Marine Environmental Research 24 (1988) 51-56

Interaction of Xenobiotics with Mercury-adsorbed Phospholipid Monolayers

A. Nelson & N. Auffret

Institute for Marine Environmental Research, Prospect Place, The Hoe, Plymouth, UK

The experimental system of lecithin films adsorbed on mercury electrodes has been described in detail previously.1 One of the applications of this system is to study the interaction of toxic organic substances with lipid monolayers. This study examines the following groups of toxic compounds: polycyclic aromatic hydrocarbons (PAH), DDT and other chlorinated pesticides, and the phenothiazines. The effect of these substances on the monolayer is' measured b)' monitoring the capacitance-potential curves of the adsorbed phospholipid. The response to these compounds is manifested as a change in the potential and height of two capacitance peaks.

PA H penetrate the film without disrupting its fundamental structure. The extent g[ interaction is dependent on the hydrophobicity and aromaticity of the PA H. DD T interacts with the layer depressing the capacitance peaks. The heterocyclic 3-ringed phenothiazines also affect the membrane; the response depends on the N- and ring substituents on these compounds. These findings are part of continuing studies in which the effects of xenobiotics on the monolayer and their biological effects are being compared. In addition, these adsorbed membrane systems have analytical uses in marine chemistry.

In previous papers, the formation and properties of adsorbed phospholipid monolayers on mercury electrodes has been described? Their utility in measuring the interaction ofxenobiotic compounds with membrane systems has also been assessed. Preliminary studies on this aspect have been reported. 2 In this paper, we discuss the interaction of mercury-adsorbed monolayers of dioleoyl lecithin (di-O-PC) with various groups of organic compounds. It.is shown that compounds known to be toxic at the general biomembrane level have a strong effect on this adsorbed lipid membrane

51 Marine Environ. Res. 0141-1136/88/$03"50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

52 A. Nelson, N. Auffret

U vs Ag /AgCI (V)

-0,3

I

- 0 . 9

!

-I,S

I

/-,0

Cd

(~Flcrn 2 )

20

2

\ 0

3

L

Fig. 1. Characteristic differential capacity (Cd)-potential (U) curve for a monolayer ofdi-O- PC on a mercury electrode. Potential regions are indicated: (1) region of capacitance minimum; (2) anodic capacitance increase; (3) reversible capacitance peaks; (4) final - 1.25 V

peak.

system. The lipid membrane can distinguish between different groups of compounds and is sensitive to the hydrophobicity, electronic structure and stereochemical properties of the molecule.

The experimental system in which phospholipid monolayers are adsorbed on to mercury electrodes has been reported in detail before. 1 Figure 1 displays the characteristic capacitance-potential curve for a di-O-PC monolayer. The regions of interest which are affected by interaction with xenobiotics are.indicated. The most significant, reproducible and useful response arises from the effect of organic compounds on the reversible capacitance peaks. These capacitance peaks coincide with sharp changes in permeability of the monolayer to aquated ions as measured by cadmium and zinc voltammetry. 1

In order to show that the interaction of the mercury-adsorbed monolayer with organic xenobiotics has some biological meaning, we have to consolidate evidence from the experiments to establish:

1. The electrode is measuring phospholipid monolayer-organic compound interaction and not organic compound-mercury interaction.

2. The organic compound-phospholipid monolayer interaction is related in some way to the biological activity of the compound.

Interaction of xenobiotics with mercury-adsorbed phospholipid monolayers 53

The experimental technique for testing the effect of the organic compounds on the monolayer is to add a small quantity of the compound dissolved in a suitable solvent (acetone or methanol) to the electrolyte. After a period of stirring which ensures equilibrium between the membrane and the compound, the capacitance-potential curve is recorded. Previous studies have shown that the small amount of solvent (1-10/A) added to the electrolyte has no effect on the monolayer.

The effects of PAH penetration on this membrane system has already been discussed in detail. 2'3 All PAH, including substituted PAH and heterocyclic PAH, tested effect the same kind of response from the membrane. The PAH cause a negative potential shift of the reversible capacitance peaks. The extent of shift is in part dependent on the hydrophobicity as expressed in the log octanol-water partition coefficient (log Kow ). If a series of molecules-- acridine, anthracene and 1-chloranthracene--are considered, they possess almost identical aromaticity but their hydrophobicity increases within the series. The log Kow values are, respectively, 3.254 and 4"545 for the first two compounds. The log Kow of 1-chloranthracene will be further increased by 0.9 due to the presence of the chloro-substituent. 4 In a 2 × 10 -7 mol /dm 3 aqueous solution of these compounds, the order of negative shift of the second capacitance peak is, respectively, 30, 160 and >220mV.

The aromaticity is a limiting factor determining the observed xenobiotic- lipid membrane interaction. Alkanes of similar hydrophobicity to and in the same electrolyte concentrations as the 4-ringed PAH do not effect a response from the membrane. The penetration of the monolayer by PAH is reversible; PAH can be expelled at more negative potentials, leaving the lipid film in its original form. Thus, the PAH do not disrupt the lipid monolayer structure. The reversibility of penetration indicates that the PAH shows no direct interaction with the mercury. If the PAH was interacting with the mercury it would compete with the lipid for the interface and displace the lipid from the mercury surface.

Considering now the chlorinated pesticides, DDT and its congeners have the strongest effect on the adsorbed membrane. The effect of these compounds is to suppress the reversible capacitance peaks (Fig. 2(a)). There is no increase in the capacitance minimum. The lack of increase in the capacitance minimum indicates that these compounds adsorb but do not penetrate the monolayer, since DDT has a higher dielectric constant due to the presence of the chloro-groupings. Adsorption of these compounds on the surface as opposed to penetration is commensurate with their bulky structure and She depression of the capacitance peaks. The monolayer is sensitive to levels of DDT down to 10-9 mol /dm 3 in the electrolyte. The interaction with DDT is irreversible; DDT cannot be expelled from the monolayer system. The chlorinated cyclodiene insecticides (dieldrin, endrin

54 A. Nelson, N. Auffret

Cd

(pF/cm ~ )

[a)

20

0

50 (b)

- 2 1

:/A I I I I

-0,~ -1,0

U vs Ag/AgCI(V)

Fig. 2. Influence of xenobiotics in the electrolyte on the capacitance-potential curves of a di-O-PC monolayer on mercury. (a) DDT in electrolyte, 0-11 #mol/dm3: (1) after 15min stirring; (2) after 60rain stirring. (b) Chlorpromazine in electrolyte: (1) 1.1/~mol/dm3; (2)

2.0 pmol/dm 3.

and heptachlor) and lindane have comparatively little effect on the membrane system. Unlike the PAH, DDT changes the form of the capacity- potential curve and decreases the potential window of the capacity minimum domain. The membrane electrode can distinguish between DDT and its congeners. Methoxychlor, which possesses two methoxy groups rendering it more hydrophilic, has a faster action on the adsorbed membrane. DDT is known to exert its toxic action by acting on the sodium channel protein of the nerve membrane. 6 It is plausible that this lipophilic insecticide partitions into the membrane lipid and binds at the protein-lipid interfacefl The contrasting effect of DDT and the other chlorinated insecticides on black lipid membranes has already been shown. 8 It can be speculated that there is some analogy between the action of DDT on the capacitance peaks which represent a voltage-induced sharp change in aquatic ion permeability and the effect of DDT on sodium channels in the

Interaction of xenobiotics with mercury-adsorbed phospholipid monolayers 55

0

%depression

of f i r s t

capacitance

peak

L.O

80

X

X X

X X • •

X

I I i I I

0 2 4 6 8

Fig. 3. CPZ, PZ concentrat ion ( I Jmol /d2)

Influence of chlorpromazine (X) and promethazine (O) in the electrolyte on the first reversible capacitance peak.

biological membrane. The investigation of these links is the subject of continuing studies.

The cationic phenothiazine neuroleptic drugs, compounds which are recognised to be active at the membrane level, 9'1° have been tested on the lipid-coated electrode. As expected, these compounds have a strong effect on the membrane (Fig. 2(b)). They adsorb on the surface without penetrating; there is no change in the capacity minimum. The interaction is reversible. Thus the compound can be desorbed by transferring the membrane-coated electrode with adsorbate to a clean solution and stirring. Each of these compounds gives a characteristic sensitivity of response in relation to the concentration of the drug in solution. Significantly, chlorpromazine and promethazine, compounds with very well characterised biological activity (chlorpromazine is more active than promethazinel°), exhibit the same differences in activity on the lipid-coated electrode (Fig. 3).

Unsubstituted phenothiazine produces a similar response to PAH (comparable with acridine). However, substitution at the nitrogen atom with cationic side chains alters this response. Additional substituents on the aromatic ring, such as the chlorine atom in chlorpromazine, have a strong effect.

With reference to the uses of this electrode system to marine environmental studies, we define two clear applications:

1. As an analytical detector. Present studies have shown that the response of the membrane system is sufficiently reproducible to enable it to be used to measure PAH in aqueous systems. The detection limit is 2 nmol /dm 3. Only soluble free PAH penetrates the

56 A. Nelson, N. Auffret

.

adsorbed lipid monolayer giving a response. As a result the electrode can be used in organic speciation studies. In toxicological studies. A structure-act ivi ty relationship is being developed concerning the effect of organic c o m p o u n d s on the monolayer . It has been found that hydrophilicity, aromatici ty and steric factors are impor tan t in determining the type and extent of interact ion with the lipid monolayer. In some cases there is a correlat ion between biological activity and interaction with the lipid monolayer as with D D T and the phenothiazine neuroleptic drugs. Studies are cont inuing in which the adsorbed lipid layer is being developed as a detector to predict the toxicity of compounds and to assay toxic activity in environmenta l samples.

R E F E R E N C E S

1. Nelson, A. & Benton, A. J. Electroanal. Chem., 202, 253-70 (1986). 2. Nelson, A. Mar. Environ. Res., 17, 306-9 (1985). 3. Nelson, A. Anal. Chim. Acta, 194, 139-49 (1987). 4. Rekker, R. F. & de Kort, H. M. Eur. J. Med. Chem.--Chimica Therapeutica,

14(6), 479-88 (1979). 5. Mackay, D., Bobra, A., Shin, W. Y. & Yalkousky, S. H. Chemosphere, 9, 701-11

(1980). 6. Vijverberg, H. P. M., van der Zalm, J. M. & van den Bercken, J. Nature, Lond.,

295, 601-3 (1982). 7. Kerkut, G. A. & Gilbert, L. I. (eds). Comprehensive Insect Physiology

Biochemistry and Pharmacology, Pergamon Press, New York, Vol. 12, Insect Control p. 25, 1985.

8. Hilton, B. D. & O'Brien, R. D. Science, 168, 841-3 (1970). 9. Hewlett, E. L. & Pearson, R. D. Science, 230, 1063-4 (1986).

10. Seeman, P. & Weinstein, J. Biochem. Pharmacol., 15, 1737-52 (1966).