Effects of methylparathion on membrane fluidity and its implications for the mechanisms of toxicity

Toxicology

in Vitro 11 (1997) 337-345

Effects of Methylparathion on Membrane Fluidity and its Implications for the Mechanisms of Toxicity V. I. C. F. LOPES, Centro

de NeurociBncias,

M. C. ANTUNES-MADEIRA Departamento

and

de Zoologia, Universidade Codex, Portugal

V. M. C. MADEIRA de Coimbra,

3049 Coimbra

(Accepted 12 February 1997) Ah&act-As probed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (DPH-PA), methylparathion decreases the phase transition midpoint of dimyristoylphosphatidylcholine (DMPC) bilayers and broadens the transition profile. Furth&more, the insecticide orders tb some extent the fluid phase of DMPC, in either the hydrophobic core or in the outer regions of the membrane, as evaluated by DPH and DPH-PA, r&ectively. These condensing effects-of methylparathion were further confir&d in fluid models of egg-yolk phosphatidylcholine. The insecticide increases to some extent the ordering promoted by cholesterol in fluid bilayers of DMPC, but high cholesterol concentrations ( > 30 mol%) prevent methylparathion interaction. In agreement with the data in models of synthetic lipids, the condensing effects of methylparathion in fluid native membranes of mitochondria, sarcoplasmic reticulum and erythrocytes are depressed with the increase in intrinsic cholesterol. Therefore, the effects of methylparathion are modulated, to a great extent, by membrane cholesterol concentration. Consequently, it can be suggested that the fluidity effects of methylparathion would be preferentially exerted in biomembranes scarce in cholesterol, e.g. mitochondria and sarcoplasmic reticulum. The perturbations promoted by methylparathion in these highly functional membranes will certainly induce bioenergetic alterations endangering cell and tissue functions, since membrane fluidity is a crucial parameter in the control of basic membrane mechanisms and, consequently, in cell homoeostasis. @ 1997 Elseuier Science Ltd DMPC = dimyristoylphosphatidylcholine; PA = 3-[p-(6-phenyl)-1,3,Shexatrienyl] phenylpropionic TM = transition midpoint. Abbreviations:

INTRODUCTION

Insecticides have been used exhaustively since World War II to control a wide range of pests, for reasons of health and to help in food production with significant benefits to the economy (Ware, 1983). Unfortunately, however, the insecticides are poorly selective and toxic effects have been detected in useful insects and other animals, either invertebrates or vertebrates (Metcalf, 1971; Ware, 1983). Negative impacts at higher levels of organization, particularly, at populations, communities and ecosystems often occur. Additionally, humans are often subjected to the toxic effects of these compounds by either handling or ingesting contaminated products. Therefore, it is a major aim in insecticide toxicology to identify the molecular mechanisms of action, that can help in the development of pesticides with improved biological selectivity. Extensive work has been devoted over the last few decades to identify the precise biochemical mechanisms underlying insecticide toxicity. Defined acute 0887-2333/97/S17.00 + 0.00 0 PII: X1887-2333(97)00024-6

1997 Elsevier

DPH = 1,6-diphenyl-1,3,5-hexatriene; DPHacid; egg-PC = egg-yolk phosphatidylcholine;

biochemical interactions have been only assigned to organophosphorus and carbamate compounds, recognized as powerful inhibitors of acetylcholinesterase (Eto, 1974; Kuhr and Dorough, 1976). However, the chronic toxicity resulting from continuous exposure to these compounds (Ohkawa, 1982) cannot be explained by anticholinesterase effects. Therefore, the molecular mechanisms underlying this form of toxicity are essentially unknown. The strong lipophilicity of most insecticides promotes incorporation in biomembranes. Owing to the crucial functions of membranes, the insecticide either acute or chronic are certainly effects, membrane related. Accordingly, previous studies indicate that insecticide compounds induce perturbations of membrane permeability and enzyme dynamics (Antunes-Madeira and Madeira, 1979 and 1982; Antunes-Madeira et al., 1981). In some circumstances, a correlation between the perturbations in membrane mechanisms and insecticide toxicity could be established. Since basic membrane

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V. I. C. F. Lopes ef al.

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mechanisms are greatly influenced by the membrane physical state and organization (Sikkema et al., 1995), the primary effects of insecticides may be related to physical changes at the level of lipid-lipid and lipid-protein interactions. Additionally, partition studies (Antunes-Madeira and Madeira, 1989) indicate that membrane organization modulates to a great extent insecticide incorporation. Consequently, the order gradient across the bilayer thickness (Chefurka et al., 1987) suggests domain distributions of the insecticides and, consequently, discrete physical effects across the width of the bilayer. Our recent work has been directed along with these lines. using appropriate fluorophores that probe the physical state of the outer regions of the bilayer and the hydrophobic core. The effects of several insecticide compounds, namely lindane (AntunesMadeira et al., 1990) DDT (Antunes-Madeira et al., 1991), DDE (Antunes-Madeira and Madeira, 1993) parathion (Antunes-Madeira et al., 1994), malathion (Videira et al., 1994) and azinphos (Videira ef al., 1996), on the fluidity of model and native membranes have been studied and their localization along the bilayer thickness has been tentatively predicted. On the sequence of these studies the effects of methylparathion [O,O-dimethyl 0-(p-nitrophenyl) phosphorothioate] on the physical state of model and native membranes have been studied, to collect additional data, that can be useful in the future to identify the toxicity of the compounds on the basis of their localization along the bilayer thickness and consequent physical perturbations. MATERIALS AND METHODS

Chemicals

Cholesterol, dimyristoylphosphatidylcholine (DMPC), egg-yolk phosphatidylcholine (egg-PC) and 1,6-diphenyl-1,3,5hexatriene (DPH) were purchased from the Sigma Chemical Co. (St Louis, MO, USA). 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (DPH-PA) was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Methylparathion, represented in Fig. 1, was obtained from Supelco, Inc. (Bellefonte, PA, USA). All these compounds were of the highest commercially available quality.

solutions of pure phospholipids in CHCll were taken in round-bottomed flasks and the solvent was evaporated to dryness. The resulting lipid film on the wall was hydrated with an appropriate volume of 50 rnM KC], 10 mM Tris-maleate (pH 7), and dispersed under N, atmosphere by hand shaking in a water-bath 7-10°C above the transition temperature of the phosphohpids. Phospholipid-cholesterol bilayers were obtained by supplementing original phospholipid solutions with required amounts of cholesterol. Several native membranes, for instance, erythrocytes, sarcoplasmic reticulum and mitochondria, were prepared as described elsewhere (AntunesMadeira and Madeira, 1984). In all cases, the final nominal concentration of membrane lipids was 345 /lM. Model and native membranes were briefly sonicated in a low energy water sonifier. This procedure does not distort the transition of lipid bilayers, but disperses aggregates, facilitating the readings of fluorescence and decreasing the scattered light. Sonication has been applied for a controlled period of time to avoid the turbidity to decrease below 0.15 absorbance at 600 nm. Incorporation of probes membranes

and methylparathion into

DPH and DPH-PA in dimethylformamide were injected (few ~1) into membrane suspensions (345 PM in total lipid) to give a final lipid/probe molar ratio of about 200. The mixture was initially vigorously vortexed for 10 set, and then methylparathion was added from concentrated ethanolic solutions (50 mM). The mixture was allowed to equilibrate in the dark for 18-20 hr. It should be emphasized that: (1) the probes by themselves, at the used ratios (1:200 relatively to lipids), induce minimal perturbations of lipid geometry (Lentz, 1989) and, consequently, can be used to report alterations promoted by xenobiotics used at much higher stoichiometric ratios relative to bilayer phospholipids (see legends to the figures); (2) it was ascertained that added concentrations of the insecticide were within the solubility range and (3) the solvents by themselves (a few ~1) had no detectable effects.

Preparation of membranes

Synthetic membranes were prepared as described elsewhere (Antunes-Madeira ef al.. 1990). Briefly,

Fig. 1. Structure

of methylparathion

Fluorescent polarization measurements

Fluorescence spectra were recorded in a PerkinElmer spectrofluorometer, model MPF-3, provided with a thermostated cell holder. The excitation was set at 336 nm and the emission at 450 nm. The excitation and emission slits were 4 and 6 nm, respectively. The temperature of the sample was checked with an accuracy of + O.l”C, using a thermistor thermometer. The degree of fluorescence polarization (P) was calculated according to Shinitzky and Barenholz (1978) and Litman and

Membrane fluidity as affected by methylparathion Barenholz

(1982) from the equation:

p = II, - 1-G

41+ I,G where I,, and I, are the intensities of the light emitted with its polarization plan parallel (,,) and perpendicular (,) to that of exciting beam. G is the correction factor for the optical system, given by the ratio of vertically to the horizontally polarized emission components when the excitation light is polarized in the horizontal direction (Litman and Barenholz, 1982). A high degree of polarization reflects a limited rotational diffusion of the probes and, therefore, represents a high structural order or low membrane fluidity, and vice versa. The term ‘fluidity’ is used here as being inversely proportional to the polarization of diphenylhexatriene probes and refers only to the rate of motion of the phospholipid acyl chain. In bulky isotropic fluids, fluidity is given by the reciprocal of the viscosity. Thus, the fluidity used in the present work is related but not identical with the physical definition of fluidity. According to Shinitzky and Barenholz (1978), the upper theoretical limit for fluorescence polarization (PO) in isotropic solutions is 0.5 All the fluorescence measurements were corrected for the contribution of light scattering by using controls with membranes, but without added probes.

RESULTS AND DISCUSSION

Phospholipid and phospholipid-cholesterol bilayers

Fluorescence polarization of DPH, a probe buried in the bilayer core (Shinitzky and Barenholz, 1978) and of DPH-PA, a probe anchored in the bilayer surface by its charged propionic group (Trotter and Starch, 1989) was used to determine the perturbations induced by methylparathion across the bilayer thickness. DPH and DPH-PA polarizations report the rotational diffusion of the probes which strongly depends on the relative internal motions of phospholipid acyl chains, that is, on the degree of bilayer fluidity (Antunes-Madeira et af., 1994). Fluorescence polarization of DPH and DPH-PA embedded into fluid bilayers of egg-yolk PC, a lipid with a phase transition at - 5°C (Bittman and Blau, 1976). is displayed in Fig. 2. It is clear from the figure that polarization values of DPH are lower than those of DPH-PA, that is, membrane structural order decreases from the outer to the central bilayer observations regions, according to classical (Chefurka et al., 1987). Over the temperature range under study (from 12°C to 4O”C), methylparathion (50 PM) induces ordering effects along all the thickness of the bilayer, as evaluated by DPH and DPH-PA (Fig. 2, dotted lines). Furthermore, the difference in fluorescence polarization in the presence

339

and in the absence of methylparathion is independent of temperature and is about 0.028, as detected by DPH, or 0.012 as detected by DPH-PA. These ordering effects promoted by methylparathion are similar to those induced by a temperature decrease of about 8°C that is, a significant change in temperature is necessary to mimic the effect of the insecticide. However, in oiuo, compensatory physiological regulatory mechanisms may limit the effects of temperature changes. However, if the temperature change is too fast and the organisms are not able to adapt structurally-related toxicity-like effects may occur. Cossins et af. (1977) studied the behavioural effects in goldfish after a sudden change in temperature. They observed that a downshift of 10°C induces hyperexcitability, a shift of 13°C results in equilibrium lost and a 14°C shift promotes coma-like insensitivity. Since the physical state of lipids is affected by small changes in temperature, studies by Cossin et al. suggest that membrane fluidity perturbations may be involved in the physiological behaviour. Also interesting is the fact that the behaviour of the fish submitted to temperature stress resembles the behaviour of organisms intoxicated with methylparathion and with other organophosphorus compounds (Eto, 1974). Thus, fluidity perturbations in membranes may be relevant to the toxicity mechanisms. The effects promoted by methylparathion in the fluid phase of egg-lecithin bilayers are qualitatively similar to those described for parathion (AntunesMadeira et al., 1994). However, the effects of parathion are quantitatively stronger than those obtained for methylparathion, in correlation with its higher toxicity (Metcalf, 1971). Figure 3 summarizes the effects of increasing concentrations of methylparathion (O-100 PM) on the thermotropic phase transition of DMPC, detected by fluorescence polarization of DPH and DPH-PA. A broadening of the phase transition profile, which is dependent on the insecticide concentration, is apparent. This effect indicates, according to Jain and Wu (1977), interaction of the xenobiotic with the co-operativity region of the bilayer, namely, the region of C,C, carbon atoms of the acyl chains, which modulates the sharpness of the transition. Also apparent in Fig. 3 is the shift of the phase transition midpoint (Tm) to lower temperature values. The intensity of this effect, detected by DPH and DPH-PA, is also concentration dependent. Thus, Tm is shifted by 0.5, 1.1 and 1.4”C for methylparathion concentrations of 25, 50 and 100 PM, respectively, as detected by DPH. These disordering effects induced by methylparathion are only noticed in the temperature range from 20 to 24°C. Below and above this range, the insecticide increases the structural order of the lipid bilayers, the effects being more pronounced in the fluid than in the gel phase. The ordering effects in the fluid phase are comparable to those observed in egg-yolk lecithin bilayers (Fig. 2).

V. 1. C. F. Lopes et al

340

0.051 IO

15

20

25

Temperature

30

35

40

35

40

(“C)

0.28 r

10

15

20

25

Temperature

30 (“C)

Fig. 2. Fluorescence polarization (P) of (A) DPH and (B) DPH-PA in egg-yolk PC bilayers as a function of temperature, in the absence (solid symbols and lines) or in the presence (open symbols and dotted lines) of 50 PM methylparathion, corresponding to a molar stoichiometry of 0.145 relative to lipids. Regression lines were calculated by means of the least-squares method. Correlation coefficients vary from 0.992 to 0.995.

Methylparathion-lipid interactions in the transition range differ from those occurring in the gel and fluid phases. In the transition range the lateral density fluctuations are particularly important and structural defects between gel and fluid domains occur (Lee, 1977) increasing the free volume of the bilayer. Consequently, it may be predicted that partition of methylparathion would increase in the range of the phase transition, as previously measured for parathion and other insecticides (Antunes-Madeira and Madeira, 1984 and 1989; Videira et al., 1995 and 1996). Therefore, the phospholipid acyl chains must

conform to accommodate the extra amount of incorporated insecticide, resulting in weakening of lipid-lipid interactions, and a consequent downshift of the Tm, as detected by DPH and DPH-PA. The bilayers in gel and fluid phases are more stable relatively to the state of the transition where ordered and disordered domains oscillate at a high rate. Therefore, hydrogen bonding or dipoltiipole interactions may take place between the nitrophenylphosphorotioate group of methylparathion and the headgroups of phospholipids with a consequent decrease in the headgroup spacing,

Membrane fluidity as affected by methylparathion

341

P 0.25

10

15

-0A

0.15 10

10

IS

I 15

20

20

25

30

Temperature

(“C)

25

50

‘c 20

35

35

40

I

I 40

40

I 25

Temperature

I 30

35

(“C)

Fig. 3. Fluorescence polarization (P) of (A) DPH and (B) DPH-PA in DMPC bilayers, in the absence (solid symbols and lines) or presence (open symbols and dotted lines) of increasing concentrations of methylparathion (0, 0; 0, 25 PM; a, 50 PM; 0, 100 p(M), which correspond to insecticide:lipid stoichiometries of 0.072, 0.145 and 0.290, respectively. The insets represent differential plots of the data in the main plots.

inducing condensation of the bilayer structure, particularly in the fluid phase, as detected by the fluorescent probes. The stronger ordering effects promoted by parathion (Antunes-Madeira et al., 1994) relative to methylparathion are certainly related to the chemical structure. The two additional methylene groups of parathion render it more hydrophobic and higher partition is expected. stronger effects are induced by Accordingly, parathion since its core structure is identical to

methylparathion. These and previous data (AntunesMadeira et al., 1990, 1991 and 1994; Videira et al., 1994 and 1996) indicate that the physical state of membrane lipids and the molecular structure of the insecticides are important parameters that may modulate toxicity in terms of alteration of the membrane structural organization. Native membranes are putatively composed of domains or patches of lipids differing in composition and fluidity (Welti and Glaser, 1994). Therefore, membrane

342

domains

V. 1. C. F. Lopes with higher

fluidity

would

affected by insecticide compounds

be particularly

of the parathion

type. Figure 4 shows the effects of 50 pM methylparathion on DMPC bilayers enriched with cholesterol, in the temperature range from 12 to 40-C. The insecticide induces limited ordering effects at temperatures below the phase transition of DMPC, as detected either by DPH or DPH-PA. Above the phase transition of DMPC, both probes detect

et al.

ordering effects promoted by methylparathion, which gradually fade at cholesterol concentrations equal or

higher to 20 mol% (Fig. 4). Similar effects have been described, previously, for the organophosphorus compounds parathion (Antunes-Madeira et al., 1994) malathion (Videira et al., 1994) and azinphos (Videira et al., 1996) and for the organochlorine compound lindane (Antunes-Madeira et al., 1990). The present and previous data suggest a strong exclusion of these insecticides from the bilayer at high

0.40 -

P 0.25 0.20 0.15-

10

15

20

25

Temperature

30

35

40

(“Cl

0.40 r ti

0.35 -

50 30 20

P 0.30 -

0.25 -

1 10

Temperature

(“C)

Fig. 4. Fluorescence polarization (P) of (A) DPH and (B) DPH-PA in DMPC/cholesterol bilayers, in the absence (solid lines) or presence (dotted lines) of 50 PM methylparathion (0.145 stoichiometry relative to lipids). The numbers adjacent to the curves represent mol% of cholesterol incorporated into DMPC bilayers. Each curve was drawn across 19-22 experimental points which were removed for the sake of clarity.

Membrane

343

fluidity as affected by methylparathion

cholesterol concentrations. studies (Antunes-Madeira

Indeed, previous partition and Madeira, 1989) for parathion, malathion and lindane, in egg-lecithin membranes, at 24”C, indicate an apparent inverse linear relationship between the partition of these insecticides and the molar ratio of cholesterol. A complete exclusion of the insecticides is observed for cholesterol concentrations of 50 mol%, that is, at a stoichiometry of 1: 1 relative to phosphoIipids

allowing for maxknal van der Waals contacts in the hydrocarbon region (Presti et al., 1982). Assuming that partitioning of methylparathion is similar to that of parathion, as previously determined (AntunesMadeira and Madeira, 1984), the effect of cholesterol is related to the exclusion of the insecticide from the bilayer that is almost complete at 50 mol% cholesterol for parathion (Antunes-Madeira and Madeira, 1984). Therefore, it can be suggested that

A

0.40

0,35

SR M O.lOl

10

I

15

I

I

20

25

Temperature

0.40 -

I

I

I

30

35

40

(“C)

6

0.35 -=++=+-&

.

--h .h . . A-k&#+_+

-‘..I.--.

MAE

10

15

20 25 Temperature

30 (“C)

35

40

Fig. 5. Fluorescence polarization (P) of (A) DPH and (B) DPH-PA in several native membranes differing in intrinsic cholesterol, as a function of temperature, in the absence (solid symbols and lines) and presence (open symbols and dotted lines) of 50 PM methylparathion (insecticidezlipid stoichiometry of 0.145). Cholesterolfphospholipid molar ratios for mitochondria (M), sarcoplasmic reticulum (SR) and erythrocytes (E) are 0, 6 and 37 moi%, respectively. Regression lines were calculated as in Fig. 2. The correlation cuetiients vary from 0.887 and 0.991. As in the previous figs, error bars are not represented, since for most experimental points they are encompassed by the size of the symbol.

344

V. I. C. F. Lopes et al.

cholesterol and the insecticide compete for the same membrane domains. Since it is generally believed that cholesterol causes an increase in the density of lipid bilayers or, in other words, a decrease in free volume (Almeida et al., 1992), it can be concluded that cholesterol decreases the free volume for methylparathion incorporation and interaction. Thus, cholesterol would change the membrane organization in such a way that the free volume for insecticide incorporation and interaction decreases, since insecticide and cholesterol compete for similar sites in the membrane. Theoretical and experimental approaches (Ipsen et al., 1987; Vist and Davis, 1990) concur with this suggestion. Fluid phospholipid membranes can exist in a liquid-disordered phase at very low cholesterol concentrations, a liquid-ordered phase at high cholesterol concentrations, or in two phases at intermediate sterol concentrations; apparentiy, only the liquid-disordered phase allows the incorporation and interaction of the insecticide and the liquid-ordered phase prevents insecticide incorporation and interaction. This phase permits only the incorporation and interaction of compounds that localize in the hydrophobic core of the membrane, for example DDT (Antunes-Madeira et al., 1991) and DDE (Antunes-Madeira and Madeira. 1993). Native membranes

The fluorescence polarization studies were extended to native membranes, with low and high cholesterol contents, to collect pertinent information with biochemical significance (Fig. 5). Thus, the fluorescence polarization of DPH and DPH-PA, incorporated into native membranes of mitochondria, sarcoplasmic reticulum and erythrocytes, was monitored in the temperature range from 12 to 40°C. As shown in Fig. 5, the fluorescence polarization of native membranes changes linearly with the temperature and significantly depends on the intrinsic cholesterol content. The effects of methylparathion (50 PM), probed by DPH and DPH-PA, are qualitatively similar to those described for the models (Figs 4 and 5, dotted lines). Thus, the effects on membrane organization are very limited in cholesterol-rich membranes, for example erythrocytes, but apparent ordering effects of the insecticide are observed in cholesterol-poor membranes, for example mitochondria and sarcoplasmic reticulum. The relative effects dependent on cholesterol content are essentially related with insecticide partitioning highly affected by cholesterol (Antunes-Madeira and Madeira, 1989). Similar conclusions have been previously drawn for parathion, malathion and azinphos. Furthermore, the perturbations induced by the organophosphorus compounds in either native membranes or the models follow the sequence: parathion > methylparathion 3 azinphos > > malathion, a sequence that correlates with their effects on membrane-linked functions (Antunes-Madeira and Madeira, 1979 and 1982; Antunes-Madeira et al.,

1981) and with their toxicities (Metcalf, 1971). Since membrane fluidity is a crucial factor in basic membrane mechanisms and, consequently, in cell homoeostasis, the above data suggest that fluidity perturbation may contribute to insecticide toxicity. Concluding

remarks

The present data for methylparathion and previous data for parathion (Antunes-Madeira et al., 1994), malathion (Videira et al., 1994) and azinphos (Videira et al., 1996) suggest that these compounds and the organophosphorus compounds in general, locate along the co-operativity region and would extend to the bilayer surface by their polar phosphate group. Such a localization results in the perturbation of electrostatic and hydrophobic forces among membrane components and, consequently, perturbation of normal membrane fluidity along all the thickness of the bilayer. The acute toxicity of organophosphorus compounds is primarily related with inhibition of acetylcholinesterase (Eto, 1974). On the other hand, the perturbations of lipid-lipid and lipid-protein interactions and related alterations of membrane permeability and enzyme activities may be associated with chronic toxicity. Furthermore, it is clearly established that the activity of integral membrane proteins depends on the physicochemical properties of boundary domains (Lee, 1991). Therefore, the present data for methylparathion and previous data for parathion, azinphos and malathion suggest that physical perturbations at the boundary lipids may also affect acetylcholinesterase activity and may concur for the acute toxicity. Acknowledgements-This work was supported by grants Praxis/Z/Z. 1/BIO/ll56/94. Praxis/2/2.1/SAU/1400/95 and EC n&work ERBCHRXCT-946666. ‘Virginia i. C. F. Lopes is a recipient of a grant from JNICT (PBICT/BIO/ 1997195). REFERENCES

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