: Dimyristoyl- and dipalmitoyllecithin. Biochemistry 1976;15:4575–80. [6] Lange Y, Gough A, Steck TL. Role of the bilayer in the shape of the isolated erythrocyte membrane. J Membr Biol 1982;69:113 – 23. [7] Lundbaek JA, Birn P, Girshman J, Hansen AJ, Andersen OS. Membrane stiffness and channel function. Biochemistry 1996;35:3825 – 30. [8] McCarthy J, Shugart LR. Biological markers of environmental contamination. In: McCarthy J, Shugart LR, editors. Biomarkers of Environmental Contamination. USA: Lewis, 1990:3 – 14. [9] Noble A. Partition coefficients (n-octanol-water) for pesticides. J Chromatogr 1993;642:3–14. [10] Norris B, Quevedo L. Pentachlorophenol inhibits ion transport in the isolated toad cornea. Gen Pharmacol 1993;24:867 – 72. [11] Quevedo LL, Neumann E, Schmidt E, Cardenas H. Action of lycorine on noradrenergic response of a nerve-skin preparation. Cell Mol Biol 1988;34:295–302. [12] Sa´nchez-Martin MJ, Sanchez-Camazano M. Adsorption of chloridazon by soils and their components. Weed Sci 1991;39:417 – 22. [13] Sheetz MP, Singer SJ. Biological membranes as bilayer couples. A molecular mechanism of drug induced interactions. Proc Natl Acad Sci USA 1974;71:4457–61.
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[14] Stehr-Green PA, Farrar JA, Burse VW, Royce WG, Wohlleb JC. A survey of measured levels and dietary sources of selected organochlorine pesticide residues and metabolites in human sera from rural population. Am J Public Health 1988;78:828–30. [15] Suwalsky M, Buguen˜o N. X-ray studies on phospholipid bilayers. V. Interactions with DDT. Z Naturforsch 1985;40c:567–75. [16] Suwalsky M, Espinoza MA, Bagnara M, Sotomayor CP. X-ray studies on phospholipid bilayers. IX. Interactions with pentachlorophenol. Z Naturforsch 1990;45c:265 – 72. [17] Suwalsky M, Villena F, Montoya G, Garrido C, Sa´nchez I, Neira F. X-ray studies on phospholipid bilayers. XII. Interaction of pentachlorophenol with myelin. Z Naturforsch 1992;47c:601– 7. [18] Suwalsky M, Benites M, Villena F, Aguilar F, Sotomayor CP. Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes. Biochim Biophys Acta 1996;1285:267– 76. [19] Suwalsky M, Benites M, Villena F, Aguilar F, Sotomayor CP. The organochlorine pesticide heptachlor disrupts the structure of model and cell membranes. Biochim Biophys Acta 1997;1326:115– 23. [20] Suwalsky M, Benites M, Villena F, Aguilar F, Sotomayor CP. Interaction of the organochlorine pesticide dieldrin with phospholipid bilayers. Z Naturforsch 1997;52c:450 – 8. [21] Suwalsky M. Structural studies on phospholipid bilayers by X-ray diffraction methods. In: Hidalago C, editor. Physical properties of biological membranes and their functional implications, chapter 1. USA: Plenum, 1988:3 – 19. [22] Thompson IG, Mills JW. Isoproterenol-induced current changes in glands of frog skin. Am J Physiol (Cell Physiol) 1981;241 (10):C250 – 7. [23] Videira RA, Antunes-Madeira RC, Madeira MCM. Interaction of ethylazinphos with the physical organization of model and native membranes. Biochim Biophys Acta 1996;1281:65–72.
The organochlorine herbicide chloridazon interacts with cell membranes Mario Suwalsky a,*, Maritza Benites a, Fernando Villena b, Beryl Norris b, Ladislao Quevedo b a b
Faculty of Chemical Sciences, Uni6ersity of Concepcion, Casilla 3 -C, Concepcion, Chile Faculty of Biological Sciences, Uni6ersity of Concepcion, Casilla 3 -C, Concepcion, Chile Received 7 May 1997; accepted 14 November 1997
Abstract Chloridazon is a widely used organochlorine herbicide. In order to evaluate its perturbing effect on cell membranes it was made to interact with human erythrocytes, frog adrenergic neuroepithelial synapse and molecular models. These consisted in multilayers of dimyristoylphosphatidylethanolamine (DMPE) and of dimyristoylphosphatidyltidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that chloridazon interacted preferentially with DMPC multilayers. Scanning electron microscopy revealed that 0.1 mM chloridazon induced erythrocyte crenation. According to the bilayer couple hypothesis, this is due to the preferential insertion of chloridazon in the phosphatidylcholine-rich external moiety of the red cell membrane. Electrophysiological measurements showed that nerve stimulation was followed immediately by a transient increase in short-circuit current (SCC) and in the potential difference (PD) of the neuroepithelial synapse. Increasing concentrations of chloridazon caused a dose-dependent and reversible decrease of the responses of both parameters to 76% of their control values. The pesticide induced a similar (28%) significant time-dependent decrease in the basal values of the SCC and of PD. These results are in accordance with a perturbing effect of chloridazon on the phospholipid moiety of the nerve fibre membrane leading to interference with total ion transport across the nerve skin junction. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Chloridazon; Herbicide; Phospholipid bilayer; Erythrocyte membrane; Neuroepithelial synapse; X-ray diffraction; Scanning electron microscopy
1. Introduction Organochlorine pesticides, residues and metabolites, are ubiquitous in the environment because of their widespread use. Thus, the risk for human exposure and contamination is considerable [14]. Chloridazon (5 - amino - 4 - chloro - 2 - phenyl - 3(2H) - pyridazinone), also
Abbre6iations: DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidylethanolamine; PD, potential difference; SCC, short-circuit current; SEM, scanning electron microscopy. * Corresponding author. Tel.: + 56 41 234985; fax: + 56 41 240280; e-mail: [email protected]. 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)00002-4
known as pyrazon [4], is an organochlorine herbicide used to control broad-level weeds, particularly in sugar beet crops [12]. In general, the molecular mechanisms of pesticide toxicity are poorly understood. However, the lipophilicity of most compounds makes lipid-rich membranes a plausible target of their interaction with living organisms [1]. It has been suggested that some effects related to their toxicity could be mediated by changes in membrane fluidity [23]. This is consistent with the hypothesis that alterations in the organization of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein functions [7]. Thus, the effect of dieldrin upon the activities of phospholipase C has been explained as a perturbation
30
M. Suwalsky et al. / Comparati6e Biochemistry and Physiology, Part C 120 (1998) 29–35
of the lipidic environment of the enzyme by the pesticide [3]. However, in spite of the putative toxic effects of pesticides in the perturbation of the membrane lipid phase, a clear relationship between changes in fluidity elicited by pesticides and their toxicity requires further support. In the course of in vitro systems search for the toxicity screening of chemicals, different cellular models have been applied to examine the adverse effects of pesticides in isolated organs [8]. This article describes the interaction of chloridazon with cell membranes of human erythrocytes and frog synapse as well as with molecular models of phospholipid multilayers. These systems have been used to determine the interaction and perturbing effects on membranes by the pesticides DDT [15], pentachlorophenol [16,17], 2,4-D [18], heptachlor [19] and dieldrin [20]. The multilayers consisted of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the human erythrocyte membrane, respectively [2]. Considering the lipophilic nature of chloridazon and the amphiphilic character of phospholipids, the interactions were assayed in hydrophobic and aqueous media in a wide range of concentrations. The capacity of chloridazon to perturb the multilayer structure of DMPC and DMPE was determined by X-ray diffraction. A simple model for ion movements across epithelia is the frog nerve-skin preparation. Electric stimulation of the synapse between sympathetic nerve endings and skin mucous glands has long been known to induce a transient rise in the bioelectric parameters. These are the transmembrane potential difference (PD) and the short-circuit current (SCC) across the skin [10], whose rise is due to an increase in active Cl − transport by the mucous gland [22]. The effects of chloridazon on the response of the frog neuroepithelial synapse to electrical stimulation and on the basal values of the bioelectric parameters of the frog skin were examined. The interaction of chloridazon with human erythrocytes was observed by scanning electron microscopy (SEM) to detect shape changes induced by the herbicide.
2. Materials and methods
2.1. X-ray diffraction analysis of phospholipid multilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma and chloridazon (99%, MW 221.6) from Riedel de Haen were used without further
purification. About 3 mg of each phospholipid was mixed with the corresponding weight of chloridazon in order to attain DMPC:chloridazon and DMPE:chloridazon powder mixtures in the molar ratios of 10:1, 5:1, 2:1 and 1:1. Each mixture was dissolved in chloroform:methanol 3:1 v/v and left to dry. The recrystallized samples were placed in special glass capillaries. They were diffracted in Debye-Scherrer cameras of 114.6 mm diameter and flat-plate cameras with 0.25 mm diameter glass collimators provided with rotating devices. The same procedure was followed with samples of each phospholipid and chloridazon. The aqueous specimens were prepared in glass capillaries mixing each phospholipid and chloridazon in the same proportions as described above. Each capillary was then filled with 200 ml of distilled water. These specimens were X-ray diffracted 2 days after preparation in flat-plate cameras. Specimen-tofilm distances were 8 or 14 cm, standardized by sprinkling calcite powder on the capillary surface. Ni-filtered CuKa radiation from a Philips PW1140 X-ray generator was used. The relative reflection intensities were obtained from films by peak-integration in a Joyce-Loebl MKIIICS microdensitometer interfaced to a PC. No correction factors were applied. The experiments in water were performed at 179 2°C, which is below the main transition temperatures of both DMPC and DMPE.
2.2. Scanning electron microscope (SEM) studies on human erythrocytes Chloridazon was made to interact in vitro with erythrocytes by incubating blood samples of five human healthy male adult donors. For this purpose, the samples were obtained by puncture of the ear lobule. One drop was aspirated with a plastic tuberculin syringe without the needle, 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) 0.1 ml of blood stock mixed with 0.9 ml of saline solution (control); and (b) 0.1 ml of blood stock mixed with saline chloridazon suspension to obtain concentrations equivalent to 0.1 and 1 mM. All the samples were incubated for 1 h at 37°C. Then, they were fixed with glutaraldehyde by adding one drop of each sample to plastic tubes containing 1 ml of 2.5% glutaraldehyde in saline solution, reaching a final fixation concentration of 2.4%. After resting overnight at 5°C, the fixed samples were washed twice with saline solution, placed directly on Al stubs, air dried at 37°C for 30 min to 1 h and finally gold coated for 3 min at 10 − 1 torr in a S 150 Edwards sputter device. The observations and photographic records were performed in an ETEC Autoscan scanning electron microscope.
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2.3. Electrophysiological measurements on frog ner6e-skin preparation The experiments were performed on frogs of the species Caudi6erbera Caudi6erbera (180 – 350 g) which were kept in tap water at room temperature (18 –22°C) for at least 24 h before use. The frogs were pithed and the cutaneous branch of the tibial nerve was isolated together with the attached piece of skin and mounted in Ussing chambers as previously described [11]. An area of 1.33 cm2 was exposed to 3.5 ml of phosphatebuffered (pH 7.5) Ringer’s solution on both surfaces and gassed with a stream of air. The SCC was monitored with non-polarizable Ag/AgCl electrodes placed at 15 mm from the epithelium and connected to a voltage-clamp circuit (C. Me´traux Electronique) set to keep the PD across the skin at 0 mV. The PD was measured with calomel-agar electrodes at intervals of 2 min for 4 s. Both parameters were displayed on a two-channel Cole-Parmer recorder. Experiments were started 30 min after the bioelectric parameters of the preparation had reached a steady level. For electric stimulation, the nerve was placed on a pair of Ag electrodes connected to the isolation unit of a Grass S44 stimulator. Square wave pulses of 4 ms duration at a rate of 10 Hz and 10 V for 30 s were used. Preparations were stimulated at regular intervals (30 min). For each preparation the control responses were stable. Since each experiment lasted 8 – 10 h, usually only one set of readings was made for each neuroepithelial synapse. The effect of the pesticide on the steady basal values of the bioelectric parameters (non-stimulated preparations) was also assessed. The pesticide in aqueous suspensions was added to the solution bathing the inner (serosal) surface of the skin in the final equivalent concentrations indicated in the text. Statistical analysis was performed using Student’s paired ttest. Values throughout the work refer to means 9 SEM for each neuroepithelial synapse and for each skin.
3. Results
3.1. X-ray studies on phospholipid multilayers The molecular interaction of chloridazon with multilayers of the phospholipids DMPC and DMPE was studied in aqueous and hydrophobic media. Fig. 1 shows a comparison of the diffraction patterns of DMPC, chloridazon and of their 1:1 molar mixture after interacting and being recrystallized from chloroform:methanol 3:1 v/v solutions. The pattern of DMPC was affected by the herbicide: the lipid reflection intensities decreased, followed by the consequent disappearance of the weakest. However, several new reflections appeared, all due to chloridazon strongest reflections.
Fig. 1. Microdensitograms from X-ray diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v). Flat-plate cameras. Specimen-to-film distance 8 cm.
˚ DMPC bilayer width On the other hand, the 54.5 A remained practically constant (Table 1). These results indicated that part of the herbicide interacted with DMPC, penetrating into the phospholipid bilayer core and perturbing its structure. Fig. 2 illustrates the diffractograms obtained after DMPC, chloridazon and their 1:1 molar mixture were immersed in an excess of distilled water. Water produced an expansion of ˚ when dry to 63.1 A ˚. DMPC bilayer width from 54.5 A The observed reflections were reduced to only the first three orders of the bilayer width and a relatively intense ˚ . The latter arose from the stiff reflection of 4.2 A and fully extended hydrocarbon chains organized with rotational disorder in an hexagonal lattice [5]. Chloridazon produced a marked decrease in the phospholipid reflection intensities. On the other hand, the herbicide strongest reflections again showed up in the X-ray diagram of DMPC. These results meant that chloridaTable 1 DMPC and DMPE bilayer widths Phospholipid
Physical state
˚) Bilayer width (A
DMPC Dry DMPC:chloridazon 1:1 Dry DMPC Immersed in water DMPC:chloridazon 1:1 Immersed in water
54.5 54.5 63.1 63.1
DMPE Dry DMPE:chloridazon 1:1 Dry DMPE Immersed in water
51.4 51.4 51.4
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Fig. 2. Microdensitograms from X-ray diagrams of DMPC, chloridazon and of their 1:1 molar mixture in water. Flat-plate cameras. Specimen-to-film distance 8 cm.(The DMPC low-angle reflection of ˚ was observed in diagrams obtained at 14 cm specimen-to-film 63.1 A distance).
zon produced a perturbation in the DMPC bilayer structure, despite the fact that it is not very soluble in water. Fig. 3 exhibits the X-ray patterns obtained after DMPE was made to interact with chloridazon in the
Fig. 4. Microdensitograms from X-ray diagrams of DMPE, chloridazon and of their 1:1 molar mixture in water. Flat-plate cameras. Specimen-to-film distance 8 cm.
same way as described for DMPC in a hydrophobic medium. The perturbing effect of the pesticide upon the structure of DMPE multilayers was similar to that observed in DMPC. In fact, at their 1:1 molar ratio, a decrease of the lipid reflection intensities and the presence of several reflections from the herbicide were observed. Finally, the results of the interaction of DMPE and chloridazon in the presence of water are presented in Fig. 4. As may be observed, the X-ray pattern of DMPE, including its bilayer width (Table 1), is similar to the dry pattern shown in Fig. 3, although its reflections are weaker. On the other hand, at the 1:1 molar ratio, chloridazon produced a moderate decrease of DMPE reflection intensities. As in the previous experiments, many chloridazon reflections also showed up. These results indicated that the pesticide was also able to interact with DMPE bilayers, although the extent of the structural perturbation was less than that induced in DMPC under the same physicochemical conditions.
3.2. Scanning electron microscopy (SEM) studies on human erythrocytes
Fig. 3. Microdensitograms from X-ray diagrams of samples recrystallized from chloroform methanol 3:1 (v/v). Flat-plate cameras. Specimen-to-film distance 8 cm.
Blood samples were incubated with chloridazon suspensions equivalent to 0.1 and 1 mM. The SEM examinations indicated that chloridazon in both concentrations induced erythrocyte crenation, i.e. blebs or protuberances developed on their surface (Fig. 5b).
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In all cases, the cell population showed extensive crenation; only a few red cells were unchanged.
Table 2 Inhibitory effect of increasing concentrations of chloridazon (inner solution) on frog neuroepithelial synapse response to electric stimulation
3.3. Electrophysiological measurements on the frog ner6e-skin preparation
Chloridazon concentration % Decrease in (Mm) PD
Stimulation of the nerve was followed by a transient increase in the bioelectric parameters of the skin. The increase in SCC usually consisted of two main components. The first was a rapid rise in current from 36.49
0.01 0.10 1.00 1.00
2.6 90.5 16.7 92.1 24.5 93.6* (30.3 94.1) 24.5 93.6* (30.3 94.1)
% Decrease in SCC 1.5 90.5 13.6 92.1 23.6 9 3.2* (41.2 9 6.0) 23.6 9 3.2* (41.2 9 6.0)
Results are expressed as % decrease (means 9 SEM) in the skin response of the PD and of the SCC over the basal values of the non-stimulated skin; n =9. Figures in parentheses refer to basal bioelectric values in non stimulated synapses. * Significantly different from the response in the absence of pesticide: PB0.05 (Student’s paired t-test).
Fig. 5. Scanning electron microscope (SEM) images of human erythrocytes; × 2500. (a) control; (b) incubated with 1 mM chloridazon.
4.4 to 45.8 94.3 mA/cm2. The peak was reached in 0.399 0.03 min and the duration of the rapid rise was 1.029 0.07 min (n=22). The second component consisted of a slow rise when the rapid component was declining; the peak was variable, usually smaller than that of the first rise. The profile of the rise in PD was similar, although always smaller in magnitude than that of the rise in SCC: a rapid initial component rose from 33.69 3.1 to 36.2 9 3.2 mV (n= 22). Since the slow component was nearly continuous with the rapid component and very difficult to measure, it was not further analysed. The values throughout the work refer to the initial rapid rise in SCC and in PD. In 22 neuroepithelial synapses, stimulation of the nerve every 30 min for a period of 8–10 h induced repetitive responses which did not decline significantly in magnitude. The addition of aliquots of Ringer’s solution to the inner surface of the skin had no effect on the response to nerve stimulation in 12 preparations. Increasing concentrations of the pesticide, equivalent to 0.01 and 1.0 mM, produced a concentration-dependent reduction in the nerve-skin response to electric stimulation. Smaller concentrations had no effect. Table 2 shows that the decrease of the response to nerve stimulation at maximal concentration was 23.49 3.0 % in 140.09 20.6 min (n=9). By this time, the peak of the rapid rise in SCC was reached in 0.45 9 0.06 min and the duration of the rapid component was 1.489 0.11 min, values which were significantly different from control (PB 0.05 and B 0.01, respectively). The magnitude of the second component was not significantly reduced. All these effects were usually reversible after removal of chloridazon by a 3-fold washout. During the time course of the experimental runs, a gradual and significant (PB0.02) decline in the basal values of the PD and SCC was observed, both in synapses untreated
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and treated with the pesticide. The observed decrease was 27.59 3.1%; the slope of the line was significantly different from that of untreated synapses (r =0.89, P B 0.001, n= 8).
4. Discussion The widespread use of the herbicide chloridazon has stimulated interest in the study of its toxic effects on cell membranes. As described in the Section 1, the lipophilic character of most pesticides makes the phospholipid moiety of cell membranes a very plausible target for their interaction. The resulting changes in their fluidity should affect vital functions of membrane proteins. However, due to the comparatively low lipophilicity of chloridazon (log Ko/w 1.14) [9] as compared to other pesticides, slight or no interaction with phospholipid bilayers was expected. In fact, it has been reported that it is not significantly hazardous to fish and no symptoms of poisoning have been noted in humans [4]. The results indicate that chloridazon interacted with phospholipid bilayers present in the membranes of human erythrocytes, frog neuroepithelial synapses and molecular models. In fact, human erythrocytes exposed to chloridazon at concentrations as low as 0.1 mM developed spiculated shapes (echinocytes). According to the bilayer couple hypothesis [13], the shape changes induced in red cells by foreign molecules are due to differential expansion of the two monolayers. Thus, echinocytes are observed when the added molecules insert into the outer monolayer, whereas cup shapes (stomatocytes) are induced when they locate into the inner monolayer [6]. It can therefore be concluded that chloridazon preferentially interacted with phospholipids in the outer monolayer of the erythrocytes. Our studies on the membrane models support this conclusion. In fact, the X-ray studies on DMPC and DMPE multilayers showed that chloridazon preferentially interacted with the former. As previously described, DMPC and DMPE are representative of phospholipid classes located in the outer and inner monolayers of the red cell membrane, respectively [2]. The different type and degree of perturbation induced by chloridazon in DMPC and DMPE multilayers can be explained by differences in their packing arrangements. Chemically they only differ in their terminal amino groups, being + NH3 in DMPE and + N(CH3)3 in DMPC. 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 them. However, DMPE molecules pack tighter than those of DMPC. This effect, due to the DMPE smaller polar group and higher effective charge, makes for a very stable multi-
layer arrangement which is not significantly perturbed by the presence of water [21]. On the other hand, the gradual hydration of DMPC bilayers leads to water filling the highly polar interbilayer spaces. Consequently, there is an increase in its bilayer width from ˚ when dry up to nearly 63 A ˚ when fully hydrated 54.5 A at a temperature below that of its main transition. This condition promoted the incorporation of chloridazon into DMPC bilayers, its penetration into the acyl chain region and the ensuing molecular perturbation of the phospholipid bilayer structure. Electrophysiological measurements of an adrenergic synapse of the frog C. Caudi6erbera showed significant inhibition of the bioelectric response to nerve stimulation and a decrease of the basal bioelectric parameters. Since adrenergic synapse transmission is dependent on Ca2 + entry through the presynaptic nerve terminal membrane, and on active ion transport, principally Cl − , it may be concluded that the effect of the pesticide might be due to a change in noradrenaline activity and interference with total ion transport across the neuroepithelial membrane. The time-dependent reduction in the values of the basal bioelectric parameters of the neuroepithelial synapse induced by chloridazon might be ascribed to interference with active Na + transport across the skin. These results confirmed the fact, as previously reported [7], that the structural perturbation of membrane lipid bilayers affects the functioning of ion channels. A comparison of the effects produced by chloridazon with those induced by other organochlorine pesticides studied by the same methods indicates a low toxicity for chloridazon. In fact, heptachlor [19], 2,4-D [18] and dieldrin [20] affected the structure of DMPC multilayers at considerably lower concentrations than those of chloridazon. On the other hand, the first two pesticides produced greater perturbation in human erythrocytes than that induced by chloridazon. These differences might be due to dissimilar lipophilicities; the partition coefficients of heptachlor, 2,4-D, dieldrin and chloridazon expressed as log Ko/w, are 5.38, 4.32, 2.81 and 1.14, respectively [9].
Acknowledgements This work was supported by grants from FONDECYT (1960680) and DIUC (95.24.09-1 and 94.33.75-1).
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