Partition coefficient of organophosphorus insecticides evaluated by fluorescence quenching

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A C T A U N I V E R S I T A T I S L O D Z I E N S I S

FOLIA BIOCHIMICA ET BIOPHYSICA 11, 1996

Janusz Błasiak

PARTITION COEFFICIENT OF ORGANOPHOSPHORUS INSECTICIDES EVALUATED BY FLUORESCENCE QUENCHING

The partition coefficient of two organophosphorus insecticides: malathion and methylparathion was evaluated in membranes of various cholesterol content. Fluores cence quenching studies revealed that methylparathion bonded strongly (in a do se-dependent manner) to liposomes formed from phosphatidylcholine. The binding of the insecticide was weaker when the vesicles were prepared from equimolar phosphatidylcholinexholesterol mixtures. Quenching was more pronounced when a fluorescence probe localized in deep core of the phospholipid (perylene) was used than when a probe binding to more outer region of the membrane (ANS) was employed. The results indicate that binding of some insecticides to membrane can be modulated by cholesterol content.

1. INTRODUCTION

O rganophosphorus insecticides are known as powerful inhibitors o f acetylcholinesterase [1]. T he consequences o f this effect are increased excitability, convulsions and m uscular paralysis which take place before death o f poisoned animals [2-6], However, there are some symptom s of organophosphorus insecticide toxicity, such as memory and visual disturbances, the immune system alternation which cannot be related to the inhibition o f acetylcholinesterase [2, 6, 7]. The exact molecular mechanisms of insecticide action are poorly understood in general.

D ue to their lipophilic character insecticides can accum ulate in the lipid rich biomembranes which can be taken into account as possible target and sites o f immediate and chronic insecticide action.

T o fu rther understan d the m em brane m echanism s affected by the insecticides, it is essential to relate the m em brane effects to the actual membrane concentrations, i.e. to the partion coefficient. Insecticide partitioning is affected by m ultiple param eters, namely tem perature, cholesterol content, fluidity, m em brane geometry and the intrinsic m olecular properties o f the com pounds [8-12],

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In the present work the partition coefficient o f m alathion (0,0-dimethyl S - (1,2-dicarboxyethyl) phosphorodithioate) and methylparathion (0,0-dimethyl 0 - (p-nitrophenyl) phosphorothioate) has been evaluated for m em branes of different cholesterol content.

Fragm ented pig erythrocyte m em branes were obtained according to the m ethod of A u [13]. Liposomes were formed either of pure phosphatidylocholi- ne or from equimoral amounts of phosphatidylcholine and cholesterol. A thin film of desired lipid was hydrated with 50 mM KC1, 10 mM Tris-m aleate buffer (pH 8.0) and dispersed under a nitrogen atmosphere shaking by hand at a room temperature. The liposomes was dispersed by vortexing and sonication. The final concentration of lipid was 300 ¿¿M in each kind o f preparation.

Protein was estimated by the protein-dye binding m ethod o f B r a d f o r d [14] with bovine serum album in as the standard.

Steady-state fluorescence m easurem ents were m ade with a Perkin-Elm er LS-5B luminescence spectrometer. The theory of the quenching of fluorescence was applied to determine qualitatively the affinity o f organophosphorus insecticides to m em branes. The quenching of an excited fluorophore in free solution is expected to follow the Stern-Volmer equation

where I and I D are the fluorescence intensities in the presence and absence of quencher, respectively, K sv is the Stern-Volmer quenching constant and [Q] is the concentration of quencher. The distribution o f a quenching molecule between aqueous (A) and lipid (L) phase m ay be described by a partition coefficient K p = [Q J /[Q J [15]. Thus the Stern-Volmer equation becomes

2. MATERIALS AND METHODS

— - 1 = K sv [Q] (1) I

(

2

)

I Since Vt[Qt ] = Va[ Q J + L J Q J (3) where V denotes volume and subscript T referees to either total volume or total concentration, equation (2) becomes

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I. I

O K svK pVT[QT]

VA + VLK p

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The first derivative o f I 0/I as a function o f K p is always positive, so the higher value o f K p, the higher coefficient I0/I. Thus, the value o f 1^1 m ay be considered as a measure o f the affinity o f a quencher to m em brane. The insecticides malathion and methylparathion were assumed to be quenchers. Tw o fluorescent probes were used, perylene for very hydrophobic region of membrane and ANS (8-anilino-l-napthalene sulfonate) for more hydrophilic dom ains. Perylene, at a final concentration o f 2.5 /iM , was excited at 410 nm and emission was m easured at 468 nm. F or ANS at 25 /¿M, those param eters were 366 and 480 nm, respectively. Both excitation and emission slits were 5 nm in either case. One-way analysis of variance was used to analyze obtained results. The differences between m eans were com pared by m eans o f the Scheffe’s m ultiple com parison test [16].

T hree kinds o f preparation were used: fragments of the pig erythrocyte m em brane, liposomes formed from phosphatidylcholine and liposomes from phosphatidylcholine and cholesterol in equim olar ratio. These preparations were labeled with two fluorescent probes: perylene, which binds to the hydrophobic core of lipids and ANS, which localized in m ore hydrophilic region of the membrane.

M alathion did not change the ratio Io/I o f any preparation, when either perylene or ANS was used (Figs. 1A and B).

M ethylparation increased the value o f Ic/I for liposomes o f phosp hatidylcholine (PC) and erythrocyte m em brane as m easured w ith the fluorescent probe perylene (Fig. 2A). The changes caused in liposomes from PC and cholesterol (PC + ChE) were not significant. A n increase in the ratio was also observed with ANS-labeled PC liposomes and erythrocyte m em branes (Fig. 2B). There were no significant changes for PC + ChE liposomes. F o r each probe m ethylparathion increased the value I 0/I in a dose-dependent m anner. The changes observed for PC liposomes were m ore pronounced than for erythrocyte membranes.

In general, it follows from the results that m ethylparathion has an ability to quench the fluorescence o f used probes, whereas m alathion either has no such ability or does not bind to m em brane at all. The latter statem ent does not seem to be true in the light o f the results o f A n - t u n e s - M a d e i r a and M a d e i r a [8], who showed that m alathion did bind to liposomes prepared from PC.

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maJathlon concentration [fxM]

malathlon concentration [p.M]

Fig. 1. Quenching of fluorescence of perylene (A) and ANS (B) in the presence of malathion. I0 denotes the fluorescence in the absence of the insecticide, whereas I - in the presence. (O ) - erythrocyte membranes, ( • ) - liposomes o f phosphatidylcholine, (V ) - liposomes from phosphatidylcholine and cholesterol mixed in equimolar ratio. Each point represents the mean

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methylparathion concentration [jiM]

methylparathion concentration [|iM]

Fig. 2. Quenching of fluorescence of perylene (A) and ANS (B) in the presence of methyl parathion. I0 denotes the fluorescence in the absence of the insecticide, whereas I - in the presence. (O ) - erythrocyte membranes, ( • ) - liposomes of phosphatidylcholine, (V ) - liposomes from phosphatidylcholine and cholesterol mixed in equimolar ratio. Each point

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O btained results indicate th at cholesterol can m odify the binding of organophosphorus insecticides to membranes and influence on the interaction of these agents with membranes. The above statem ent is in agreement with the results o f the study o f A n t u n e s - M a d e i r a and M a d e i r a [8, 9], who showed that cholesterol incorporation in m em branes prevents the binding o f parathion to them. They also showed that partition coefficient o f m alathion in egg phosphatidylcholine bilayers decreased linearly with tem perature, over a range (10-40°C) at which the lipid was in a liqu id-crystalline state; addition o f 50 m ol% cholesterol severely decreased partition and practically abolished the tem perature dependence. Partition values in native m em branes decreased sequentially as follows: sarcoplasm ic reticulum, m itochondria, brain microsomes, myelin and erythrocytes; this dependence reflects the relative content of cholesterol.

Protective action o f cholesterol against exposure of m em branes to m alathion and m ethylparathion was also dem onstrated by B ł a s i a k and W a l t e r [17] and B ł a s i a k [18, 19].

Cholesterol action m ay be a result either o f com petition for similar interaction sites or a consequence o f changes in structural organization of phopspholipids. The results obtained in this work seem to indicate the first possibility, but changes in the structure o f the m em brane m ay also be taken into consideration as a possible source of the observed changes.

It follows also from the obtained results that, in the case of m ethyl parathion, the quenching o f fluorescence was m ore effective when a probe was situated in the cooperative region (ANS) than it was in deep core of lipids (perylene). This observation allows one to draw a conclusion that m ethylparathion preferentially binds to the outer region of the bilayer.

There is a need o f further work to establish clear relationship between insecticides partition and their toxicity.

Acknowledgements

The spectrofluorimetric m easurem ents were conducted in the D epartm ent of Biophysics, University of Lodz. The author thanks Professor M. Bryszewska for her help in this research. Technical assistance o f M. Spychała and A. Łuczyńska is appreciated.

4. REFERENCES

[1] M a t s u m a r a F. (1985), Toxicology o f Insecticides, Plenum Press, New York, p. 161-172. [2] K u h r R. J., D o r o u g h H. W. (1976), Carbamate Insecticides: Chemistry, Biochemistry and

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[3] O h k a w a H. (1982), [in:] Insecticide Mode o f Action, ( C o a t s J. R., ed.), Academic Press, New York, p. 163-185.

[4] D o h e r t y J. D. (1979), Pharmacol. Ther., 7, 123.

[5] R e v z i n A. M. (1983), [in:] Pesticide Chemistry: Human Welfare and the Environment, ( M a t s u n a k a S., H u s t o n D. H. and M u r p h y S. D., eds), vol. 3, Pergamon Press, Oxford, p. 419-424.

[6] D e v e n s B. H., G r a y s o n M. H., I m a m u r a T., R o d g e r s K. E. (1985), Pestic. Biochem. Physiol., 24, 251.

[7] R o d g e r s K. E., G r a y s o n M. H., I m a m u r a T., D e v e n s B. H. (1985), Pestic. Biochem. Physiol., 24, 260.

[8] A n t u n e s - M a d e i r a M. C., M a d e i r a V. M. C. (1987), Biochim. Biophys. Acta, 901, 61.

[9] A n t u n e s - M a d e i r a M. C„ M a d e i r a V. M. C. (1984), Biochim. Biophys. Acta, 778, 49.

[12] A n t u n e s - M a d e i r a M. C., M a d e i r a V. M. C. (1989), Pest. Sd., 26, 167. [13] A u K. S. (1987), Biochim. Biophys. Acta, 905, 273.

[14] B r a d f o r d M. M. (1976), Anal. Biochem., 72, 248.

[15] J o n e s O. T., L e e A. G. (1985), Biochim. Biophys. Acta, 812, 731.

[16] Z a r J. H. (1974), Biostatistical Analysis, Prentice Hall, New Jersey, p. 159-161. [17] B ł a s i a k J., W a l t e r Z. (1992), Acta Biochim. Polon., 39, 49.

[18] B ł a s i a k J. (1993), Pestic. Biochem. Physiol., 45, 72. [19] B ł a s i a k J. (1993), Acta Biochim. Polon., 40, 35.

Came in editorial office Department of Molecular

„Folia biochimica et biophysica” Genetics, University of Łódź

22.11.1993 Poland

Janusz Błasiak

OCENA WSPÓŁCZYNNIKA PODZIAŁU INSEKTYCYDÓW FOSFOROORGANICZNYCH METODĄ TŁUMIENIA FLUORESCENCJI

Badając tłumienie fluorescenęji przez malation i metyloparation stwierdzono, że pierwszy związek nie tłumi iluorescencji żadnego ze stosowanych w badaniach znaczników (perylen i ANS). Opisując tłumienie za pomocą równania Stema-Volmera stwierdzono, że metyloparation wiązał się z liposomami utworzonymi z fasfatydylocholiny. Wiązanie to wykazywało silną zależność od stężenia insektycydu. Wiązanie insektycydu było znacznie słabsze, gdy liposomy tworzone były z różnych ilośd molowych fosfatydylocholiny i cholesterolu. Tłumienie iluorescencji było wyraźniejsze, gdy stosowano znacznik wiążący się z rdzeniem dwuwarstwy lipidowej (perylen), niż wówczas, gdy stosowano znacznik wiążący się z bardziej zewnętrznym obszarem dwuwarstwy (ANS). Uzyskane wyniki wskazują, że wiązanie niektórych insektycydów fosforo organicznych może być zmieniane przez cholesterol.