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Interaction of triclosan with eukaryotic membrane lipids. Henning Lygre1, Grete Moe1, Rita. Skålevik1, Holm Holmsen2. 1Department of Odontology – Oral.
Copyright  Eur J Oral Sci 2003

Eur J Oral Sci 2003; 111: 216–222 Printed in UK. All rights reserved

European Journal of Oral Sciences ISSN 0909-8836

Interaction of triclosan with eukaryotic membrane lipids

Henning Lygre1, Grete Moe1, Rita Sklevik1, Holm Holmsen2 1

Department of Odontology – Oral Pharmacology, and 2Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, Norway

Lygre H, Moe G, Ska˚levik R, Holmsen H. Interaction of triclosan with eukaryotic membrane lipids. Eur J Oral Sci 2003; 111: 216–222.  Eur J Oral Sci, 2003 The possibility that triclosan and PVM/MA (polyvinylmethyl ether/maleic acid) copolymer, additives to dentrifrices, could interact with eukaryotic membrane lipids was studied by two methods: first, by determining the pressure/molecular area isotherms at 37C of glycerophospholipid monolayers, using the Langmuir technique; and second, by phase-transition parameters in liposomes of the same lipids, using differential scanning calorimetry (DSC). Triclosan interacted, in a concentrationindependent manner, with monolayers of saturated phosphatidylcholines (PC; i.e. markers of the outer membrane leaflet of eukaryotic cells). Triclosan and PVM/MA copolymer mixtures were shown to clearly interact in a concentration-dependent manner with PC. Triclosan was found to interact with liposomes of saturated and unsaturated phosphatidylcholines and phosphatidylserines (PS; i.e. markers of the inner membrane leaflet of eukaryotic cells), and saturated ethanolamines (PE; i.e. markers of the inner membrane leaflet of eukaryotic cells), resulting in a decrease of the lipid melting temperature (Tm). PVM/MA copolymer changed the Tm of PS, PC, and PE in different manners. By adding PVM/MA or triclosan–PVM/MA copolymer mixtures to 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS) no lipid transitions were detected. A biphasic change of the PC transition temperature resulted when triclosan or triclosan PVM/MA copolymer mixtures were added, indicating domain formation and change of the lipid polymorphism.

Triclosan (C12H7Cl3O2), 2,4,4¢-trichloro-2¢-hydroxy-diphenyl-ether, a bisphenol with molecular wt of 289.53 (Fig. 1), exhibits pharmacological effects towards prokaryotic and eukaryotic cells. Based on methods for detecting molecular interactions several aspects of triclosan activity in prokaryotic cells has been revealed (1). Triclosan has demonstrated broad-spectrum antimicrobial activity against prokaryotes (2). It is active at very low concentrations, and long-lasting activity has been demonstrated. Triclosan acts mainly by inhibiting fatty acid biosynthesis (1), through blocking lipid biosynthesis by specifically inhibiting the enzyme enoyl-acyl carrier protein reductase (ENR) (3). An electron density map has revealed the mode of binding of triclosan adjacent to the nicotinamide ring of the nucleotide cofactor in the enzyme’s active site (1). The phenol ring of triclosan forms a face-to-face interaction with the nicotinamide ring, allowing extensive p–p stacking interactions. Principally, molecular interactions encompass van der Waals interactions, hydrogen bonds and electrostatic interactions. The van der Waals contacts are made by both rings of the triclosan with residues lining the active site and the substrate-binding pocket of ENR, and with the nucleotide cofactor. Hydrogen bonds are formed by the phenolic hydroxyl group of triclosan with the 2¢-OH of the nicotinamide ribose of the nucleotide, and with the phenolic oxygen of tyrosine-156, which is believed to

Henning Lygre, Department of Odontology – Oral Pharmacology, University of Bergen, Armauer Hansens Hus, N)5021 Bergen, Norway Telefax: +47–55974605 E-mail: [email protected] Key words: calorimetry; cell membrane; lipids; triclosan Accepted for publication January 2003

function as a proton donor during the catalytic cycle of ENR (4). In eukaryotes it has been suggested that the primary effects from triclosan are on the membranes (5). Biological membranes are complex and well-organized multimolecular assemblies, composed of a lipid bilayer and a variety of proteins. Eukaryotic cells have numerous membrane systems, the best characterized being endoplasmic reticulum, Golgi membranes, plasma, mitochondrial, lysosomal, and nuclear membranes (6). The major classes of lipids in biological membranes are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), sphingomyelin, diphosphatidylglycerol (DPG), and phosphatidylinositol (PI). In eukaryotic membranes the glycerol-based phospholipids are predominant, particularly PC, PE, PS, and PI. Glycerolipids make up the essential milieu of cellular membranes, and act as a barrier for the entry of compounds into cells (6). With the aim of studying interactions between a given effector and cell membranes, several approaches are possible. One can work with living cells and observe what happens in situ, or one can isolate cellular membranes. Natural membranes are very complex entities with a great variety of lipids and proteins. If one is interested in specific aspects of a given biological phenomenon occurring at membrane level, the best choice is to use membrane models (7). A lipid monolayer of glycerophospholipids spread at the air–water interface

Interaction of triclosan with lipids

Wilhelmy Cl

plate

Cl

O Cl

217

OH

Fig. 1. Structural formula of triclosan.

has been shown to represent a simple model of a single membrane leaflet of cells (8). The aim of the present study was to reveal potential molecular interactions between triclosan and PVM/MA copolymer, and different kinds of glycerophospholipids found in eukaryotic cells. Model membranes made of lipids were used in two manners, as monolayers and as liposomes (i.e. concentric bilayers).

barrier

mono

layer

barrier

subphase Trough Fig. 2. Schematic representation of the device for the monolayer technique.

Material and methods Chemicals

the film. In recent versions, the trough and the barriers are often made of Teflon (Fig. 2). The whole device rests on a antivibration plate and is placed inside a thermostatically controlled box (7). Our experiments were performed using a M 1000 Minitrough (75 · 364 · 5 mm), made of Teflon (KSV instruments, Helsinki, Finland). All experiments were performed at 37C with two movable barriers, constant speed (5 mm min)1) on a Ringer solution (0.15 m NaCl, 5.6 mm KCl, 1.7 mm CaCl, pH 7.4). The surface pressure was measured using the Wilhelmy plate method. The lipids were applied in droplets on either side of the Wilhelmy plate using a Hamilton pipette, and the solvent allowed to evaporate before compression of the monolayer. The lipids used in these experiments were dissolved in chloroform to a concentration of 1 mg ml)1. Control samples of 20 ll were used. Samples containing lipids and triclosan (25 lm, 50 lm, 75 lm and 100 lm) were mixed 1 : 2 prior to application. Ringer solution and Ringer with 30 lg/ml PVM/MA copolymer were used as a subphase solution. Samples of 40 ll were used. All experiments were repeated five times. The median value was chosen for statistics.

The term C18 : 0 denotes a fatty acid with 18 carbon atoms and with no double bonds, whereas 18 : 2 signifies that there are two double bonds. Fatty acids in biological systems usually contain an even number of carbon atoms, typically between 14 and 24. The 16- and 18-carbon fatty acids are the most common. Fatty acids have different physiological roles, and they are building blocks of glycerophospholipids. These molecules are important components of biological membranes, and are composed of a polar region and a hydrophobic region. The latter, containing two long hydrocarbon tails a saturated acyl in sn-1 position and an unsaturated acyl group in the sn-2 position, are usually found in biological systems. 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS, 16 : 0/18 : 1), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS, 18 : 0/18 : 1), 1-stearoyl-2-oleoyl-sn-glycero-3phosphocholine (SOPC, 18 : 0/18 : 1), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 16 : 0/16 : 0), were purchased from Sigma (St Louis, MO, USA). 1,2-Distearoyl-sn-glycero3-phosphocholine (DSPC), 18 : 0/18 : 0), 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine (DPPE), 16 : 0/16 : 0), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS, 18 : 0/ 18 : 0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Triclosan (C12H7Cl13O2), 2,4,4¢-trichloro-2¢hydroxy-diphenyl-ether (Irgacare DP 300) was purchased from Ciba Specialty Chemicals (Basle, Switzerland). The 2% PVM/MA copolymer (C4H4O4.C3H6O)n, MW 216.000 (Gantrez S-97 Powder) was purchased from International Speciality Products (Guildford, UK).

Liposomes were made of glycerophospholipids dissolved in chloroform. Triclosan (25 lm) was added, lyophilized and allowed to dry overnight. Samples of dry powder were suspended in 1.5 ml Ringer solution and Ringer solution with 30 lg/ml PVM/MA copolymer to give a 4 mm lipid suspension, containing multilamellar liposomes. Unilamellar vesicles were obtained by freeze–thawing seven times.

Monolayer technique

Differential scanning calorimetry

In 1917, Irving Langmuir introduced the experimental and theoretical modern concepts on monolayers. The main parameters which characterize the film state of a given substance spread on a aqueous subphase are the temperature, T, the surface pressure, p, the surface area and the number of molecules, these two last parameters being expressed as area per molecule, A. Therefore, at constant T, an equation of state of the film has the general form:

Differential scanning calorimetry (DSC) measures the heat absorbed (or released) by a sample as it undergoes an endothermic (or exothermic) phase transition. Three parameters of interest in such traces are (Table 1): (a) the area under the transition peak, which is proportional to the enthalpy of the transition (DH); (b) the width of the transition, which gives a measure of the ÔcooperativetyÕ of the transition (TC1/2); and (c) the transition temperature (Tm) itself. The enthalpy of the transition reflects the energy required to melt the acyl chains, whereas cooperativity reflects the number of molecules that indergo a transition simultaneously.

p ¼ pðAÞT The surface pressure-area (p-A) isotherm of a monolayer constitutes the essential characterization of the properties of

Liposome preparation

Lygre et al.

Results

Differential scanning calorimetry values of transition temperature (Tm), half-width temperature (TC1/2), and enthalpy-change (DH) for the liposomes studied Sample POPS Control (16 : 0/18 : 1) Triclosan Copolymer Triclosan + copolymer SOPS Control (18 : 0/18 : 1) Triclosan Copolymer Triclosan + copolymer SOPC Control (18 : 0/18 : 1) Triclosan Copolymer Triclosan + copolymer DSPC Control (18 : 0/18 : 0) Triclosan Copolymer Triclosan + copolymer DPPC Control (16 : 0/16 : 0) Triclosan Copolymer Triclosan + copolymer DPPE Control (16 : 0/16 : 0) Triclosan Copolymer Triclosan + copolymer DSPS Control (18 : 0/18 : 0) Triclosan Copolymer Triclosan + copolymer

Tm (C)

TC1/2 (C)

DH (kJ/mol)

15.76 12.80 17.69 13.67

2.63 5.76 4.13 5.51

2.25 2.23 0.88 1.04

26.38 23.71 – –

4.75 5.00 – –

1.37 2.48 – –

1.25 – 1.37 –

6.20 2.33 6.13 4.26

5.92 3.29/5.51 6.14 3.44/5.65 51.78 50.48 55.02 53.11

2.50 4.00 0.62 2.63

22.08 24.16 11.97 16.54

41.66 41.60 41.83 40.62/42.92

0.50 0.74 0.62 2.11/5

3.69 1.46 7.10 6.49

67.06 66.95 64.42 67.08

0.75 1.25 0.63 0.88

9.54 3.15 7.55 6.36

64.32 64.18 42.74 40.46

0.63 1.00 6.38 5.14

7.38 0.33 5.57 3.31

Two numbers indicate biphasic compounds. POPS, 1-palmitoyl2-oleyl-sn-glycero-3-phosphoserine; SOPS, 1-stearoyl-2-oleoylsn-glycero-3-phosphoserine; SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine; DSPC, 1,2-distearoyl-sn-glycero-3phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DSPS, 1,2-distearoyl-sn-glycero-3-phosphoserine.

Measurements were performed on a Microcal VP-DSC differential scanning calorimeter (MicroCal, Northampton, MA, USA) with cell volumes of 0.5 ml at the indicated scan rates. A Ringer solution was used in all experiments. All samples were degassed and the calorimetric cells were kept under an excess pressure of 30 kPa to prevent degassing during the scan. A scanning rate of 1.5C min)1 was used for all samples. The original scans were processed by subtraction of the buffer baseline and, further correction by defining a progress baseline from the pre- to the posttransition regions using the origin software (Microcal) provided with the instrument. Triclosan was applied in a concentration of 25 lm. Statistics The median, the non-parametric measure of central tendency, was used.

Monolayer technique

Both DPPC and DSPC were used for the monolayer technique (Figs 3 and 4). Triclosan interacted with monolayers of DSPC in a concentration-independent manner and decreased the molecular area of the membrane lipids. When a mixture of triclosan and PVM/MA copolymer was added, the effect of triclosan on the molecular area of DSPC was found to be concentrationdependent (Fig. 4). Differential scanning calorimetry

Both triclosan, copolymer and triclosan combined with copolymer had marked effects on the thermograms 85 DPPC in Ringer with 30 ug/ml copolymer

Difference in molecular area MMA

Table 1

DPPC in Ringer

80 75 70 65 60 55 50 0

20

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Fig. 3. The effect of triclosan on 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) monolayers. Results are presented as the difference in molecular area between control and added samples, and different concentrations of triclosan. The figure represents the median value of five repetitions. Minimum and maximum values are indicated. 80

Difference in molecular area MMA

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DSPC in Ringer with 30ug/ml copolymer DSPC in Ringer

75

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50 0

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Triclosan (µM)

Fig. 4. The effect of triclosan on 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) monolayers. Results are presented as the difference in molecular area between control and added samples, and different concentrations of triclosan. The figure represents the median value of five repetitions. Minimum and maximum values are indicated.

Interaction of triclosan with lipids 0.020

Control Triclosan Copolymer Triclosan+Copolymer

0.015

Cp (cal ˚C–1)

(Figs 5–11) and the transition temperatures (Tm), transition interval (TC1/2) and the corresponding enthalpies (DH) (Table 1) of the phospholipid liposomes studied. A typical thermogram of pure 16 : 0/16 : 0-PC (DPPC) is given in Fig. 5 (control), showing a main transition at 41.66C and a pretransition at 34.65C. Incorporation of triclosan in the liposomes drastically altered the thermogram by lowering Tm, causing disappearance of the pretransition and inducing another transition above Tm for pure DPPC; both transitions have higher transition intervals than pure DPPC. The copolymer lowered Tm only slightly, gave no secondary

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0.010

0.005

0.000 0.012

Control Triclosan Copolymer Triclosan+Copolymer

0.010

60

70

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Temperature (˚C)

Fig. 7. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and DPPE with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) copolymer and a mixture of triclosan and PVM/MA copolymer.

0.008

Cp (cal ˚C–1)

65

0.006

0.004

0.002

0.008

0.000 Control Triclosan Copolymer Triclosan+Copolymer

0.006

–0.002 35

40

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Temperature (˚C)

Fig. 5. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) copolymer and a mixture of triclosan and PVM/MA copolymer.

Cp (cal ˚C–1)

30

0.004

0.002

0.000

Control Triclosan Copolymer Triclosan+Copolymer

0.020

Cp (cal ˚C–1)

0.015

–0.002 20

40

60

80

Temperature (˚C)

Fig. 8. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1,2-distearoyl-sn-glycero3-phosphoserine (DSPS) and DSPS with added triclosan, PVM/ MA (polyvinylmethyl ether/maleic acid) copolymer and a mixture of triclosan and PVM/MA copolymer.

0.010

0.005

0.000

–0.005 30

40

50

60

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Temperature (˚C)

Fig. 6. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1,2-distearoyl-sn-glycero3-phosphocholine (DSPC) and DSPC with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) copolymer and a mixture of triclosan and PVM/MA copolymer.

transitions, but drastically reduced DH. With both copolymer and triclosan present in the DPPC liposomes, a thermogram closely resembling that of pure DPPC was produced, although with higher DH (Fig. 5). By increasing the saturated acyls in PC by two carbons each, i.e. 18 : 0/18 : 0-PC (DSPC), the thermogram of pure DSPC was similar to that of DPPC, although with higher Tm and pretransition temperature and greater transition intervals and DH (Fig. 6, control; Table 1). Triclosan lowered Tm, copolymer increased it, while triclosan in combination with copolymer produced a Tm which was intermediate between that of triclosan and copolymer (Fig. 6).

Lygre et al. 0.0002 Control Triclosan Triclosan + Copolymer Copolymer

0.0000 –0.0002

Cp (cal ˚C–1)

Keeping the two acyl groups saturated and changing the head group from phosphocholine to phosphoethanolamine (eliminating three N-methyl groups), as in DPPE, produced a similar control thermogram as DPPC but with distinctly higher Tm and pretransition temperature (Fig. 7, control; Table 1). The effects of triclosan and copolymer on the thermograms of DPPE were very different from on those of DPPC. With DPPE, triclosan had only a slight effect on Tm, while copolymer alone drastically lowered this parameter, and triclosan combined with polymer gave little change in Tm (Fig. 7). Triclosan with or without copolymer lowered DH (Fig. 7, Table 1). Changing the head-group to phosphoserine, but keeping the acyls saturated and identical, as in DSPS, gave a thermogram of basically the same appearance as those with phosphocholine and phosphoethanolamine, except for an additional transition above the 64.32C Tm (Fig. 8, control). The effects of triclosan and copolymer separately and in combination produced quite different alterations in the thermogram of this saturated, acidic PS species compared with the saturated neutral phospholipids discussed above. Triclosan lowered the DH drastically without changing Tm in the DSPS liposomes (Fig. 8, Table 1) and the copolymer separately and in combination with triclosan lowered Tm drastically, as well as lowering DH (Fig. 8, Table 1). It is evident that both triclosan and copolymer, separately or in combination, alter both Tm and DH in the thermograms of saturated glycerophospholipids, and that the alterations are dependent on both the acyl chain length and, particularly, whether the head groups are neutral or acidic. Thus, the additions alter the physicochemical characteristics of the liposomes, although whether these are due to domain formations and/or changes in phospholipid packing cannot be probed with thermograms alone.

–0.0004 –0.0006 –0.0008 –0.0010 –0.0012 –0.0014 0

20

40

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Temperature (˚C)

Fig. 10. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1-palmitoyl-2oleyl-sn-glycero-3-phosphoserine (POPS) and POPS with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) and a mixture of triclosan and PVM/MA copolymer.

0.0000

Control Triclosan Copolymer Triclosan+Copolymer

–0.0002

Cp (cal ˚C–1)

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–0.0004

–0.0006

–0.0008

–0.0010

–0.0012

Cp (cal ˚C–1)

0

Control Triclosan Copolymer Copolymer+Triclosan

0.004

10

20

30

40

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Temperature (˚C)

Fig. 11. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1-stearoyl-2oleoyl-sn-glycero-3-phosphoserine (SOPS) and SOPS with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) and a mixture of triclosan and PVM/MA copolymer.

0.002

0.000

–0.002

0

5

10

15

20

Temperature (˚C)

Fig. 9. Differential scanning calorimetry thermograms obtained from aqueous liposome suspensions of 1-stearoyl-2-oleoyl-snglycero-3-phosphocholine (SOPC) and SOPC with added triclosan, PVM/MA (polyvinylmethyl ether/maleic acid) copolymer and a mixture of triclosan and PVM/MA copolymer.

The neutral 18 : 0/18 : 1-PC (SOPC) had a low Tm (5.92C), and any possible pretransition phase could not be measured with the calorimeter used (Fig. 9, control; Table 1). With triclosan alone or with copolymer the thermogram became biphasic with an additional transition around 3C and a slight lowering in the original Tm, while copolymer alone had almost no effect on the thermogram (Fig. 9). The corresponding acidic phospholipids, POPS (Fig. 10, control) and SOPS (Fig. 11, control), gave biphasic thermograms, similar to the di-isoacyl PS DSPS (Fig. 8, control). Inclusion of triclosan with or without copolymer had drastic effects

Interaction of triclosan with lipids

on the thermograms. With both POPS and SOPS, triclosan lowered Tm, while the copolymer increased Tm in POPS (Fig. 10, Table 1), but caused all phase transitions to disappear completely with SOPS, also in the presence of triclosan (Fig. 11). Copolymer alone increased Tm in POPS (Fig. 10). Thus, both triclosan and copolymer, separately or in combination, had marked effects on the physicochemical characteristics of naturally occurring glycerophospholipids. These effects were dependent on the head group as well as the length of the saturated and unsaturated acyls studied.

Discussion Although much information is available on the in vivo pharmacology and toxicology of triclosan, studies on the effects at the eukaryotic cellular level are sparse. Damage to the integrity of the plasma membrane has been confirmed, as assessed by the leakage of lactic acid dehydrogenase from human gingival epithelial cells (9). A prerequisite for cellular damage to happen is that an interaction occurs between the foreign compound and the biological system. Cellular membranes are complex systems, with functions that reach far beyond the physical separation between the cell and its surrounding or the delimitation of the cell’s different compartments. Interactions between the cell and its environment is governed by processes taking place at the plasma membrane. Lipids are diverse in their forms and show great variations between different membranes. The apparent specialization of the lipids in different tissues, plasma membranes and organelles might be of importance for the biological function of different cells. For example, drug delivery to a cell may occur via different processes at the plasma membrane of different cells. Investigations of molecular interactions at membranes still represent a major challenge. Triclosan has been found to destabilize structures which comprise the functional integrity of cell membranes (10). Interactions through lipid components in the cell membrane may result in a disturbance of signal transduction mechanisms (11). In our study, the potency of triclosan to associate with, and to act on the physical properties of phospholipid membranes, representative of those found in eukaryotes, was demonstrated. Triclosan has been shown to be targeting fatty acid synthesis in prokaryotes (3). Difference Fourier analysis has been used to demonstrate a tight binding between the enzyme ENR and triclosan. By using the monolayer technique, it is possible to explore a potential relationship between the surface pressure and the molecular area of membrane lipids (Fig. 2). In our study, PC membranes were used to model the eukaryotic outer membrane cell leaflet, and PS and PE were used as models of the inner membrane leaflet. A saliva-like Ringer solution was used as a subphase. The acyl chains of fatty acids were altered, with regard to chain length and degree of saturation, in order to determine the importance of the acyl chains for possible interactions. Unsaturated and

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saturated acyl chains were present in the sn-2-position and sn-1-position, respectively, just as in naturally occurring phospholipids. Adding a compound at low concentration in a monolayer, it is assumed to affect the surface pressure exclusively by its interaction with lipid molecules (12). Accordingly, interactions of triclosan or triclosan–PMV/MA copolymer and membrane lipids may be demonstrated as an altered relationship between surface pressure and molecular area (Figs 3 and 4). In eukaryotic cell membranes the bilayer equivalence pressure has been reported to be 30–35 mN m)1 (12). Thus, we have calculated the difference in corresponding molecular area between glycerophospholipid monolayer, with triclosan and triclosan-PMV/MA copolymer at this level (Figs 3 and 4). We have used concentrations between 25 lm and 100 lm of triclosan. Higher amounts than 100 lm results in damage to the integrity of the plasma membrane, as assessed by the leakage of lactic acid dehydrogenase (LDH) (9). However, the molecular weight of LDH is 140 000, implying that membranes must be grossly affected. The fluidity of membranes depends on the nature of the acyl chain region comprising the hydrophobic domain of most membrane lipids. Membrane lipids can exist in a frozen gel state or a fluid liquid–crystalline state, depending on temperature. Transitions between the gel and liquid-crystalline phases can be monitored by a variety of techniques, including nuclear magnetic resonance (NMR), electron spin resonance, fluorescence and (DSC). When the fatty acyl chains of lipid molecules in bilayer membranes exist in an ordered, rigid state, all of the C-C bonds have a trans conformation, whereas in the disordered state, some are in the gauche conformation. The transition from the rigid (all trans) to the fluid (partly gauche) state occurs rather abruptly as the temperature is raised above Tm, the melting temperature (Table 1). The small peak (the pretransition) (Figs 6 and 7) comes from a change in tilt of fatty acyl chains with respect to the bilayer plane (13) and represents a small endothermic reorganization in the packing of the gelstate lipid molecules prior to melting (6). The major peaks (Figs 5–11) arise from a phase transition in which crystalline fatty acyl chains become disordered because of the introduction of kinks (13). Phosphatidyl choline and serine bilayers undergo a phase transition when heated, as detected here by DSC, which measures the rate of uptake of heat on warming a sample (Figs 5–11). We have used DSC in order to characterize the influence of triclosan or triclosan–PVM/MA copolymer on the thermotropic properties of the phospholipids. Adding a foreign molecule to a phospholipid system would normally be expected to change the transition temperature of the phospholipid if both molecules are miscible (14). For all the tested phospholipids, except SOPC (Fig. 9), we observed a broadening of the transition peak and a shift of the Tm to lower temperatures, when triclosan was added (Figs 5–8, 10 and 11). Triclosan perturbs the cooperative behavior of the phospholipids. This could be explained by a molecular interaction between the phospholipid acyl chains and the aromatic rings of the triclosan molecule. These findings are in

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accord with the effects of abietic acid on DPPC (14). Abietic acid is a major compound of the oleoresin synthesized by many conifers, and constitutes a major class of environmental toxic compounds with potential health hazards to animal, human and plant life. When triclosan was added to SOPC (Fig. 9), two peaks appeared in the thermograms. This could be due to lipid polymorphism. The biological significance of lipid polymorphism appears in membrane fusion processes (15) or in mitochondrial structures (16). Two peaks in the thermograms indicate that incorporation of triclosan with SOPC resulted in two transitions. Accordingly, two different lipid structures are made, bilayer and a hexagonal HII phase. This is in accord with the findings of Aranda & Villalain (14) for abietic acid. Two peaks in the thermograms also resulted from the incorporation of triclosan–PVM/PA copolymer into SOPC and DPPC (Figs 5 and 9). Lipids with small head groups (i.e. choline) tend to form the inverted hexagonal phase HII (15). When PVM/PA-copolymer or triclosan–PVM/PAcopolymer was added to SOPS (Fig. 11) no transitions were detected and, consequently, the lipids remain in a rigid phase. Adding of PVM/MA resulted in an increased melting temperature in most of the lipids. However, when added to DSPS (Fig. 8) a dramatic decrease in lipid melting temperature resulted, suggesting a marked increase in membrane fluidity. In summary, the main result of this study is that triclosan and triclosan–PVM/MA copolymer are able to be incorporated in model membrane phospholipids using lipid components that are known to exist in eukaryotic cells. Triclosan also seems to be able to affect lipid polymorphism. The perturbing effects of triclosan on membrane structures suggest that this molecule would alter membrane functions, affecting not only lipids, but also indirectly the proteins of the membrane, the functions of which are highly dependent on membrane structure. Hence, molecular research is necessary to gain insight into the mechanisms whereby these compounds exert effects at the cellular level.

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