Changes Induced by Malathion, Methylparathion and Parathion on Membrane Lipid Physicochemical Properties Correlate with Their Toxicity

Biochimica et Biophysica Acta 1511 (2001) 360^368 www.bba-direct.com

Changes induced by malathion, methylparathion and parathion on membrane lipid physicochemical properties correlate with their toxicity

Romeu A. Videira, Maria C. Antunes-Madeira, Virg|¨nia I.C.F. Lopes, V|¨tor M.C. Madeira *

Centro de Neurocieªncias, Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal

Received 23 October 2000; received in revised form 15 January 2001; accepted 8 February 2001

Abstract

Perturbations induced by malathion, methylparathion and parathion on the physicochemical properties of dipalmitoylphosphatidylcholine (DPPC) were studied by fluorescence anisotropy of DPH and DPH-PA and by differential scanning calorimetry (DSC). Methylparathion and parathion (50 WM) increased the fluorescence anisotropy evaluated by DPH-PA and DPH, either in gel or in the fluid phase of DPPC bilayers, but mainly in the fluid phase. Parathion is more effective than methylparathion. On the other hand, malathion had almost no effect. All the three xenobiotics displaced the phase transition midpoint to lower temperature values and broadened the phase transition profile of DPPC, the effectiveness following the sequence: parathion s methylparathionEmalathion. A shifting and broadening of the phase transition was also observed by DSC. Furthermore, at methylparathion/lipid molar ratio of 1/2 and at parathion/lipid molar ratio of 1/7, the DSC thermograms displayed a shoulder in the main peak, in the low temperature side, suggesting coexistence of phases. For higher ratios, the phase transition profile becomes sharp as the control transition, but the midpoint is shifted to the previous shoulder position. Conversely to methylparathion and parathion, malathion did not promote phase separation. The overall data from fluorescence anisotropy and calorimetry indicate that the degree of effect of the insecticides on the physicochemical membrane properties correlates with toxicity to mammals. Therefore, the in vivo effects of organophosphorus compounds may be in part related with their ability to perturb the phospholipid bilayer structure, whose integrity is essential for normal cell function. ß 2001 Published by Elsevier Science B.V.

Keywords: Organophosphorus compounds; Physicochemical properties of lipids; Fluorescence anisotropy; Di¡erential scanning calorimetry

1. Introduction ther invertebrates or vertebrates, including humans [1,2]. Therefore, it is of major interest to identify Organophosphorus insecticides are widely used, the molecular mechanisms of action that can help for agricultural and domestic purposes, in the control in the development of insecticides with improved se- of insect pests. However, most of the above com- lectivity. pounds are highly toxic to non-target organisms, ei- The acute toxicity of organophosphorus insecticides is predominantly mediated by the inhibition of acetylcholinesterase [3]. However, a current under- * Corresponding author. Fax: +351-239826798; standing of how these compounds interact with the E-mail: [email protected] acetylcholinesterase is incomplete, since the capacity

0005-2736 / 01 / $ ^ see front matter ß 2001 Published by Elsevier Science B.V. PII: S0005-2736(01)00295-4

BBAMEM 78071 22-3-01 R.A. Videira et al. / Biochimica et Biophysica Acta 1511 (2001) 360^368 361 to phosphorylate the enzyme appears to decrease as the organophosphorus concentration increases [4]. On the other hand, the chronic toxicity resulting from continuous exposure to these compounds [5,6] escapes a rational understanding on the basis of molecular mechanisms. The lipophilicity of organophosphorus insecticides favors their incorporation in membranes [7,8]. The incorporated compounds elicit physical and chemical perturbations and presumably dysfunction of the membranes. In previous work of our own [9^12] and of others [13^16] perturbations of basic membrane mechanisms have been identi¢ed. A correla- tion between the perturbations in membrane basic mechanisms and insecticide toxicity is often apparent. On the other hand, evidence is accumulating that Fig. 1. Structure of malathion, O,O-dimethyl S-(1,2-dicarboxy- lipid bilayer structure and dynamics are implicated in ethyl) phosphorodithioate, methylparathion, O,O-dimethyl-O- membrane functionality [17^20]. Therefore, the pri- (p-nitrophenyl) phosphorothioate, and parathion, O,O-diethyl- O-(p-nitrophenyl) phosphorothioate. mary e¡ects of insecticides may result from physicochemical changes at the level of membrane lipid structure and organization. Consequently, the aim pounds were of the highest commercially available of the present work is to further elucidate a putative purity. relationship between changes in the physicochemical properties of the membrane and insecticide toxicity, 2.2. Methods attempting to clarify the molecular mechanisms underlying the toxic e¡ects of insecticides. Thus, 2.2.1. Preparation of membranes for £uorescence changes in the physicochemical properties of model anisotropy studies dipalmitoylphosphatidylcholine (DPPC) bilayers in- Model membranes were prepared as described duced by malathion, methylparathion and parathion, elsewhere [21]. Brie£y, solutions of pure DPPC in evaluated by di¡erential scanning calorimetry (DSC) CHCl3 were evaporated to dryness in round-bot- and by £uorescence anisotropy of 1,6-diphenyl-1,3,5- tomed £asks. The resulting lipid ¢lm on the wall of hexatriene (DPH) and [p-(6-phenyl)-1,3,5-hexatrienyl] the round-bottomed £ask was hydrated with an ap- phenylpropionic acid (DPH-PA) have been studied. propriate volume of 50 mM KCl, 10 mM Tris^mal- eate (pH 7.0) and dispersed under N2 atmosphere by handshaking in a water bath set 7^10³C above the 2. Materials and methods transition temperature of DPPC and multilamellar vesicles were obtained. The ¢nal nominal concentra- 2.1. Materials tion of the lipid was 345 WM. The model membranes were brie£y sonicated in a low-energy water soni¢er DPPC was obtained from Sigma Chemical Co., St. to disperse aggregates, in order to decrease the light Louis, MO, USA. The probes DPH and DPH-PA scattering and to improve the readings of £uores- were purchased from Molecular Probes, Inc., Eu- cence. Sonication was applied for a controlled period gene, OR, USA. The insecticides malathion, O,O-di- of time to avoid the turbidity decreasing below 0.15 methyl S-(1,2-dicarboxyethyl) phosphorodithioate, absorbance units at 600 nm. methylparathion, O,O-dimethyl-O-(p-nitrophenyl) phosphorothioate and parathion, O,O-diethyl-O-(p- 2.2.2. Incorporation of the probes and insecticides into nitrophenyl) phosphorothioate (Fig. 1) were supplied membranes by Supelco, Inc., Bellefonte, PA, USA. All the com- Few Wl of the £uorescence probes in dimethylform-

BBAMEM 78071 22-3-01 362 R.A. Videira et al. / Biochimica et Biophysica Acta 1511 (2001) 360^368 amide were injected into membrane suspensions (345 bling of membrane vesicles which is very slow as WM in lipids) to give a ¢nal lipid/probe molar ratio compared with molecular motion [24]. of about 300/1. The mixture was initially vigorously vortexed for 10 s and then the insecticides were 2.2.4. DSC studies added from concentrated ethanolic solutions. The Multilamellar vesicles for DSC were prepared as mixture was incubated in the dark to protect the described above. The lipid concentration was nomi- probes, for a period of 18^20 h, i.e. the time required nally 666 WM in all the samples examined. Insecti- for equilibrium, since the insecticides have to pene- cides were added from concentrated ethanolic solu- trate multiple lipid bilayers. The incubation was per- tions to give the insecticide/lipid molar ratios formed at the phase transition temperature of DPPC, indicated in the ¢gures. The mixtures (lipid plus in- at which the incorporation of the insecticides is max- secticide) were allowed to equilibrate for 18^20 h, imal [7]. Control samples received equivalent vol- and then centrifuged at 45 000Ug, for 45 min, at umes of dimethylformamide and ethanol. Added sol- 4³C. The wet pellets were hermetically sealed into vents (few Wl) had no detectable e¡ects on the assays. aluminum pans and placed in a Perkin-Elmer Pyris 1 di¡erential scanning calorimeter. The DSC mea- 2.2.3. Fluorescence anisotropy measurements surements were performed at a scan rate of 5³C/ Fluorescence spectra were recorded in a Perkin- min, over the temperature range from 20 to 60³C. Elmer spectro£uorometer, model MPF-3, provided This rate yielded thermograms essentially identical with a thermostated cell holder. The excitation was to those obtained at 1³C/min, meaning that thermo- set a 365 nm and the emission was detected at 450 dynamic equilibrium is maintained at scanning rates nm. The excitation and emission slits were 4 and 6 of 5³C/min. To check the reproducibility of the re- nm, respectively. The temperature of the sample was sults, three di¡erent samples were measured for each checked with an accuracy of þ 0.1³C, using a ther- insecticide/lipid ratio. For each sample, three heating mistor thermometer. The anisotropy coe¤cient (r) and two cooling scans were recorded. Cooling scans was calculated according to Shinitzky and Barenholz yielded thermograms very similar to the heating [22] from the equation: scans, but, and accordingly to the literature [27], I 3I G the transitions in cooling curves are shifted, by about r e P 1³C, to lower temperatures. Therefore, due to the I 2I G e P supercooling phenomenon, accurate thermotropic where Ie and IP are the intensities of the light emitted transitions are evaluated from heating curves. There- with its polarization plane parallel (e) and perpendic- fore, heating scans have been used in the present ular (P) to that of exciting beam. The grating cor- work. rection factor for the optical system (G) is given by To determine the total phospholipid content of the the ratio of vertically to the horizontally polarized samples, the aluminum pans were opened and the emission components, when the excitation light is samples dissolved in chloroform/methanol (5/1) mix- polarized in the horizontal plane [23]. Blanks were tures. Then, phospholipids were quanti¢ed by mea- always recorded without added probes to correct for suring inorganic phosphate [28] released after hydro- light scattering, which was always very low in our lysis of the extracts at 180³C in 70% HClO4 [29]. assay conditions. The degree of £uorescence anisotropy of DPH and DPH-PA re£ects rotational di¡usion of the probes, 3. Results which depends on the relative mobility of phospholipid acyl chains, i.e. on the degree of bilayer £uidity 3.1. Fluorescence anisotropy studies [24^26]. Therefore, a high degree of anisotropy re- £ects a limited rotational di¡usion of the probes Fluorescence anisotropies of DPH, a probe buried and, consequently, represents a high structural order in the bilayer core [22], and of DPH-PA, a probe or low membrane £uidity, and vice-versa. Only lipid anchored in the bilayer surface by its charged pro- motion a¡ects the probe anisotropy, not the tum- pionic group [30], provide the relative perturbations

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Fig. 2. Fluorescence anisotropy (r) provided by DPH (A) and DPH-PA (B), in DPPC bilayers, as a function of temperature, in the absence (solid symbols and lines) and presence (open Fig. 3. Fluorescence anisotropy changes, represented as % of symbols and dotted lines) of 50 WM malathion (O), methyl- control, induced by malathion (M), methylparathion (MP) and parathion (E) and parathion (a). Insets represent the ¢rst deriv- parathion (P) in £uid membranes of DPPC (a), mitochondria atives of anisotropy data of the main plots. The derivative (O) and sarcoplasmic reticulum (E), as a function of the LD50 curves 1, 2, 3 and 4 correspond to the main curves with the of the above insecticides to mammals. A and B represent the symbols b, O, E and a, respectively. The standard deviations results obtained with DPH and DPH-PA, respectively. Fluores- (S.D.) of the average of three independent experiments, for cence anisotropy values are the means þ S.D. of at least three each temperature, are too small to be displayed by error bars. independent experiments. Fluorescence anisotropy values for mitochondria and sarcoplasmic reticulum were taken from [26^28].

BBAMEM 78071 22-3-01 364 R.A. Videira et al. / Biochimica et Biophysica Acta 1511 (2001) 360^368 induced by the organophosphorus compounds across the DPPC bilayer thickness. Thus, the e¡ects promoted by 50 WM of malathion, methylparathion and parathion on the thermotropic pro¢le of DPPC are displayed in Fig. 2. The insecticides under study broaden the transition pro¢le and shift the phase transition midpoint (Tm) to lower temperature values. These e¡ects provided by both probes are more apparent for parathion and less apparent for malathion. Methylparathion has intermediate e¡ects. Therefore, the order of the e¡ects correlates reason- ably with their toxicity to mammals [1]. Additionally, methylparathion and parathion increase the £uorescence anisotropy of DPPC bilayers, in gel and £uid phase, but mainly in £uid phase (Fig. 2). Again the e¡ects of parathion are stronger than those of methylparathion. Malathion has almost no e¡ect. The ordering e¡ects, which are more apparent when detected with DPH, are consistent with previous data obtained in other models and in several £uid native membranes [31^33]. Considering the £uid phase of DPPC, where the ordering e¡ects are more apparent and representing the £uorescence anisotropy changes versus the toxicity of the insecticides to mammals (represented by LD50), data of Fig. 3 are obtained. Also, in the same ¢gure are represented £uorescence anisotropy changes induced by the insecticides under study in £uid native membranes [31^33], as a function of toxicity to mammals. Either in models or in native membranes it is apparent that parathion, the most toxic insecticide, has the strongest e¡ect, whereas malathion, the less toxic, has the lowest e¡ect; methylparathion, with intermediate toxicity, exerts also intermediate e¡ects.

3.2. DSC

The e¡ects of malathion, methylparathion and parathion on the thermotropic pro¢les of DPPC were also searched by DSC (Fig. 4). The thermotropic pretransition of DPPC is strongly a¡ected by low amounts of insecticide, and can no longer be detected for parathion/lipid molar ratio of 1/20 or Fig. 4. DSC thermograms for mixtures of insecticide/DPPC. The insecticide/phospholipid molar ratios are indicated on the for methylparathion or malathion/lipid molar ratio curves. The DSC pro¢les correspond to heating scans. of 1/7, as shown in Fig. 4. In agreement with £uorescence anisotropy data, the main transition peak broadens and shifts to lower temperature values, an e¡ect that depends on insecticide concentration.

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Conversely to malathion, methylparathion and para- 6.01 and 290 mg/kg, respectively [34]. Considering a thion, at insecticide/lipid molar ratios of 1/2 and 1/7, total £uid volume of 58 ml/kg [35], the lethal serum respectively, induce a shoulder in the main peak, in concentrations estimated for parathion, methylpara- the low temperature side, suggesting coexistence of thion and malathion are 118, 394 and 15 100 WM, di¡erent phases. At higher insecticide/lipid molar ra- respectively. Therefore, the concentration of 50 WM, tios, i.e. 1/1 for methylparathion and 1/2 for para- used in £uorescence studies, is far from the lethal thion, the transition is again sharp and displaced to doses for these compounds. In DSC studies, the the position of the previous shoulder, whereas the bulk insecticide concentration varied between 0 and main peak completely disappears. Therefore, only 666 WM for methylparathion and malathion and be- one peak, with a Tm of about 36³C or 37.4³C for tween 0 and 333 WM for parathion. Therefore, for mixtures of methylparathion/DPPC or parathion/ parathion and methylparathion the studies were ex- DPPC, respectively, is obtained (Fig. 4). tended above the lethal doses. However, the second The calorimetric enthalpies of the thermotropic peak in DPPC thermograms appears at 333 WM for events described in Fig. 4 are depicted in Fig. 5. methylparathion and at 95 WM for parathion, i.e. at Concentrations of malathion up to 0.5 insecticide/ concentrations close to the lethal toxic concentra- lipid molar ratio decrease vH, but higher insecti- tions in vivo. cide/lipid molar fractions have a limited e¡ect on vH. Conversely to malathion, methylparathion and parathion increase vH up to a molar ratio of 0.5. 4. Discussion Further increase of the ratio has a limited e¡ect on vH. These results suggest di¡erent interactions be- The e¡ects of malathion, methylparathion and tween malathion and lipids as compared with meth- parathion on the physicochemical properties of ylparathion and parathion. DPPC dispersions were assessed by £uorescence ani- The above insecticide concentrations were used on sotropy and by DSC. To determine the modi¢cations the basis of the following ¢ndings. LD50 values (de- in lipid packing across the bilayer thickness, £uores- termined in mammmals, oral administration) for cence anisotropies of DPH and DPH-PA were mea- parathion, methylparathion and malathion are 2.0, sured. Lateral heterogeneities or domains were searched by DSC. This technique also provides val- uable information on the phase transition as well as on the enthalpy of melting of membrane components. Therefore, £uorescence anisotropy and DSC provide complementary information about the biophysical modi¢cations occurring in DPPC bilayers as a result of insecticide interaction. Fluorescence anisotropy indicates that the insecticides under study, at concentrations of 50 WM, corresponding to insecticide/lipid stoichiometry of 1/7, shift the phase transition midpoint to lower temperature values and broaden the phase transition (Fig. 2). These e¡ects suggest, accordingly to Jain and Wu [36], a displace- ment of the xenobiotics into the vicinity of the ¢rst eight carbons of the acyl chains, i.e. in the cooperativity region. This is the region where the rigid rings of cholesterol also locate [37]. Interesting is also the fact that an inverse linear relationship between the Fig. 5. Enthalpy changes (vH, kJ/mol) for the gel to liquid- crystalline phase transition of insecticide/DPPC mixtures, at dif- partition of parathion and malathion and the molar ferent insecticide lipid molar ratios. Each point represents the ratio of cholesterol has been observed [7]. Further- average of at least three independent experiments þ S.D. more, a complete exclusion of the insecticides from

BBAMEM 78071 22-3-01 366 R.A. Videira et al. / Biochimica et Biophysica Acta 1511 (2001) 360^368 the membrane is observed at 50 mol% cholesterol. versely to methylparathion and parathion, malathion On the basis of theoretical and experimental ap- has almost no e¡ect either in the gel or the £uid proaches [38,39], the way by which cholesterol a¡ects phase, which may re£ect its low partition, since its the physical state of lipids and, consequently, the maximum in DPPC bilayers is 244, whereas the max- incorporation of insecticides into the bilayers can imal partition for parathion is 1050 [7]. On the other be discussed as follows. Fluid phospholipid mem- hand, the molecular geometry of malathion is more branes can exist in a liquid-disordered phase, at similar to the molecular geometry of individual phos- very low cholesterol concentrations, a liquid-ordered pholipids and, consequently, a good accommodation phase, at high cholesterol concentrations, or the two and miscibility is expected without extensive pertur- phases, at intermediate sterol concentrations. Appar- bation of the membrane packing and organization, ently, only the liquid-disordered phase allows the in- as compared with the other insecticides (Fig. 2). The corporation and interaction of this type of insecti- results of Fig. 4 also indicate limited perturbation of cides, but the liquid-ordered phase, induced by the membrane biophysical properties in mixtures of packing of cholesterol with lipids in the cooperativity malathion/DPPC as compared with perturbations ex- region [37], prevents insecticide incorporation and pected by methylparathion or parathion/DPPC. On interaction. Therefore, high cholesterol concentra- the other hand, a decrease in vH for mixtures of tions increase the density of lipid bilayers in such a malathion/DPPC (Fig. 5) re£ects decreasing of van way that the free volume for insecticide incorpora- der Waals interactions and indicates that lipid chain tion and interaction decreases as a consequence of a packing is di¡erent from that of methylparathion or membrane localization similar to that of sterol. parathion/DPPC mixtures, where an increasing of Conversely to malathion, methylparathion and vH is observed. Also, ethylazinphos, another orga- parathion, at insecticide/lipid molar ratios of 1/2 nophosphorus insecticide, induces a new transition in and 1/7, respectively, induce a shoulder in the main DPPC thermograms [40] and increases vH. Further- peak, in the low temperature side (Fig. 4). Therefore, more, additional work with this insecticide (article at these ratios, the insecticides are non-randomly dis- submitted) supports the phase heterogeneity. Ethyl- tributed in the membrane plane. According to Jain azinphos increases the passive proton permeability of and Wu [36], a compound that promotes a new lipid bilayers reconstituted with DPPC and mito- phase transition speci¢cally interacts with the polar chondrial lipids. A signi¢cant enhancement of proton headgroups of the phospholipids. Therefore, methyl- permeability was detected at insecticide/lipid molar parathion and parathion are probably positioned in ratios identical to those inducing phase separation the membrane with their polar moieties intercalated in the plane of DPPC bilayers, as revealed by between the polar groups of the phospholipids and DSC. The organochlorine insecticides of the cyclo- their hydrophobic moieties buried in the cooperativ- diene family K- and L-endosulfan also a¡ect the ther- ity region. This location facilitates hydrogen bonding motropic properties of DPPC bilayers. Conversely to or dipole^dipole interactions between the nitrophe- L-endosulfan, K-endosulfan, with higher toxicity, nylphosphorothioate group of the insecticides and promotes a new peak in the thermograms [41] and the headgroups of phospholipids with a consequent increases the proton permeability in DPPC and mi- decrease in the headgroup spacing. Therefore, a con- tochondrial membranes (results to be published). densing e¡ect is expected to occur, particularly in the Lindane, another organochlorine insecticide, also £uid phase, extending through the bilayer thickness, creates lateral heterogeneities in lipid membranes as probed by DPH and DPH-PA (Fig. 2). The stron- [42]. However, this e¡ect does not promote increase ger e¡ect promoted by parathion relatively to meth- in the permeability but the opposite due to a sealing ylparathion is certainly related to the chemical struc- e¡ect of the interfaces by the insecticide [43]. Other ture. The two additional methylene groups of drugs [44^47] with the ability to increase the dynamic parathion render it more hydrophobic and higher membrane heterogeneity will also compromise mem- partition is expected. Accordingly, stronger e¡ects brane permeability. Moreover, the existence of dis- are induced by parathion since its core structure is continuous regions not percolated [48] in a continu- identical to that of methylparathion (Fig. 1). Con- ous membrane phase would di¤cult certain

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