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Toxic effects of the fungicide benomyl on cell membranes
Comparative Biochemistry and Physiology Part C 125 (2000) 111 – 119
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Toxic effects of the fungicide benomyl on cell membranes Mario Suwalsky a,*, Maritza Benites a, Beryl Norris b, Patricio Sotomayor c a b
Faculty of Chemical Sciences, Uni6ersity of Concepcio´n, Casilla 160 -C, Concepcio´n, Chile Faculty of Biological Sciences, Uni6ersity of Concepcio´n, Casilla 160 -C, Concepcio´n, Chile c Institute of Chemistry, Catholic Uni6ersity of Valparaiso, Valparaiso, Chile
Received 7 May 1999; received in revised form 15 September 1999; accepted 27 September 1999
Abstract This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse and on membrane models. The latter consisted of large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE). Results showed that benomyl at concentrations as low as 10 mM decreased the stimulatory response of the potential difference (PD) and the short-circuit current (SCC) of the frog sympathetic junction. It is concluded that benomyl caused a dose-dependent reduction in the response of a sympathetic junction of the frog to stimulation leading to Cl− channel perturbation. This finding might be explained from those obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, similar (0.01–1.0 mM) concentrations induced structural perturbations in DMPC large unilamellar vesicles and multilayers, respectively. Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by the phospholipid bilayers in the interaction of the pesticide with cell membranes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Fungicide; Benomyl; Cell membrane; Neuroepithelial synapse; Phospholipid bilayer
1. Introduction The widespread use of pesticides in an attempt to increase crop production has generated a series of toxicological and environmental problems, in particular the appearance of resistant species of pests, toxic effects in man and animals, and perAbbre6iations: DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidiylethanolamine; DPH, 1,6diphenyl-1,3,5-hexatriene; GP, general polarization; laurdan, 6-dodecanoyl-2 dimethylaminonaphthalene; LUV, large unilamellar vesicles; PD, potential difference; r, fluorescence anisotropy. * Corresponding author. Tel.: + 56-41-204171; fax: + 5641-245974. E-mail address: [email protected] (M. Suwalsky)
sistence of pesticides in the environment. The use of organochlorine pesticides has created the greatest concern as they are designed precisely to be persistent and are manufactured inexpensively. Due to their lipophilic character they can be potentially hazardous because of their transport through the food chain and accumulation in the lipid phase of cellular and subcellular membranes. Benomyl is a benzimidazole fungicide widely used on a variety of food crops and ornamental plants (Lim et al., 1997). It is known for its ability to induce structural changes in chromosomes (Zelesco et al., 1990) and hepatocyte microtubule cytoskeleton (Urani et al., 1995), testicular dysfunction (Lim et al., 1997), inhibition of protein synthesis (Marinovich et al., 1996) and neuronal
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cell differentiation (McLean et al., 1998), lipid peroxidation and glutathione depletion (Urani et al., 1995; Banks et al., 1997). However, we were unable to find any reference in the literature concerning the interaction of benomyl with cell membranes. In general, the molecular mechanisms of pesticide toxicity are poorly understood. Nonetheless, the lipophilicity of most compounds makes lipid-rich membranes sensitive target sites for their interactions with living organisms. It has been suggested that some effects related to their toxicity could be mediated by changes in membrane fluidity (Videira et al., 1996). 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 (Lundbaek et al., 1996). 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. This article describes the interaction of benomyl with cell membranes of frog synapse and molecular models. The latter consisted in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), and large unilamellar vesicles (LUV) of DMPC. They represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. These systems have been used to determine the interaction with, and perturbing effects on, membranes by the pesticides DDT (Suwalsky et al., 1985), pentachlorophenol (Suwalsky et al., 1990), 2,4-D (Suwalsky et al., 1996), heptachlor (Suwalsky et al., 1997a), dieldrin (Suwalsky et al., 1997b), chloridazon (Suwalsky et al., 1998a) and lindane (Suwalsky et al., 1998b). A suitable model for ion movements across epithelia is the frog nerve-skin preparation. Electric stimulation of the synaptic junction between noradrenergic nerve endings and skin mucous glands (Quevedo et al., 1988; Norris et al., 1995) is followed by a rise in the potential difference (PD) and in the short-circuit current (SCC) across the skin; this response is probably due to an increase in active chloride transport across the mucous glands (Thompson et al., 1981; Gonza´lez et al., 1989). The bioelectric parameters are kept at a steady basal level by active Na+ transport (see review by Ussing et al. (1994)). The capacity of benomyl to perturb the phospholipids under the forms of multilayers and vesicles was deter-
mined by X-ray diffraction and fluorescence spectroscopy, respectively. Considering the lipophilic nature of benomyl and the amphiphilic character of the phospholipids, the interactions with DMPC and DMPE multilayers were carried out in hydrophobic and aqueous media in a range of concentrations.
2. Materials and methods
2.1. Electrophysiological measurements on frog ner6e-skin preparation The experiments were performed on frogs of the species Caudi6erbera caudi6erbera (200–350 g) which were kept in tap water at room temperature (18–22°C) at least 24 h prior to use. After pithing, the cutaneous branch of the tibial nerve supplying part of the skin of the hindleg was isolated together with the attached piece of skin and mounted in Ussing chambers. An area of 1.33 cm2 was exposed to 3.5 ml of phosphate-buffered (pH 7.5) Ringer’s solution on both surfaces and oxygenated with a stream of air. The composition of the solution was (mM): Na+ 114, K+ 2.5, Cl− 2.0, HCO− 2.3 and glucose 11. The SCC was 3 monitored with non-polarizable Ag/AgCl electrodes placed at 15 mm from the skin and connected to a voltage-clamp circuit (G. Metraux Electronique) set to keep the PD across the skin at 0 mV. The PD was measured with calomel electrodes at intervals of 2 min for 4 s. Both parameters were displayed on a two-channel ColeParmer 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). The effect of the pesticide on the steady basal values of the bioelectric parameters (non-stimulated preparations) was also assessed. Benomyl 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. Noradrenaline hydrochloride from Sigma was used in some experiments (serosal surface). Values throughout the work refer to means9S.E.M. for each neuroepithelial
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synapse and for each non-stimulated skin. Statistical analysis was performed using Student’s paired t-test.
2.2. X-ray diffraction studies of phospholipid multilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma and benomyl (analytical reference grade, MW 290.3) from Shell Research Ltd were used without further purification. About 3 mg of each phospholipid were mixed with the corresponding weight of the pesticide in order to attain DMPC:benomyl and DMPE:benomyl powder mixtures in the molecular 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 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 colimators provided with rotating devices. The same procedure was used with samples of each phospholipid and benomyl. The aqueous specimens were prepared in glass capillaries mixing each phospholipid and the pesticide in the proportions as described above. Each capillary was then filled with about 200 ml of distilled water. These specimens were X-ray diffracted 2 days after preparation in flat-plate 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 Philips PW 1140 X-ray generator was used. The relative reflection intensities on film were measured by peak integration using a Bio-Rad GS-700 densitometer and Molecular Analyst/PC image software; no correction factors were applied. The experiments in water were performed at 17 92°C, which is below the main transition temperatures of both DMPC and DMPE. Higher temperatures would have induced transitions in more fluid phases making it harder to detect the structural changes induced by benomyl.
2.3. Fluorescence measurements of large unilamellar 6esicles (LUV) DMPC LUV suspended in water were prepared by extrusion of frozen and thawed multilamellar liposome suspension through two stacked poly-
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carbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp.) employing nitrogen pressure at 10°C over the lipid transition temperature, to a final concentration of 0.3 mM. DPH and Laurdan were incorporated into LUV by addition of small aliquots of concentrated solutions of the probe in tetrahydrofurane and ethanol (being 12 and 17 mM their final respective concentrations) to LUV suspensions and gently shaken for ca. 30 min. Fluorescence spectra and anisotropy measurements were respectively performed in a Spex Fluorolog (Spex) and in a phase shift and modulation Greg-200 steady-state and time-resolved spectrofluorometer (I.S.S.), both interfaced to computers. Software from I.S.S. were used for data collection and analysis. Measurements of LUV suspensions were made at 18°C employing 10 mm path-length square quartz cuvettes. Sample temperature was controlled by an external bath circulator (Cole-Parmer) and measured prior and after each measurement using a digital thermometer (Omega). Anisotropy measurements were made in the ‘L’ configuration using prism polarizers (Glan Thompson) in both exciting and emitting beams. The emission was measured with the aid of a high pass filter (Schott WC-420) which showed negligible fluorescence. Laurdan fluorescence spectral shifts were quantified through the general polarization (GP) concept which was evaluated by GP= (Ib− Ir)/(Ib + Ir), where Ib and Ir are the intensities at the blue and red edges of the emission spectrum, respectively. These intensities were measured at 440 and 490 nm, corresponding to the emission maxima of Laurdan in the gel and liquid crystalline phases, respectively (Parasassi et al., 1990). Benomyl was incorporated into LUV suspensions by addition of small aliquots of a concentrated ethanol solution and incubated at 40°C for ca. 15 min. Samples with probes but without benomyl showed no variation in the measured parameters during periods longer than those employed in the experiments. Blank subtraction was performed in all measurements using unlabelled samples without probes. Benomyl 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. Ethanol and tetrahydrofurane effects were considered in control experiments measuring both fluorescence parameters as functions of ethanol and tetrahydrofurane additions with no significant effects at the maxima
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Fig. 1. Single experiment illustrating effects of increasing concentrations of benomyl (B) applied in the inner bathing solution, on the response of the neuroepithelial synapse of the frog Caudi6erbera caudi6erbera to electric stimulation. (1) 0.01 mM; (2) 0.1 mM; (3) 1.0 mM. PD, potential difference; SCC, short-circuit current; S, stimulation.
used concentrations respectively.
of
150
and
12
mM,
3. Results
3.1. Electrophysiological measurements on frog ner6e-skin preparation 3.1.1. Response to ner6e stimulation For each preparation, the responses consisted of transient increases in the bioelectric parameters, although they differed slightly in form and amplitude in a few synapses. Therefore the electric changes were expressed as percentages of the control values. Each experiment lasted 8 – 10 h and only one set of readings could be made for each synapse. Tracings of the responses showed that the increase in SCC consisted of two main components. The first component was a rapid rise in current from 33.393.6 to 43.5 9 3.3 mA/cm2 (Fig. 1). The peak was reached in 0.37 9 0.02 min and the duration of the rapid rise was 1.11 90.07 min (n= 12). The second component consisted of a slow rise when the first component was declining: the peak was very 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 26.09 3.0 to 29.093.1 mV (n= 12). 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 12 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.
3.1.2. Effect of benomyl on the responses to ner6e stimulation Increasing concentrations (equivalent to 0.01 up to 1.0 mM) produced a concentration-dependent reduction in the response to stimulation; Table 1 Table 1 Inhibitory effect of increasing concentrations of benomyl (inner solution) on the frog neuroepithelial synapse response to electric stimulationa Benomyl (mM)
% Decrease in the PD response
% Decrease in the SCC response
0.01 0.1 1.0
15.0 9 3.0 24.0 9 2.5* 34.0 9 4.4*
2.5 9 0.8 9.1 9 2.4 24.8 93.0*
a Results are expressed as percent decrease (means 9S.E.M., n = 12) in the skin response of the potential difference (PD) and of the short-circuit current (SCC) over the basal values of the non-stimulated skin. These values were: PD 26.0 9 3.0 mV and SCC 33.3 93.6 mA/cm2. Significantly different from the responses to nerve stimulation in the absence of pesticide (Student’s paired t-test). * PB0.05.
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shows a 25 and 34% decrease in the magnitude of the SCC and PD responses, respectively; this decrease was reached 152.0 913.0 min after initiation of the exposure to benomyl. This effect was usually reversible after removal of the chemical by a threefold washout. Observation of the records showed that the peak of the rapid component after addition of the third concentration of the pesticide was reached in 0.47 91.10 min and the duration of the rapid rise was 1.98 9 0.12 min, values which were significantly different from control (P B0.01 and B0.05, respectively). The magnitude of the second component was not significantly reduced. During the experimental runs a gradual, non significant decline in the basal values of the PD and SCC was observed.
3.1.3. Effect of benomyl on noradrenaline stimulation of the ner6e-skin preparation Noradrenaline (1.0 mM) applied to one skin half increased SCC from 21.0 94.5 to 63.8 9 5.8 mA/cm2; when this agent was added to the other skin half after exposure of the skin to 1.0 mM benomyl, SCC increased from 19.09 3.0 to only 53.5 96 mA/cm2, a 23% reduction (n = 6, P B 0.05). 3.2. X-ray diffraction studies of phospholipid multilayers The molecular interactions of benomyl with DMPC and DMPE multilayers were determined in both hydrophobic and aqueous media. Fig. 2 shows a comparison of the diffracting patterns of DMPC, benomyl and of their 1:1 molar mixture after interacting and recrystallizing from chloroform:methanol 3:1. It can be seen that benomyl induced limited structural perturbation of DMPC, decreasing its reflection intensities; its bilayer width remained practically constant at 55.4 A, . On the other hand, nearly all the benomyl reflections were present in that pattern, although slightly decreased in intensity. Fig. 3 shows that the results were different when these interactions took place in an aqueous medium: water expanded DMPC bilayer width from 55.0 A, when dry to 64.4 A, ; the reflections were reduced to only the first three orders of the bilayer width and a relatively intense one of 4.2 A, . The latter arose from the stiff and fully extended acyl chains organized with rotational disorder in an
Fig. 2. Microdensitograms from X-ray diffraction diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v). Flat-plate cameras; specimen to film distance: (A) 14 cm, (B) 8 cm.
hexagonal lattice. Addition of 0.01 mM benomyl did not significantly alter DMPC structure; however, 1.0 mM benomyl induced considerable perturbation in DMPC since the phospholipid reflection intensities decreased by about 70%. As no reflections from the pesticide were present in this pattern it may be concluded that it was entirely bound to DMPC. Addition of 4 mM benomyl practically destroyed the bilayer organization of DMPC, as none of the lipid reflections were present. On the other hand, the strongest benomyl reflections showed up weakly in this X-ray diagram and they became more intense when its concentration increased to 13.2 mM. The results of the interaction of benomyl with DMPE in the hydrophobic medium are shown in Fig. 4: those obtained in the aqueous medium are presented in Fig. 5. It may be appreciated that the effects of the pesticide upon the structure of DMPE were practically nil in the highest assayed concentrations as the X-ray patterns of DMPE were not significantly affected by benomyl.
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3.3. Fluorescence measurements of large unilamellar 6esicles (LUV) The influence of benomyl upon the bilayer of DMPC LUV was evaluated at the deep phospholipid acyl chain hydrophobic core and at the hydrophilic/hydrophobic interface, i.e. the phospholipid polar head level. This was achieved evaluating DPH steady state fluorescence anisotropy (r) and Laurdan fluorescence spectral shifts, respectively. The latter was quantified through the general polarization parameters (Parasassi et al., 1995). As shown in Table 2 0.01 mM benomyl moderately affected these parameters; however, higher concentrations produced a marked decrease in DPH fluorescence anisotropy and Laurdan GP. These results imply a disordering effect induced by the pesticide on the acyl chain packing and an enhancement of molecular dynamics or water penetration at the phospholipid polar head level, respectively. Fig. 4. Microdensitograms from X-ray diffraction diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v), Flat-plate cameras; specimen to film distance: (A) 14 cm, (B) 8 cm.
4. Discussion
Fig. 3. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPC and benomyl. Flat-plate cameras; specimen-to-film distance: (A) 14 cm, (B) 8 cm. Benomyl:DMPC molecular ratios: (1) 2.5 × 10 − 4, (2) 0.025, (3) 0.10, (4) 0.30.
The toxicity of pesticides is a matter of great concern in view of their widespread use in pest control. Despite this fact, the molecular mechanisms that underlie their toxic effects are still not understood. This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse, and on membrane models constituted by phospholipid multilayers and large unilamellar vesicles. Results show that benomyl, even at a concentration as low as 10 mM, decreased the stimulatory response of the PD and the SCC of the frog sympathetic junction. This finding tends to agree with those obtained from the studies on membrane models. In fact, experiments carried out by fluorescence spectroscopy on DMPC LUV showed that 10 mM benomyl induced a reduction in the anisotropy (r) of DPH and the general polarization (GP) of laurdan, implying a disordering effect at the acyl chain and polar group level, respectively. On the other hand, X-ray analysis of
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Fig. 5. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPE and benomyl. Flat-plate cameras; specimen-to-film distance, (A) 14 cm, (B) 8 cm. Benomyl:DMPE molecular ratio: (4) 0.30.
DMPC and DMPE multilayers showed that benomyl preferentially interacted with the former. This can be explained by differences in their packing arrangements. Chemically DMPC and DMPE 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 DMPE smaller polar group and higher effective charge, makes for a Table 2 Effect of benomyl on the anisotropy (r) of DPH and the general polarization (GP) of laurdan embedded in large unilamellar DMPC vesiclesa Benomyl (mM)
Benomyl/ DMPC ratio
(r) DPH
(GP) laurdan
0.00 0.01 0.10
0.00 0.03 0.33
0.272 0.258 0.126
0.511 0.480 0.270
a
Probe:lipid ratio 1:600.
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very stable multilayer arrangement which is not significantly perturbed by water (Suwalsky et al., 1988). 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 55.4 to 64.4 A, when fully hydrated at 17°C, a temperature below that of its main transition. These conditions promote the incorporation of benomyl into DMPC bilayers, its penetration into the acyl chain region and the ensuing perturbation of the phospholipid molecular arrangement. In fact, 1 mM benomyl considerably affected its bilayer structure and 4 mM benomyl completely destroyed the molecular organization of DMPC.These toxic concentrations of benomyl are of the same order of magnitude of those reported in the literature for other biological systems (Marinovich et al., 1996; Lim et al., 1997). Were these experiments carried out at higher temperatures, e.g. 37°C, it would have been hard to detect the resulting structural perturbation by X-ray diffraction. Under these conditions, high temperature and water excess, DMPC presents the highly fluid La phase, i.e. a one dimensional lamella with acyl chains in a liquid-like conformation, characterized by a very broad and difuse X-ray diffraction band centered at about 4.6 A, . However, the results obtained by fluorescence spectroscopy on the highly fluid DMPC LUV confirmed those attained by X-ray diffraction. The structural information gathered from these two methods was useful to explain the effects caused by benomyl on the neuroepithelial membrane of the frog skin. Although it has been demonstrated that benomyl is an anticholinesterase agent acting at the neuromuscular synapse (Baron et al., 1991), its effect on the membrane properties could also be due to one or more of four mechanisms: (a) entirely lipid with no protein interaction involved, i.e. a loss of bilayer integrity and a decreased resistance across the bilayer; (b) a lipid-perturbing effect, changing the bulk properties of the bilayers in a way that alters the lipid-protein interaction such as to favour certain protein conformational states; (c) specific interaction with a part of a receptor facing the surrounding bilayer and (d) specific interaction with a part of a receptor facing the cytoplasm. The first mechanism is not involved in the synaptic membrane since addition of benomyl did not change the resistance significantly. On the
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other hand, the finding that benomyl strongly interacted with DMPC, representative of a phospholipid class located in the outer leaflet of cells membranes, and did not interact with DMPE, which represents another class of phospholipids located in the cytoplasmic moiety of membranes, tends to disregard the third and particularly the fourth mechanism. Thus, the second mechanism may be the most relevant, that is, an effect on the lipid-protein interface. Therefore, the synaptic mechanisms must be considered. Noradrenergic transmission is dependent on Ca2 + entry through the presynaptic nerve terminal and on active ion (principally Cl−) transport; the pesticide-induced decrease of the skin response to noradrenaline might be due to alteration of adrenergic receptors and interference with ion transport across the neuroepithelial membrane. The reduced synaptic response to stimulation also agrees with interference of benomyl with Cl− secretion in the mucous glands. Unpublished data from our laboratories show that isethionate Ringer’s solution blocks the response to electrical stimulation. Gonza´lez et al. (1989) showed that Cl− transport could be largely accounted for by cAMP-dependent processes and that it is contingent on the Na+ -K2-2Cl co-transport system in the basolateral membrane. This process is driven by Na+/K+ ATPase (Butt et al., 1994) and has been clarified for the frog gland by Ussing et al. (1996) and Videira et al. (1996). Begenisich et al. (1998) found that epithelial cells harbour a wide variety of Cl− channels with an ample spectrum of biophysical and regulation characteristics, some of which have not yet been identified, and it may be suggested that benomyl leads to such putative channel dysfunction. Among important pathways are those regulated by G proteins and cAMP. If the pesticide induces changes in the lipid-protein interface, any ensuing integral membrane protein conformational change will alter receptor and channel functions, in accordance with the second mechanism. Amiloride treatment did not change the rapid component of the response (Norris et al., 1993); in contrast, the slow component often disappeared. This effect, which points to decreased Na+ transport, was also observed in the presence of 2,4-D (Suwalsky et al., 1999), but not after addition of benomyl. This finding suggests that the pesticide had only a negligible effect on Na+ transport. It is concluded that benomyl caused a dose-dependent reduction
in the response of a sympathetic junction of the frog to stimulation leading to Cl channel perturbation. One way to achieve it might be through the ‘membrane bilayer pathway’ (Mason, 1993) by which benomyl would first partition into the lipid bilayer which would assist it by optimizing its location, orientation, and concentration with respect to the protein-binding site. Alternatively, the protein functions may be affected by the perturbation induced by benomyl in the lipid bilayer surrounding the proteins (Lundbaek et al., 1996). Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by phospholipid bilayers in the pesticide interaction with cell membranes.
Acknowledgements This work was supported by grants from FONDECYT (1990289), Andes Foundation (C12302), DIUC (98.24.19-1) and DGIUCV.
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model membranes. Biochim. Biophys. Acta. 1285, 267 – 276. Suwalsky, M., Benites, M., Villena, F., Aguilar, F., Sotomayor, C.P., 1997a. The organochlorine pesticide heptahclor disrupts the structure of model and cell membranes. Biochim. Biophys. Acta 1326, 115 – 123. Suwalsky, M., Benites, M., Villena, F., Aguilar, F., Sotomayor, C.P., 1997b. Interaction of the organochlorine pesticide dieldrin with phospholipid bilayers. Z. Naturforsch. 52c, 450 – 458. Suwalsky, M., Benites, M., Villena, F., Norris, B., Quevedo, L., 1998a. The organochlorine herbicide chloridazon interacts with cell membranes. Comp. Biochem. Physiol. 120C, 29 – 35. Suwalsky, M., Rodr’geuz, C., Villena, F., Aguilar, F., Sotomayor, C.P., 1998b. The organochlorine pesticide lindane interacts with the human erythrocyte membrane. Pest. Biochem. Physiol. 62, 82 – 95. Suwalsky, M., Quevedo, L., Norris, B., Benites, M., 1999. Toxic action of the herbicide 2,4-D on the neuroepithelial synapse and on the non-stimulated skin of the frog Caudiverbera caudiverbera. Bull. Environ. Contam. Toxicol. 1999 (in press). Thompson, I.G., Mills, J.W., 1981. Isoproterenol-induced current changes in glands of frog skin. Am. J. Physiol. 241, C250 – C257. Urani, C., Chiesara, E., Galvani, P., Marabini, L., Santagostino, A., Camatini, M., 1995. Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes. Toxicol. Lett. 76, 135 – 144. Ussing, H.H., 1994. Does active transport exist? J. Membr. Biol. 137, 91 – 98. Ussing, H.H., Lind, F., Larsen, E.H., 1996. Ion secretion and isotonic transport in frog skin glands. J. Membrane Biol. 152, 101 – 110. Videira, R.A., Antunes-Madeira, R.C., Madeira, V.C.M., 1996. Interaction of ethylazinphos with the physical organization of model and native membranes. Biochim. Biophys. Acta 1281, 65 – 72. Zelesco, P.A., Barbieri, I., Marshall Graves, J.A., 1990. Use of a cell hybrid test system to demonstrate that benomyl induces aneuploidy and polyploidy. Mutat.Res. 242, 329 – 335.
www.elsevier.com/locate/cbpc
Toxic effects of the fungicide benomyl on cell membranes Mario Suwalsky a,*, Maritza Benites a, Beryl Norris b, Patricio Sotomayor c a b
Faculty of Chemical Sciences, Uni6ersity of Concepcio´n, Casilla 160 -C, Concepcio´n, Chile Faculty of Biological Sciences, Uni6ersity of Concepcio´n, Casilla 160 -C, Concepcio´n, Chile c Institute of Chemistry, Catholic Uni6ersity of Valparaiso, Valparaiso, Chile
Received 7 May 1999; received in revised form 15 September 1999; accepted 27 September 1999
Abstract This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse and on membrane models. The latter consisted of large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE). Results showed that benomyl at concentrations as low as 10 mM decreased the stimulatory response of the potential difference (PD) and the short-circuit current (SCC) of the frog sympathetic junction. It is concluded that benomyl caused a dose-dependent reduction in the response of a sympathetic junction of the frog to stimulation leading to Cl− channel perturbation. This finding might be explained from those obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, similar (0.01–1.0 mM) concentrations induced structural perturbations in DMPC large unilamellar vesicles and multilayers, respectively. Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by the phospholipid bilayers in the interaction of the pesticide with cell membranes. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Fungicide; Benomyl; Cell membrane; Neuroepithelial synapse; Phospholipid bilayer
1. Introduction The widespread use of pesticides in an attempt to increase crop production has generated a series of toxicological and environmental problems, in particular the appearance of resistant species of pests, toxic effects in man and animals, and perAbbre6iations: DMPC, dimyristoylphosphatidylcholine; DMPE, dimyristoylphosphatidiylethanolamine; DPH, 1,6diphenyl-1,3,5-hexatriene; GP, general polarization; laurdan, 6-dodecanoyl-2 dimethylaminonaphthalene; LUV, large unilamellar vesicles; PD, potential difference; r, fluorescence anisotropy. * Corresponding author. Tel.: + 56-41-204171; fax: + 5641-245974. E-mail address: [email protected] (M. Suwalsky)
sistence of pesticides in the environment. The use of organochlorine pesticides has created the greatest concern as they are designed precisely to be persistent and are manufactured inexpensively. Due to their lipophilic character they can be potentially hazardous because of their transport through the food chain and accumulation in the lipid phase of cellular and subcellular membranes. Benomyl is a benzimidazole fungicide widely used on a variety of food crops and ornamental plants (Lim et al., 1997). It is known for its ability to induce structural changes in chromosomes (Zelesco et al., 1990) and hepatocyte microtubule cytoskeleton (Urani et al., 1995), testicular dysfunction (Lim et al., 1997), inhibition of protein synthesis (Marinovich et al., 1996) and neuronal
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 0 9 7 - 3
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cell differentiation (McLean et al., 1998), lipid peroxidation and glutathione depletion (Urani et al., 1995; Banks et al., 1997). However, we were unable to find any reference in the literature concerning the interaction of benomyl with cell membranes. In general, the molecular mechanisms of pesticide toxicity are poorly understood. Nonetheless, the lipophilicity of most compounds makes lipid-rich membranes sensitive target sites for their interactions with living organisms. It has been suggested that some effects related to their toxicity could be mediated by changes in membrane fluidity (Videira et al., 1996). 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 (Lundbaek et al., 1996). 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. This article describes the interaction of benomyl with cell membranes of frog synapse and molecular models. The latter consisted in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), and large unilamellar vesicles (LUV) of DMPC. They represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. These systems have been used to determine the interaction with, and perturbing effects on, membranes by the pesticides DDT (Suwalsky et al., 1985), pentachlorophenol (Suwalsky et al., 1990), 2,4-D (Suwalsky et al., 1996), heptachlor (Suwalsky et al., 1997a), dieldrin (Suwalsky et al., 1997b), chloridazon (Suwalsky et al., 1998a) and lindane (Suwalsky et al., 1998b). A suitable model for ion movements across epithelia is the frog nerve-skin preparation. Electric stimulation of the synaptic junction between noradrenergic nerve endings and skin mucous glands (Quevedo et al., 1988; Norris et al., 1995) is followed by a rise in the potential difference (PD) and in the short-circuit current (SCC) across the skin; this response is probably due to an increase in active chloride transport across the mucous glands (Thompson et al., 1981; Gonza´lez et al., 1989). The bioelectric parameters are kept at a steady basal level by active Na+ transport (see review by Ussing et al. (1994)). The capacity of benomyl to perturb the phospholipids under the forms of multilayers and vesicles was deter-
mined by X-ray diffraction and fluorescence spectroscopy, respectively. Considering the lipophilic nature of benomyl and the amphiphilic character of the phospholipids, the interactions with DMPC and DMPE multilayers were carried out in hydrophobic and aqueous media in a range of concentrations.
2. Materials and methods
2.1. Electrophysiological measurements on frog ner6e-skin preparation The experiments were performed on frogs of the species Caudi6erbera caudi6erbera (200–350 g) which were kept in tap water at room temperature (18–22°C) at least 24 h prior to use. After pithing, the cutaneous branch of the tibial nerve supplying part of the skin of the hindleg was isolated together with the attached piece of skin and mounted in Ussing chambers. An area of 1.33 cm2 was exposed to 3.5 ml of phosphate-buffered (pH 7.5) Ringer’s solution on both surfaces and oxygenated with a stream of air. The composition of the solution was (mM): Na+ 114, K+ 2.5, Cl− 2.0, HCO− 2.3 and glucose 11. The SCC was 3 monitored with non-polarizable Ag/AgCl electrodes placed at 15 mm from the skin and connected to a voltage-clamp circuit (G. Metraux Electronique) set to keep the PD across the skin at 0 mV. The PD was measured with calomel electrodes at intervals of 2 min for 4 s. Both parameters were displayed on a two-channel ColeParmer 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). The effect of the pesticide on the steady basal values of the bioelectric parameters (non-stimulated preparations) was also assessed. Benomyl 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. Noradrenaline hydrochloride from Sigma was used in some experiments (serosal surface). Values throughout the work refer to means9S.E.M. for each neuroepithelial
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synapse and for each non-stimulated skin. Statistical analysis was performed using Student’s paired t-test.
2.2. X-ray diffraction studies of phospholipid multilayers Synthetic DMPC (lot 80H8371, A grade, MW 677.9) and DMPE (lot 13H83681, A grade, MW 635.9) from Sigma and benomyl (analytical reference grade, MW 290.3) from Shell Research Ltd were used without further purification. About 3 mg of each phospholipid were mixed with the corresponding weight of the pesticide in order to attain DMPC:benomyl and DMPE:benomyl powder mixtures in the molecular 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 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 colimators provided with rotating devices. The same procedure was used with samples of each phospholipid and benomyl. The aqueous specimens were prepared in glass capillaries mixing each phospholipid and the pesticide in the proportions as described above. Each capillary was then filled with about 200 ml of distilled water. These specimens were X-ray diffracted 2 days after preparation in flat-plate 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 Philips PW 1140 X-ray generator was used. The relative reflection intensities on film were measured by peak integration using a Bio-Rad GS-700 densitometer and Molecular Analyst/PC image software; no correction factors were applied. The experiments in water were performed at 17 92°C, which is below the main transition temperatures of both DMPC and DMPE. Higher temperatures would have induced transitions in more fluid phases making it harder to detect the structural changes induced by benomyl.
2.3. Fluorescence measurements of large unilamellar 6esicles (LUV) DMPC LUV suspended in water were prepared by extrusion of frozen and thawed multilamellar liposome suspension through two stacked poly-
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carbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp.) employing nitrogen pressure at 10°C over the lipid transition temperature, to a final concentration of 0.3 mM. DPH and Laurdan were incorporated into LUV by addition of small aliquots of concentrated solutions of the probe in tetrahydrofurane and ethanol (being 12 and 17 mM their final respective concentrations) to LUV suspensions and gently shaken for ca. 30 min. Fluorescence spectra and anisotropy measurements were respectively performed in a Spex Fluorolog (Spex) and in a phase shift and modulation Greg-200 steady-state and time-resolved spectrofluorometer (I.S.S.), both interfaced to computers. Software from I.S.S. were used for data collection and analysis. Measurements of LUV suspensions were made at 18°C employing 10 mm path-length square quartz cuvettes. Sample temperature was controlled by an external bath circulator (Cole-Parmer) and measured prior and after each measurement using a digital thermometer (Omega). Anisotropy measurements were made in the ‘L’ configuration using prism polarizers (Glan Thompson) in both exciting and emitting beams. The emission was measured with the aid of a high pass filter (Schott WC-420) which showed negligible fluorescence. Laurdan fluorescence spectral shifts were quantified through the general polarization (GP) concept which was evaluated by GP= (Ib− Ir)/(Ib + Ir), where Ib and Ir are the intensities at the blue and red edges of the emission spectrum, respectively. These intensities were measured at 440 and 490 nm, corresponding to the emission maxima of Laurdan in the gel and liquid crystalline phases, respectively (Parasassi et al., 1990). Benomyl was incorporated into LUV suspensions by addition of small aliquots of a concentrated ethanol solution and incubated at 40°C for ca. 15 min. Samples with probes but without benomyl showed no variation in the measured parameters during periods longer than those employed in the experiments. Blank subtraction was performed in all measurements using unlabelled samples without probes. Benomyl 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. Ethanol and tetrahydrofurane effects were considered in control experiments measuring both fluorescence parameters as functions of ethanol and tetrahydrofurane additions with no significant effects at the maxima
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Fig. 1. Single experiment illustrating effects of increasing concentrations of benomyl (B) applied in the inner bathing solution, on the response of the neuroepithelial synapse of the frog Caudi6erbera caudi6erbera to electric stimulation. (1) 0.01 mM; (2) 0.1 mM; (3) 1.0 mM. PD, potential difference; SCC, short-circuit current; S, stimulation.
used concentrations respectively.
of
150
and
12
mM,
3. Results
3.1. Electrophysiological measurements on frog ner6e-skin preparation 3.1.1. Response to ner6e stimulation For each preparation, the responses consisted of transient increases in the bioelectric parameters, although they differed slightly in form and amplitude in a few synapses. Therefore the electric changes were expressed as percentages of the control values. Each experiment lasted 8 – 10 h and only one set of readings could be made for each synapse. Tracings of the responses showed that the increase in SCC consisted of two main components. The first component was a rapid rise in current from 33.393.6 to 43.5 9 3.3 mA/cm2 (Fig. 1). The peak was reached in 0.37 9 0.02 min and the duration of the rapid rise was 1.11 90.07 min (n= 12). The second component consisted of a slow rise when the first component was declining: the peak was very 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 26.09 3.0 to 29.093.1 mV (n= 12). 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 12 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.
3.1.2. Effect of benomyl on the responses to ner6e stimulation Increasing concentrations (equivalent to 0.01 up to 1.0 mM) produced a concentration-dependent reduction in the response to stimulation; Table 1 Table 1 Inhibitory effect of increasing concentrations of benomyl (inner solution) on the frog neuroepithelial synapse response to electric stimulationa Benomyl (mM)
% Decrease in the PD response
% Decrease in the SCC response
0.01 0.1 1.0
15.0 9 3.0 24.0 9 2.5* 34.0 9 4.4*
2.5 9 0.8 9.1 9 2.4 24.8 93.0*
a Results are expressed as percent decrease (means 9S.E.M., n = 12) in the skin response of the potential difference (PD) and of the short-circuit current (SCC) over the basal values of the non-stimulated skin. These values were: PD 26.0 9 3.0 mV and SCC 33.3 93.6 mA/cm2. Significantly different from the responses to nerve stimulation in the absence of pesticide (Student’s paired t-test). * PB0.05.
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shows a 25 and 34% decrease in the magnitude of the SCC and PD responses, respectively; this decrease was reached 152.0 913.0 min after initiation of the exposure to benomyl. This effect was usually reversible after removal of the chemical by a threefold washout. Observation of the records showed that the peak of the rapid component after addition of the third concentration of the pesticide was reached in 0.47 91.10 min and the duration of the rapid rise was 1.98 9 0.12 min, values which were significantly different from control (P B0.01 and B0.05, respectively). The magnitude of the second component was not significantly reduced. During the experimental runs a gradual, non significant decline in the basal values of the PD and SCC was observed.
3.1.3. Effect of benomyl on noradrenaline stimulation of the ner6e-skin preparation Noradrenaline (1.0 mM) applied to one skin half increased SCC from 21.0 94.5 to 63.8 9 5.8 mA/cm2; when this agent was added to the other skin half after exposure of the skin to 1.0 mM benomyl, SCC increased from 19.09 3.0 to only 53.5 96 mA/cm2, a 23% reduction (n = 6, P B 0.05). 3.2. X-ray diffraction studies of phospholipid multilayers The molecular interactions of benomyl with DMPC and DMPE multilayers were determined in both hydrophobic and aqueous media. Fig. 2 shows a comparison of the diffracting patterns of DMPC, benomyl and of their 1:1 molar mixture after interacting and recrystallizing from chloroform:methanol 3:1. It can be seen that benomyl induced limited structural perturbation of DMPC, decreasing its reflection intensities; its bilayer width remained practically constant at 55.4 A, . On the other hand, nearly all the benomyl reflections were present in that pattern, although slightly decreased in intensity. Fig. 3 shows that the results were different when these interactions took place in an aqueous medium: water expanded DMPC bilayer width from 55.0 A, when dry to 64.4 A, ; the reflections were reduced to only the first three orders of the bilayer width and a relatively intense one of 4.2 A, . The latter arose from the stiff and fully extended acyl chains organized with rotational disorder in an
Fig. 2. Microdensitograms from X-ray diffraction diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v). Flat-plate cameras; specimen to film distance: (A) 14 cm, (B) 8 cm.
hexagonal lattice. Addition of 0.01 mM benomyl did not significantly alter DMPC structure; however, 1.0 mM benomyl induced considerable perturbation in DMPC since the phospholipid reflection intensities decreased by about 70%. As no reflections from the pesticide were present in this pattern it may be concluded that it was entirely bound to DMPC. Addition of 4 mM benomyl practically destroyed the bilayer organization of DMPC, as none of the lipid reflections were present. On the other hand, the strongest benomyl reflections showed up weakly in this X-ray diagram and they became more intense when its concentration increased to 13.2 mM. The results of the interaction of benomyl with DMPE in the hydrophobic medium are shown in Fig. 4: those obtained in the aqueous medium are presented in Fig. 5. It may be appreciated that the effects of the pesticide upon the structure of DMPE were practically nil in the highest assayed concentrations as the X-ray patterns of DMPE were not significantly affected by benomyl.
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3.3. Fluorescence measurements of large unilamellar 6esicles (LUV) The influence of benomyl upon the bilayer of DMPC LUV was evaluated at the deep phospholipid acyl chain hydrophobic core and at the hydrophilic/hydrophobic interface, i.e. the phospholipid polar head level. This was achieved evaluating DPH steady state fluorescence anisotropy (r) and Laurdan fluorescence spectral shifts, respectively. The latter was quantified through the general polarization parameters (Parasassi et al., 1995). As shown in Table 2 0.01 mM benomyl moderately affected these parameters; however, higher concentrations produced a marked decrease in DPH fluorescence anisotropy and Laurdan GP. These results imply a disordering effect induced by the pesticide on the acyl chain packing and an enhancement of molecular dynamics or water penetration at the phospholipid polar head level, respectively. Fig. 4. Microdensitograms from X-ray diffraction diagrams of samples recrystallized from chloroform:methanol 3:1 (v/v), Flat-plate cameras; specimen to film distance: (A) 14 cm, (B) 8 cm.
4. Discussion
Fig. 3. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPC and benomyl. Flat-plate cameras; specimen-to-film distance: (A) 14 cm, (B) 8 cm. Benomyl:DMPC molecular ratios: (1) 2.5 × 10 − 4, (2) 0.025, (3) 0.10, (4) 0.30.
The toxicity of pesticides is a matter of great concern in view of their widespread use in pest control. Despite this fact, the molecular mechanisms that underlie their toxic effects are still not understood. This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse, and on membrane models constituted by phospholipid multilayers and large unilamellar vesicles. Results show that benomyl, even at a concentration as low as 10 mM, decreased the stimulatory response of the PD and the SCC of the frog sympathetic junction. This finding tends to agree with those obtained from the studies on membrane models. In fact, experiments carried out by fluorescence spectroscopy on DMPC LUV showed that 10 mM benomyl induced a reduction in the anisotropy (r) of DPH and the general polarization (GP) of laurdan, implying a disordering effect at the acyl chain and polar group level, respectively. On the other hand, X-ray analysis of
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Fig. 5. Microdensitograms from X-ray diffraction diagrams of aqueous suspensions of DMPE and benomyl. Flat-plate cameras; specimen-to-film distance, (A) 14 cm, (B) 8 cm. Benomyl:DMPE molecular ratio: (4) 0.30.
DMPC and DMPE multilayers showed that benomyl preferentially interacted with the former. This can be explained by differences in their packing arrangements. Chemically DMPC and DMPE 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 DMPE smaller polar group and higher effective charge, makes for a Table 2 Effect of benomyl on the anisotropy (r) of DPH and the general polarization (GP) of laurdan embedded in large unilamellar DMPC vesiclesa Benomyl (mM)
Benomyl/ DMPC ratio
(r) DPH
(GP) laurdan
0.00 0.01 0.10
0.00 0.03 0.33
0.272 0.258 0.126
0.511 0.480 0.270
a
Probe:lipid ratio 1:600.
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very stable multilayer arrangement which is not significantly perturbed by water (Suwalsky et al., 1988). 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 55.4 to 64.4 A, when fully hydrated at 17°C, a temperature below that of its main transition. These conditions promote the incorporation of benomyl into DMPC bilayers, its penetration into the acyl chain region and the ensuing perturbation of the phospholipid molecular arrangement. In fact, 1 mM benomyl considerably affected its bilayer structure and 4 mM benomyl completely destroyed the molecular organization of DMPC.These toxic concentrations of benomyl are of the same order of magnitude of those reported in the literature for other biological systems (Marinovich et al., 1996; Lim et al., 1997). Were these experiments carried out at higher temperatures, e.g. 37°C, it would have been hard to detect the resulting structural perturbation by X-ray diffraction. Under these conditions, high temperature and water excess, DMPC presents the highly fluid La phase, i.e. a one dimensional lamella with acyl chains in a liquid-like conformation, characterized by a very broad and difuse X-ray diffraction band centered at about 4.6 A, . However, the results obtained by fluorescence spectroscopy on the highly fluid DMPC LUV confirmed those attained by X-ray diffraction. The structural information gathered from these two methods was useful to explain the effects caused by benomyl on the neuroepithelial membrane of the frog skin. Although it has been demonstrated that benomyl is an anticholinesterase agent acting at the neuromuscular synapse (Baron et al., 1991), its effect on the membrane properties could also be due to one or more of four mechanisms: (a) entirely lipid with no protein interaction involved, i.e. a loss of bilayer integrity and a decreased resistance across the bilayer; (b) a lipid-perturbing effect, changing the bulk properties of the bilayers in a way that alters the lipid-protein interaction such as to favour certain protein conformational states; (c) specific interaction with a part of a receptor facing the surrounding bilayer and (d) specific interaction with a part of a receptor facing the cytoplasm. The first mechanism is not involved in the synaptic membrane since addition of benomyl did not change the resistance significantly. On the
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other hand, the finding that benomyl strongly interacted with DMPC, representative of a phospholipid class located in the outer leaflet of cells membranes, and did not interact with DMPE, which represents another class of phospholipids located in the cytoplasmic moiety of membranes, tends to disregard the third and particularly the fourth mechanism. Thus, the second mechanism may be the most relevant, that is, an effect on the lipid-protein interface. Therefore, the synaptic mechanisms must be considered. Noradrenergic transmission is dependent on Ca2 + entry through the presynaptic nerve terminal and on active ion (principally Cl−) transport; the pesticide-induced decrease of the skin response to noradrenaline might be due to alteration of adrenergic receptors and interference with ion transport across the neuroepithelial membrane. The reduced synaptic response to stimulation also agrees with interference of benomyl with Cl− secretion in the mucous glands. Unpublished data from our laboratories show that isethionate Ringer’s solution blocks the response to electrical stimulation. Gonza´lez et al. (1989) showed that Cl− transport could be largely accounted for by cAMP-dependent processes and that it is contingent on the Na+ -K2-2Cl co-transport system in the basolateral membrane. This process is driven by Na+/K+ ATPase (Butt et al., 1994) and has been clarified for the frog gland by Ussing et al. (1996) and Videira et al. (1996). Begenisich et al. (1998) found that epithelial cells harbour a wide variety of Cl− channels with an ample spectrum of biophysical and regulation characteristics, some of which have not yet been identified, and it may be suggested that benomyl leads to such putative channel dysfunction. Among important pathways are those regulated by G proteins and cAMP. If the pesticide induces changes in the lipid-protein interface, any ensuing integral membrane protein conformational change will alter receptor and channel functions, in accordance with the second mechanism. Amiloride treatment did not change the rapid component of the response (Norris et al., 1993); in contrast, the slow component often disappeared. This effect, which points to decreased Na+ transport, was also observed in the presence of 2,4-D (Suwalsky et al., 1999), but not after addition of benomyl. This finding suggests that the pesticide had only a negligible effect on Na+ transport. It is concluded that benomyl caused a dose-dependent reduction
in the response of a sympathetic junction of the frog to stimulation leading to Cl channel perturbation. One way to achieve it might be through the ‘membrane bilayer pathway’ (Mason, 1993) by which benomyl would first partition into the lipid bilayer which would assist it by optimizing its location, orientation, and concentration with respect to the protein-binding site. Alternatively, the protein functions may be affected by the perturbation induced by benomyl in the lipid bilayer surrounding the proteins (Lundbaek et al., 1996). Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by phospholipid bilayers in the pesticide interaction with cell membranes.
Acknowledgements This work was supported by grants from FONDECYT (1990289), Andes Foundation (C12302), DIUC (98.24.19-1) and DGIUCV.
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