Effects of the pyrethroid insecticide permethrin on membrane fluidity

ELSEVIER

Chemistry and Physics of Lipids 79 (1996) 21 28

Chemistry and Physic: of LIPIDS

Effects of the pyrethroid insecticide permethrin on membrane fluidity Maria Rosa Moya-Quiles, Encarnaci6n Mufioz-Delgado, Cecilio J. Vidal Departamento de Bioquimica y Biologla Molecular A, Facultad de Biologia, Edificio de Veterinaria, Campus de Espinardo, Universidad de Murcia, P.O.B 4021, 30071 Murcia, Spain

Received 3 July 1995; accepted 20 September 1995

Abstract

The interaction of permethrin with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoyl- (DSPC) bilayers has been investigated by differential scanning calorimetry (DSC) and DPH and TMA-DPH fluorescence anisotropy. In experiments performed by DSC, we show that the addition of permethrin to liposomes, in a 5:1 phospholipid/ pyrethroid ratio, decreases the phase transition temperature (Tm) of DMPC, DPPC and DSPC by 3.2, 2.3 and 1.1°C, respectively. Furthermore, DSC profiles reveal that permethrin decreases the cooperativity for the phase transition of DMPC, DPPC and DSPC membranes. DPH and TMA-DPH fluorescence anisotropy experiments show that permethrin increases membrane fluidity at temperatures below the Tin. The results are discussed in terms of a preferential localization of permethrin in the hydrophobic core of the membrane, where it diminishes the lipid packing in the gel phase and has no effect in the liquid-crystalline phase. Keywords: Pyrethroid; Permethrin; Liposomes; DSC; Membrane fluidity; DPH fluorescence anisotropy

1. Introduction

Abbreviations: GAF1A, 4-aminobutyric acid; TMA-DPH, 1[4'-(trimethylammoniu m)phenyl]-6-phenyl-1,3,5-hexatriene; DPH, 1,6-diphenyl-l,3,5-hexatriene; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; Tin, midpoint temperature of thermotropic phase transition; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MLV, multilamellar liposomes; DSC, differential scanning calorimetry. * Corresponding author, Tel.: 34 68 307100, ext. 2911; Fax: 34 68 364147; E.mail: [email protected].

The pyrethroids are a group of h y d r o p h o b i c esters with structures based on the natural pyrethrins found in the flowers of the Chrysanthem u m species and are one of the most c o m m e r cially i m p o r t a n t classes o f insecticides [1]. Pyrethroids have been subdivided into two classes based on their structural differences and toxicological and neurophysiological actions [2]. Structurally, type I pyrethroids (allethrin and permethrin) do not contain a cyano substituent, whereas type II pyrethroids (deltamethrin, cyper-

0009-3084/96/$15.00 ~-~ 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-3084(95)02503-B

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M.R. Moya-Quiles et al. / Chemistry and Physics of Lipids 79 (1996) 21 28

methrin and fenvalerate) contain the ~-cyano group. Most of the pyrethroids, i.e. allethrin, deltamethrin and permethrin, contain a cyclopropanecarboxylate residue, which is substituted by a phenyl ring in fenvalerate [3]. It has been firmly established that synthetic pyrethroids act as powerful neurotoxic agents [4]. Several molecular targets have been proposed, including the voltage-dependent sodium channels [4,5], receptor-regulated channels, such as the nicotinic [6,7] and GABA-gated chloride channels [8,9] and Ca 2+, Mg2+-ATPases [10-12]. Pyrethroids are highly hydrophobic compounds and this suggests that their action in biological membranes might be related to association with integral proteins and with phospholipids [13]. Extensive studies on the interaction of insecticides with membranes have been carried out for DDT [14,15], lindane [16,17] and parathion [18,19]. However, pyrethroids have not received similar attention and details of their membrane interactions are not well-known. In this paper, we have analyzed the effects of permethrin (Fig. 1), a pyrethroid type I, on the thermotropic properties and fluidity of lipid vesicles. The application of differential scanning calorimetry (DSC) and fluorescence anisotropy of DPH and TMA-DPH shows that the pyrethroid affects the transition temperature of the phospholipids and modifies the cooperativity for the gel to liquid-crystalline transition and the fluidity of model membranes. 2. Materials

Dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and Hepes were purchased from Sigma. EDTA was from Merck. 1,6-Diphenyl-l,3,5-hexatriene (DPH) and tetrahydrofuran were obtained from Fluka Biochemika. 1-[4'-(trimethylammonium)phenyl]-6phenyl-l,3,5-hexatriene (TMA-DPH) was from Molecular Probes (Eugene, OR, USA). Permethrin was obtained from Dr. Ehrenstorfer (Reference Substances, GmbH, Germany). Other reagents used in this study were of the highest commercially available quality.

3. Methods 3.1. Multilamellar liposomes

Multilamellar liposomes (MLV) were prepared by mixing chloroform solutions of phospholipid and permethrin in ethanol to reach an appropriate phospholipid/insecticide ratio. Then, DPH in tetrahydrofuran or TMA-DPH in tetrahydrofuran/ H20 (l:l) was added to the phospholipid/insecticide mixture to give a 200:1 phospholipid/probe ratio. A low concentration of the probes was selected to overcome possible competition between the pyrethroid and the probes in binding to the liposome [20]. The solvents were evaporated under a stream of nitrogen and then under vacuum for 4 h. The lipid film was hydrated in 0.1 mM EDTA, 100 mM NaC1, 5 mM Hepes, pH 7.4 (Hepes buffer). Hydrated samples were heated in a water bath at 10°C above the phospholipid transition temperature for 2 h, the samples being stirred in a vortex every 5 min. Control liposomes, without pesticide, were prepared with ethanol only, to a final lipid concentration of 5.5 mM. For the DSC experiments, liposomes were prepared as before, but the lipid film was hydrated with water instead of Hepes buffer, the final phospholipid concentration being 22.5 mM. 3.2. D S C experiments

Fifteen/tl of liposomes were encapsulated in an aluminium pan and measured against a reference containing 15/tl of water. Samples were heated at 5°C/min in a Perkin-Elmer DSC-7 as described before [21]. The onset and completion of the phase transition were determined from the intersections of the peak slopes with the baseline of the thermograms [22]. Total phosphorous in the pans was determined as described elsewhere [23].

OI

0 Fig. 1. Structure of permethrin.

M.R. Moya-Qui~ e t a l . / ~ e m ~ t ~ a n d ~ysics ~ L ~

23

~(1996) 2 1 - ~ i

DMPC

i

DSPC

DPPC

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E

N

b C

d e

f 15

20

25

30

30

35

40

45

50

45

50

55

60

Temperature (°C) Fig. 2. Differential scanning calorimetry thermograms of phospholipid bilayers prepared with variable concentrations of permethrin.

Liposomes were made in the absence (a) and in the presence of the insecticideat phospholipid/permethrin ratios of 50:1 (b), 25:1 (c), 10:1 (d), 5:1 (e) and 2:1 (f). The temperature was scanned at 5°C/min.

3.3. Fluorescence anisotropy measurements Fluorescence measurements were performed in a Perkin-Elmer luminescence spectrometer (Model LS-50B), The excitation and emission wavelengths were 360 nm and 430 nm, with slits of 3 and 4 nm, respectively. Aliquots (200/11) of MLV were added to 2.8 ml of Hepes buffer and the temperature was scanned between 10°C below and 10°C above the phospholipid transition temperature, at 0.5°C/min. The lipid suspension was continuously stirred and the temperature monitored during the scan. The degree of fluorescence anisotropy was calculated according to Shinitzky and Barenholz [24] from the equation: r =

Iii - - I = * G Ill + 2 * I ± * G

where I It and I i are the intensities measured with the polarization plane parallel ( II ) and perpendicular (_L) to that of the exciting beam. G is a factor used to correct the instrument's polarization and is given by the ratio of vertically to horizontally

polarized emission components when the excitation light is polarized in the horizontal direction. Permethrin showed no detectable absorbance of light at the excitation and emission wavelengths. 4. Results

4. I. DSC experiments Differential scanning calorimetry profiles for DMPC, DPPC, and DSPC multilamellar liposomes prepared with and without increasing concentrations of permethrin are shown in Fig. 2. The thermotropic gel to liquid-crystalline transition occurs at 22.7 + 0.2, 40.1 + 0.1 and 52.3 _ 0.3°C for DMPC, DPPC and DSPC, respectively, and this agrees with previous data [21,25,26]. The incorporation of permethrin in model membranes produced two effects: a notable decrease in the transition temperature of the phospholipid and a broadening of the phase transition peak. The intensity of the two effects depends on the pyrethroid concentration. At a phospholipid/

M.R. Moya-Quiles et al. / Chemistry and Physics of Lipids 79 (1996) 21-28

24

permethrin ratio of 5:1, the phase transition temperature (Tm) of DMPC, DPPC and DSPC bilayers decreased by 3.2, 2.3 and 1.1°C, respectively. The phase diagrams made with the results obtained in DSC experiments are shown in Fig. 3. In mixtures of DPPC- and DSPC-permethrin, the solid and fluid lines display a similar behavior: (i) a rapid decrease as the pyrethroid concentration

DMPC 25

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4.2. Fluidity of model membranes

I

DPPC

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r 0.0

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0.2

rises to 0.038 and 0.090 permethrin mol fraction in DPPC and DSPC, respectively, and (ii) a limited miscibility of the two components at higher concentrations. In D M P C bilayers, at low permethrin concentrations the solid line decreases more steeply than in DPPC- and DSPC-permethrin mixtures. Furthermore, permethrin is somewhat more soluble in DMPC than in DPPC or DSPC. In contrast, the fluid line shows a rather complex pattern since it falls as the pyrethroid concentration is increased up to 0.038 permethrin mol fraction, increases up to 0.17 tool fraction and remains unchanged thereafter. Furthermore, there was no significant difference between the measured change of enthalpy for the phase transition of pure phospholipids and that of the phospholipids/permethrin mixtures (Fig. 2).

0.3

Mole Fraction of Permethrin Fig. 3. Temperature-compositionphase diagrams for mixtures of DMPC, DPPC and DSPC with permethrin. The diagrams were made according to the thermograms shown in Fig. 2. Symbols correspond to the onset (A) and the completion (Q) of the main transition obtained as indicated under Methods (Section 3). S, solid line; F, fluid line.

The effects of permethrin on the fluidity of DMPC, DPPC and DSPC bilayers were assessed by measuring the changes in fluorescence anisotropy of two probes: DPH, which is preferentially located near the centre of the bilayer, and T M A - D P H , a cationic derivative of DPH that preferentially binds to the polar headgroup region in the membrane [27,28]. Thus, the alteration in fluorescence anisotropy of the probes will reflect the perturbations induced by permethrin in the bilayer structure at different depths across the bilayer thickness. The effects of increasing concentrations of permethrin on the thermotropic phase transition of multilamellar liposomes made with DMPC, DPPC and DSPC, as evaluated by fluorescence anisotropy of incorporated DPH and T M A - D P H , are shown in Figs. 4 and 5, respectively. The phase transition of phospholipids with added permethrin was broader than that of the controls, this effect being measured with the two probes. This indicated that the cooperativity of the transition was affected by the insecticide. Furthermore, permethrin shifted the phase transition midpoint to lower temperatures. The modifications of the thermotropic properties induced by permethrin in lipid vesicles were more pronounced for D M P C > DPPC > DSPC, and this was in accordance with the results obtained by DSC.

M.R. Moya-Quiles et al. / ChemiStry and Physics of Lipids 79 (1996) 21 28

25

i

DMPC

0.2

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pholipids in the fluid phase (Figs. 4 and 5). However, a comparison of the change in DPH and TMA-DPH fluorescence anisotropy produced by permethrin in lipid bilayers in the gel phase showed that DPH fluorescence anisotropy decreased to a greater extent than that of TMAi

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Fig. 4. Fluorescence anisotropy of DPH in phospholipid-permethrin mixtures. Liposomes were prepared without (O) and with permethrin at phospholipid/permethrin ratios of 50:1 (0), 25:1 (V), 10:1 (T), 5:1 (D) and 2:1 (11).

As regards the effects of permethrin on membrane fluidity, the fluorescence anisotropy of DPH and TMA-DPH in DMPC-, DPPC- and DSPC-permethrin mixtures in the gel phase notably decreased with increasing concentrations of the pyrethroid and remained unmodified in phos-

I

I

50

55

Temperature (°C) Fig. 5. Fluorescence anisotropy of TMA-DPH in phospholipid-permethrin bilayers. Liposomes were made in the absence (O) and in the presence of permethrin at phospholipid/permethrin ratios of 50:1 (O), 25:1 (V), 10:1 (V), 5:1 (D) and 2:1

(m).

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M.R. Moya-Quiles et al. / Chemistry and Physics of Lipids 79 (1996) 21-28

DPH (Figs. 4 and 5). Furthermore, the reduction in the TMA-DPH fluorescence anisotropy decreased with the length of the acyl chain of lipids, this being almost negligible in DSPC. The results suggest that although both the hydrophobic core and the surface membrane organization are disturbed by the addition of permethrin, the pyrethroid is preferentially located in the hydrophobic region of the membrane, and this agrees with its extremely low solubility in aqueous solutions. To gain an insight into the effects of permethrin on the fluidity of natural membranes, the insecticide and probes, DPH and TMA-DPH, were added to sarcoplasmic reticulum from rabbit skeletal muscle and to human erythrocyte membranes. Attempts to measure the changes in membrane fluidity were made by DPH and TMA-DPH fluorescence anisotropy. Unfortunately, the addition of the insecticide to the membrane suspensions produced turbidity in the mixture, probably due to the low solubility of permethrin in aqueous solutions. This phenomenon affected the fluorescence anisotropy measurements, preventing us from obtaining the required information. 5. Discussion

Pyrethroids can be subdivided into type I and type II, according to their structural, toxicological and pharmacological differences. Pyrethroids are more hydrophobic than other insecticides [13]. This high hydrophobicity suggests that their site of action might be in the biological membranes. Previous studies in our laboratory have shown that allethrin and deltamethrin modify the thermotropic properties and fluidity of the DMPC, DPPC and DSPC bilayers [21,29] (Moya-Quiles et al., unpublished data). In permethrin/DPPC mixtures, it has been reported that the pyrethroid lowers the phase transition temperature and decreases the DPH fluorescence anisotropy at temperatures below the T m [30].

The calorimetric measurements shown in Fig. 2 indicate that permethrin decreases the transition temperature of the phospholipids employed, the

reduction in temperature being dependent on the length of the lipid acyl chain. The transition broadening of DMPC, DPPC and DSPC induced by permethrin suggests that the insecticide modifies the cooperativity for the gel to liquidcrystalline transition. In accordance with the classification of types of alteration in calorimetric profiles of phospholipids induced by additives proposed by Jain and Wu [31], the change produced by permethrin in thermograms corresponds to an A~-type change. This is the expected modification in thermograms when the additive is preferentially located at the C1-C8 methylene region of the bilayer [31]. The partial phase diagrams for DMPC-, DPPC- and DSPC-permethrin mixtures indicate that the pyrethroid shows little miscibility in phospholipids, the solubility being higher in DMPC than in DPPC or DSPC. As regards the effects of pyrethroids on membrane fluidity, it seems likely that the dynamic properties of model membranes are affected by these plaguicides depending on their chemical structure. Previous studies have shown that allethrin, a type I pyrethroid, constituted by a cyclopentenolone alcohol residue esterified with a cyclopropane carboxylate moiety, incorporated into DMPC, DPPC and DSPC liposomes modifies the bilayer order in the temperature range of the cooperative phase transition. However, in DPPC- or DSPC-allethrin systems, the lipid order was not altered at temperatures below and above the phospholipid Tm [29]. On the other hand, deltamethrin, a type II pyrethroid with a phenoxybenzyl alcohol residue, decreased the packing order of DMPC, DPPC and DSPC bilayers in the gel state, whereas it increased the phospholipid fluidity in the liquid-crystalline state (Moya-Quiles et al., unpublished data). As regards the actions of permethrin on membrane fluidity, the results concerning DPH and TMA-DPH fluorescence anisotropy indicate that the insecticide perturbs the packing order of both the hydrophobic core and the surface membrane, particularly in the gel phase. These results partially agree with those reported by Stelzer and Gordon [30], who showed that DPH fluorescence anisotropy of DPPC liposomes in the gel phase was decreased by the addition of 10/~M perme-

M.R. Moya-Quiles et al. / Chemistry and Physics of Lipids 79 (1996) 21 28

thrin. These authors also reported that the fluorescence intensity of chlorophyll a in DPPCpermethrin mixtures remained unmodified at any temperature. In contrast, we found that permethrin modifies T M A - D P H fluorescence anisotropy when added to D M P C or DPPC (Fig. 5). It seems likely that the fluorophore of chlorophyll a is preferentially located in the membrane bilayer in a more external region than the fluorescent group of T M A - D P H . The different location of the fluorophores may explain the different results concerning changes in membrane fluidity induced by permethrin when these are monitored by chlorophyll a and by T M A - D P H fluorescence anisotropy. A comparison of the effects induced by deltamethrin (Moya-Quiles et al., unpublished data), permethrin [30] and fenvalerate [32] on model membranes reveals that the three pyrethroids share certain properties and differ in others. They decrease the phospholipid transition temperature and increase the fluidity of model membranes in the gel phase. Structurally, deltamethrin, permethrin and fenvalerate contain a common phenoxybenzyl alcohol esterified with either a cyclopropane carboxylate acid, deltamethrin and permethrin, or with a chlorobenzoxy acid derivative, fenvalerate. In addition, deltamethrin and fenvalerate possess an ct-cyano group which does not exist in permethrin. The correlation of the effects of the above pyrethroids with their structures suggests that the common phenoxybenzyl alcohol moiety, with or without the c~-cyano group, may be responsible for their preferential location in the hydrophobic core of the lipid bilayer. In contrast, allethrin, which possesses a cyclopentenolone residue instead a phenoxybenzyl alcohol, shows a preferential location at the lipid-water interphase. The conformation adopted by pyrethroids when they bind to lipid bilayers is unknown. In crystals, pyrethroids may adopt an extended or folded structure [33,34]. According to X-ray analysis, the conformation of the permethrin 1R cis single isomer in the crystal lattice is folded, with its phenoxy group in the vicinity of the CH 3 group, which is ,~.rans to the ester moiety [33]. It has been suggested that some pyrethroids might

27

adopt a 'horseshoe' conformation in the lipid bilayer with the ester group at the lipid-water interface but with both the hydrophobic acid and alcohol moieties folded back to allow penetration into the bilayer [35]. However, this does not seem to be the preferred structure for permethrin, since the pyrethroid has little effect on T M A - D P H fluorescence anisotropy in DSPC membranes and on the fluorescence intensity of chlorophyll a in DPPC vesicles [30]. The final conformation of a particular pyrethroid in a lipid bilayer will depend on its chemical composition, the length of the lipid hydrocarbon chains and the strength of the hydrophobic pyrethroid-lipid interactions. To summarize, permethrin probably interacts with model membranes by locating itself near the hydrophobic region of the lipid acyl chain, from where it perturbs the lipid order and makes the membrane more fluid in the gel phase. The results are of interest for elucidating the influence of pyrethroids on protein functions by modifying the lipid environment in which integral proteins are embedded.

Acknowledgements This work was supported by grants from the D G I C Y T (PB87/0697) and Comunidad Aut6noma de la Regi6n de Murcia (PCT 93/34). M.R. Moya-Quiles is a recipient of a scholarship from the Ministerio de Educacidn y Ciencia.

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