Effects of Polychlorinated Pesticides and Their Metabolites on

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B: Biomaterials and Membranes

Effects of Polychlorinated Pesticides and Their Metabolites on Phospholipid Organization in Model Microbial Membranes Aneta Wojcik, Marcin Pawlowski, Pawel Wydro, and Marcin Broniatowski J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08989 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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The Journal of Physical Chemistry

Effects of Polychlorinated Pesticides and their Metabolites on Phospholipid Organization in Model Microbial Membranes

Aneta Wojcik1, Marcin Pawłowski1, Paweł Wydro2, Marcin Broniatowski*1

1Department

of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University,

Gronostajowa 2, 30-387 Krakow, Poland 2Department

of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian

University, Gronostajowa 2, 30-387 Krakow, Poland

* corresponding author Email: [email protected] Tel: +48126862570

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Abstract Polichlorinated pesticides (PP) were classified as persistent organic pollutants because of their toxicity, limited degradability in the environment, bioaugmentation and accumulation in animal tissues. PP accumulate in the environment mainly in the soils and water sediments where they are toxic to the decomposer organisms including soil bacteria and fungi. Therefore, there is an urged need to search for the microorganisms capable of PP biodegradation which could be applied for soil bioremediation. The exact mechanism of PP microbial toxicity is unknown; however, there is evidence that it can be membrane related. To shed light on the interactions of PP with microbial membranes we applied Langmuir monolayers formed by phospholipids as model biomembranes. The model membranes were formed by phospholipids typical to microbial membranes: cardiolipins and phosphatidylglycerols the main components of Gram positive bacteria membranes, phosphatidylcholine typical to fungal membranes as well as phosphatidylethanolamine found in the inner membranes of Gram negative bacteria. For the studies the most ecotoxic PP and their water soluble metabolites were chosen. The monolayers were studied with the application of mutually complementary techniques: Langmuir technique, Grazing Incidence X-ray Diffraction and PM-IRRAS spectroscopy. It turned out that the cyclodiene PP are more membrane active than monocyclic PP and that the possibility of their incorporation is strictly related to the phospholipid structure. The membranes prepared of cardiolipin turned out to be especially resistant to PP incorporation. Regarding the metabolites pentachlorophenol turned out to be especially structure breaking, affecting the molecular organization of all the investigated phospholipids.


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I.

Introduction

Polychlorinated pesticides (PP) were widely applied during about three decades in the fifties, sixties and seventies of the XX century.

1

Their history started in 1939 when Hans Muller

discovered the insecticide activity of DDT which was later rewarded by the Nobel Prize in 1948.

2

PP can be divided on two vast groups: cyclodiene pesticides produced in the Diels-

Alder reaction from the common precursor hexachlorocyclopentadiene and polychlorinated aromatics as hexachlorobenzene, the congeners of polychlorinated naphthalenes and different isomers of hexachlorocyclohexane produced in the radical chlorination of benzene. 3-4 Among the cyclodiene pesticides endrin and dieldrin were produced in especially large quantities,

5

whereas in the second group the γ-cyclohexane sold under the name lindane was the main product. PP depending on their particular structure had a plethora of applications from insecticidal via fungicidal to rodenticidal.

6,7

However, already in the early sixties after the

publication of “Silent Spring” by Rachel Carson 8 it was widely accepted that these compounds are highly ecotoxic and accumulative. 9 PP were classified as persistent organic pollutants and many of them as for example: endrin, aldrin, mirex, hexachlorocyclohexane and hexachlorobenzene were included into the first list of annex A of the Stockholm Convention as members of the so called dirty dozen. 10,11 Multiple studies proved that PP not only contaminate the sites of their release but that the reverse fractionation effect takes place in the atmosphere and some of these compounds which are more volatile are displaced from the hot equatorial zones, where they were and are mainly applied, to the greater polar longitudes.

12-14

PP

accumulate in the soils and sediments because only some decomposer organisms, that is bacteria and fungi have the necessary enzymatic apparatus for their biodegradation. 6,15,16 Chlorinated organics were during decades treated as artificial xenobiotics practically absent in the environment. On the contrary, more recent studies proved that both terrestrial and 3 ACS Paragon Plus Environment


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marine organisms synthesize multiple chlorinated and polychlorinated secondary metabolites, mainly as defense compounds.

17,18

Further studies proved also that the biodegradation of

polychlorinated cycloalkanes and aromatics is possible and there is now high demand for PP degrading organisms as they can be applied in the bioremediation process of PP-contaminated land. 6,15,19,20 On the other hand, the occurrence of PP in the soils leads to the impoverishment of the decomposer communities, as for many soil bacteria and fungi PP exhibit high biocidal activity.

21-23

The exact mechanisms of the biocidal effects of PP in the soils are not known;

however, it is hypothesized that there are possible two independent options. The first is the socalled suicidal pathway: PP are acquired by the decomposer organisms and partially dehalogenated, but the less halogenated metabolites are much more toxic to the microorganisms than the parent compounds and lead to the death of the decomposer organisms. 23,24 The second pathway is connected with the membrane activity of PP.

25,26

Generally, polychlorinated

hydrocarbons are more hydrophobic than their hydrogenated counterparts.

27

They can

effectively interact with the membrane phospholipids and be retained in the cellular membrane. 26,28,29

The incorporation of PP leads to multiple adverse changes of the membrane structure and

function and finally to the death of the cell either on the apoptotic or necrotic way. In our studies we intended to shed light on the second mechanism and investigate the correlations between the PP molecular structure and the phospholipid specificity of the decomposer membranes. To achieve this goal we applied phospholipid Langmuir monolayers 30,31

as model decomposer membranes and studied the effects exerted on them by highly

hydrophobic PP differing in their molecular structure as well as by more water soluble metabolites of these substances. To emulate the decomposer membranes we investigated the anionic phospholipids: cardiolipin (CL) and phosphatidylglycerol (PG) which are the main components of Gram positive bacteria membranes

32,33

as well as zwitterionic

phosphatidylcholine (PC) which is the main phospholipid of fungal membranes. 34 To have the


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option of comparison of the Gram positive and Gram negative model bacterial membranes we have included also phosphatidyletanolamine (PE) to our studies, as the inner membranes of Gram negative bacteria are especially rich in the phospholipids of this class. 35 Regarding the polychlorinated pesticides investigated in this studies we decided to select representatives of the cyclodiene family as well as monocyclic compounds. Regarding the cyclodiene pesticides we decided to endrin, as in multiple environmental studies it was considered a super toxic pesticide

36

and mirex because of its unusually high chlorination degree. Regarding the

monocyclic pesticides we focused on hexachlorocyclohexane 6,15 and hexachlorobenzene 37,38 as both the pesticides were widely applied during many decades in agriculture and forestry, were produced in larger amounts than other pesticides, their application lead to severe pollution of multiple areas and, what is more, these compounds can migrate in the atmosphere polluting finally the polar regions. 12,13 As the water soluble metabolites of polychlorinated pesticides we selected for our studies chlorendic acid (1,4,5,6,7,7-hexachloro-5-norbornene-2,3-dicarboxylic acid) which is the common metabolite of cyclodiene pesticides and pentachlorophenol which is the common metabolite of hexaachlorocyclohexane and hexachlorobenzene. Both the compounds appear in the environment not only as pesticide decomposition products. Chlorendic acid is used as flame retardant and a precursor for the production of flame-retardant resins, and it is highly accepted that the leakages from such materials are the important source of this toxic substance in the environment. 39,40 Pentachlorophenol was (and in some countries is still) used in large quantities as wood preservative and cheap pesticide; however, in 2010 this compound was included to the Stockholm Convention list as a persistent organic pollutant, which can be especially toxic to soil bacteria and the bacteria applied in sewage treatment plants. 23,25 To study the model membranes multiple mutually complementary techniques were applied. The mechanical properties of the model membranes and their elasticity were investigated with the recording of surface pressure (π) – mean molecular area (A) isotherms



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upon the monolayers’ compression and the analysis of the compression modulus. The evolution of the model membrane textures and the effects exerted on it by the applied PP was followed with the application of Brewster angle microscopy (BAM). To obtain the information about the effects of PP on the phospholipid organization in the molecular scale the diffraction of synchrotron radiation, namely the Grazing Incidence X-ray Diffraction (GIXD) technique was applied, whereas to study the reorientations of the polar headgroups and the degree of PP inclusion into the monolayers the surface specific spectroscopy PMIRRAS and penetration tests were employed.

II.

Experimental II.1. Materials

The investigated phospholipids: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine

(DMPC),

1,2-dimyristoyl-sn-glycero-3-

phospho-(1'-rac-glycerol) (sodium salt) (DMPG) and 1',3'-bis[1,2-dimyristoyl-sn-glycero-3phospho]-glycerol (sodium salt) (tetramyristoylcardiolipin TMCL) were purchased from Avanti Polar Lipids as lyophilized powders of the purity 99%. The samples were sent packed with dry ice and were stored refrigerated at -20o C. The investigated polychlorinated pesticides: endrin (END), mirex (MX), α-hexachlorocyclohexane (HCH) and hexachlorobenzene (HCB) as well as their soluble metabolites: 1,4,5,6,7,7-hexachloro-5-norbornene-2,3-dicarboxylic acid (CHA) and pentachlorophenol (PCP) were purchased as analytical standards (purity of at least 99%) from Sigma Aldrich. The phospholipids and pesticides were applied in the experiment without any preliminary purification. The only exception is here PCP which was reacted in aqueous solution with the stoichiometric amount of NaOH to produce the sodium salt of this phenol. Pentachlorophenol has very low water solubility (about 10 mg/ L), whereas its sodium salt is very well water soluble (ca. 300 g/L). The organic solvents applied in the experiments: 6 ACS Paragon Plus Environment

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chloroform (HPLC grade, 99.5 % stabilized by ethanol), methanol (HPCL grade, 99.9%) and ethanol (HPLC grade, 98%) were purchased from Sigma Aldrich. The ultra-pure water of the resistivity 18.2 MΏ·cm was produced in our laboratory with the application of the MerckMillipore Synergy water purification system.

Scheme1. Structural formulas of the studied polychlorinated pesticides

II. 2. Solutions The samples of the investigated phospholipids were weighted on the Mettler Toledo analytical scales (accuracy of 0.01 mg) and dissolved in 10 cm3 volumetric flasks in the chloroform/methanol 9/1 v/v mixture. Routinely ca. 2 mg of the phospholipids were applied which gives the concentration of ca. 3·10-4 M. Similarly the samples of the water insoluble pesticides were dissolved in the chloroform/methanol 9/1 v/v mixture. All the stock solutions were kept refrigerated at -20o C, to prevent any decomposition of the phospholipids or 7 ACS Paragon Plus Environment


chloroform evaporation. Just before the experiments the adequate volumes of the solutions were mixed in amber glass vials to obtain the binary solution of the required mole ratio of the components. The water soluble pesticide metabolites CHA and the sodium salt of PCP were dissolved in 2 dm3 volumetric flasks in ultra-pure water to obtain aqueous solutions of the concentration of 10-4 M, which were later used as subphases in the Langmuir experiments. The pH of these subphases was measured and it turned out that at the applied concentrations it was in the limits of experimental uncertainty the same as the pH of pure MilliQ water, that is 7. For the penetration experiments ethanol solutions of CHA and the sodium salt of PCP of the concentration of 0.05 M were prepared.

II.3. Experimental techniques

II.3.1. Langmuir technique

For the preparation and compression of the monolayers three different Langmuir troughs were applied. In the routine π-A isotherm registration and penetration tests a middle size double barrier KSV NIMA LB trough (nominal area of 273 cm2) was applied. The trough was equipped with: a centrally located rectangular deposition well, an injection port and a magnetic stirrer which enabled the performance of penetration tests. BAM experiments were performed on the high-compression ratio double barrier KSV-NIMA trough (nominal area of 587 cm2). In the Sirius beamline of the Soleil synchrotron a single barrier Riegler&Kirstein (R&K) trough of the area of ca. 500 cm2 is installed. All the troughs were manufactured from single block of Teflon without any glued elements. After the experiment the surface active molecules and the subphase were removed from the troughs with vacuum aspirators. The Teflon elements of the troughs were cleaned with fibre-free tissues soaked in chloroform followed by isopropanol and


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rinsed with plentiful of ultrapure water. The surface pressure was monitored with the application of Wilhelmy-type electrobalance with a plate of filtration paper (Whatman, ashless) as the surface pressure sensor. The temperature during the experiments was kept constant 20±0.1o C with the application of Julabo water circulating bath. Only for TMCL monolayers the temperature was elevated to 25o C to enable the observation of condensed domains with the application of BAM microscope, as at 20o C TMCL monolayers are solid and homogeneous even at very low surface pressure values. In a routine experiment the Langmuir trough was filled with ultra-pure water or the 10-4 M solution of CHA or PCP and the monolayer was deposited on the subphase/air interface with the application of a gas-tight Hamilton microsyringe. Ten minutes were left for the spreading solvent evaporation, after which the monolayers were compressed with the constant compression rate of 20 cm2/min. Each experiment was repeated at least three times and the most representative π-A isotherms (medians of the data) were applied later on in the plots. The uncertainty of the mean molecular area was 1 Å2/molecule. Compression modulus CS-1 was calculated from the π-A isotherms according to its definition: 𝐶𝑆―1 = ―𝐴

( ) ∂𝜋 ∂𝐴

𝑇,𝑝,𝑛

The values of Cs-1 enable the attribution of the physical state of the monolayer. Below 50 mN/m the monolayer is in the liquid expanded (LE) state, between 50 and 100 mN/m liquid L, between 100 and 250 mN/m in the liquid condensed (LC) and above 250 mN/m in the solid state [41]. Usually the LE and L states are not differentiated and treated as the isotropic liquid or the expanded phase.



II.3.2. Penetration tests In the penetration experiments the phospholipid monolayers were deposited on pure water and compressed to the required surface pressure values (5, 10, 20 and 30 mN/m), and left for 20 min. for stabilization. 0.5 cm3 of the ethanol solutions of CHA or PCP was injected into the well of the Langmuir trough (the volume of the aqueous subphase was 250 cm3), after which the temporal dependence of surface pressure (π-t curves) was monitored. As reference π-t curves were also measured on pure water without the injection of the pesticide solution. Regarding the alcohol solutions, blank tests were also performed in which 0.5 cm3 of pure ethanol was injected below the monolayers. It turned out that the injection of such a volume of pure ethanol had no effect on the measured surface pressure values.

II.3.3. Brewster Angle Microscopy (BAM)

In these studies UltraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10x magnification objective, polarizer, analyzer and a CCD camera was used. The spatial resolution of the microscope was 2 μm. The foregoing apparatus and the Langmuir trough were placed on the table (Standa Ltd, Vilnius, Lithuania) equipped with active vibration isolation system (antivibration system VarioBasic 40, Halcyonics, Gottingen, Germany).

II.3.4. PM-IRRAS

KSV-NIMA PM-IRRAS spectrometer (PMI 550) was applied for these experiments. The spectral range of this instrument is 800-4000 cm-1 and the resolution 8 cm-1. The FTIR spectrometer equipped with polarization modulation unit (ZnSe modulator PEM-100, Hinds


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Instruments, USA) is situated on one arm of the goniometer, whereas the MCT detector is situated on the other. The IR and He-laser beams are directed out of the spectrometer to the Langmuir trough and reflected from the monolayer. The incident angle to the monolayer normal was 80° as at the grazing incidence angles the highest signal-to-noise ratio is achieved. The incoming light was continuously modulated between the p and s polarization, allowing simultaneous measurements of the spectra for both polarizations. The difference between the two signals gives surface-specific information and the sum provides the reference spectrum. The PEM-100 modulator operated at the 50 kHz frequency, the frequency of the highest amplification was set to 1500 cm-1 and the retardation was 0.5. The total acquisition time for each spectrum was 5 min, which is equivalent to 3000 interferograms (scans). The final spectrum S is defined as the δ/σ ratio, where δ = RS-RP and σ = RS+RP, where RS, RP – reflectivities of the s and p polarized beams, respectively. For each monolayer a background spectrum was measured for the bare subphase before monolayer deposition and the final normalized spectrum is defined as: ΔS = (Sπ – S0)/S0, where S0 is the background spectrum and Sπ is the spectrum obtained from a monolayer compressed to the required surface pressure π. 42,43

The FFT procedure was performed by the spectrometer software in the Mertz mode with

the application of Blackman apodization function. The spectra were baseline corrected. The spectrometer and the Langmuir trough were placed on the optical table (Standa Ltd, Vilnius, Lithuania) equipped with active vibration isolation system (antivibration system VarioBasic 40, Halcyonics, Gottingen, Germany).

II.3.5. Grazing Incidence X-ray Diffraction (GIXD)

The experiments were performed on the SIRIUS beamline at SOLEIL synchrotron (Gif-surYvette, France) using the dedicated liquid surface diffractometer. The Langmuir trough (R&K



Gmbh electronics, Germany) was mounted on the goniometer in a gas tight box with Kapton windows. Before each experiment, the canister was sealed and flushed with helium to reduce the oxygen level. Such a procedure guaranteed the reduction of the scattering background and minimized the beam damage during the experiment. After at least 30 min, the monolayer was compressed to the target surface pressure of 25 mN/m, which afterward was held constant during the entire experiment. The length of the X-ray beam was 1.565 Å. The detailed construction of the diffractometer working at the SIRIUS beamline and the parameters of the synchrotron beam applied in the GIXD experiments are described on the SOLEIL web site (www.synchrotron-soleil.fr). The scattered signal was detected using a Pilatus3 2D pixel detector (Dectris Ltd, Switzerland). This detector is used as 1D detector through the combined use of a Soller slits collimator oriented vertically to fix the in-plane 2θ resolution and an integration of the 2D image horizontally to obtain a 1D spectrum. The achieved resolution was about 0.002 Å-1. The spectra were obtained by scanning the in-plane 2θ angle. At each point, the vertically scattered intensity was recorded to obtain finally the intensity map I(Qxy,Qz) where Qxy is the scattering vector component in the monolayer plane, and Qz is the vertical component along the z-axis. The I(Qxy,Qz) diffractogrames were integrated along the vertical distribution of Qz to obtain the Bragg peaks I(Qxy). Simultaneously the spectra were integrated over the Qxy values to obtain the Bragg rods, I(Qz). The interested reader can find the principles of the GIXD method in multiple review articles.

44,45

The estimation of the full width at half

maximum (FWHM) of Bragg peak enables the calculation of the Lxy parameter, which is related to the range of 2D crystallinity, whereas from the FWHM of Bragg rod the Lz parameter related to the length of the scattering moiety can be gained. These parameters are calculated according to the Scherrer formula: Lxy ≈ 0.88・2π/FWHMpeak, Lz ≈ 0.88・2π/FWHMrod.


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III.

Results and discussion

III.1. Interactions of the water insoluble PP with the model membranes At the beginning of the studies the interactions between the water insoluble PP and two phospholipids: TMCL and DMPC were investigated with the application of the Langmuir technique. Chloroform solutions of the PP were mixed with the chloroform solutions of TMCL and DMPC at different mole ratios (0, 0.1, 0.3, 0.5 and 0.7). Appropriate volumes of the binary solutions were deposited at the air/water interface and the π-A isotherms were measured upon the monolayer compression. The π-A isotherms together with the calculated compression modulus (CS-1) – surface pressure (π) dependencies are gathered for the systems with TMCL in Fig1. The results for the systems with DMPC turned out to be qualitatively very similar to these obtained for TMCL; therefore, they are presented in the Supporting Materials (SFig1).



Fig. 1. π-A isotherms and CS-1 – π curves (insets) for the investigated binary systems: a) TMCL/END, b) TMCL/MX, c) TMCL/HCH, d) TMCL/HCB

It should be underlined that all the investigated here PP are not surface active and cannot form a Langmuir monolayer when deposited alone from their chloroform solutions at the air/water interface. This disables the thermodynamic interpretation of the data gathered in Fig. 1 and the calculation of the 2D excess functions of mixing, as there is not the reference π-A. isotherm for the one-component monolayer. In all the binary mixtures the number of the phospholipid molecules was kept constant (1016) and the number of PP molecules was increased to achieve the required mole ratio. If PP builds into the phospholipid monolayer the resultant π-A isotherm should be shifted toward greater mean molecular areas, otherwise when PP remains phaseseparated, forming 3D aggregates or add-layers, the π-A isotherm remains unchanged. As it is visible in Fig. 1a the small addition of END (X = 0.1) leads to a significant shift of the π-A isotherm toward greater mean molecular areas. and the increase of LE-LC transition surface pressure. The π-A isotherm registered at X(END) = 0.3 is shifted further to the right as compared with X(END) = 0.1; however, no further shifts were observed for higher END proportions and the isotherms at X(END) = 0.3, 0.5, and 0.7 overlap. Even at low END proportion the monolayers change their elastic properties – the maximal CS-1 values fall from 350 mN/m observed for the pure TMCL monolayer to 150 – 200 mN/m. At higher surface pressure values the π-A isotherms for the binary monolayers approach the π-A isotherm measured for pure TMCL monolayer. Thus, the data can be interpreted as follows: at X(END) = 0.1 all or most of the END molecules are incorporated into the phospholipid monolayer which leads to the shift of the π-A isotherm to higher mean molecular area and to the change of its course. The presence of END molecules in the phospholipid matrix introduces disorder intro the system which is reflected in the lowered CS-1 values. The further increase of X(END) over


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0.1 causes negligible changes into the π-A isotherm and the CS-1-π curves, so probably the additional END molecules are not located between the phospholipid molecules within the monolayer but form 3D aggregates. At high surface pressure all the isotherms overlap, so it is possible that the END molecules are successively squeezed out from the phospholipid matrix with increasing surface pressure. The described above trends are similar for the systems with MX but less pronounced. The presence of MX makes the phospholipid monolayer more compressible as the CS-1 values are considerably lower. Moreover, MX destabilizes the monolayer as from X(MX) = 0.3 the monolayers collapse at much lower surface pressures than on pure water. Thus, also for MX it can be postulated on the basis of the π-A isotherm interpretation that some molecules of this PP are incorporated between the TMCL molecules in the monolayer. The shift of the π-A isotherm at X(MX) = 0.1 is smaller than in the same conditions for END, so it can be inferred that less MX molecules are built into the TMCL matrix. The effects of HCH and HCB addition on the location and course of the TMCL π-A isotherms are negligible; however, their presence affects the monolayers’ elastic properties. Namely, the presence of HCH or HCB at the air/water interface lowers the values of CS-1. On the basis of the preliminary Langmuir experiments it can be stated that two from the investigated PP: END, MX exhibit membrane activity and that probably some part of these molecules can be built into the phospholipid matrix. However, on the basis of the sole Langmuir experiments it would be difficult to provide any in-depth interpretation of the above discussed data. If some of the PP molecules are incorporated into the phospholipid matrix they should affect the texture of the condense domains evolving upon the monolayer compression. It was also inferred from the π-A isotherms that only a rather small portion of the PP molecules is directly incorporated into the phospholipid matrix whereas the rest can remain at the interface in the form of 3D aggregates. Therefore, to verify these hypotheses, the investigated monolayers were visualized upon their compression with the application of Brewster angle microscopy and



the representative BAM images for the polychlorinated cyclodienyl pesticides (END and MX) are gathered in Fig. 2. and for HCH, HCB in Fig. 3, whereas the entire collection of the BAM images for all the investigated systems and compositions is presented in Supporting materials.

Fig. 2. Selected BAM images for the binary monolayers containing TMCL and the cyclodiene pesticides: row A) one-component TMCL monolayer, row B) system TMCL/END, X(END) = 0.1, row C) system TMCL/END, X(END) = 0.7, row D) system TMCL/MX, X(MX) = 0.1. The numbers in the photos denote surface pressure in mN/m, the scale bar is 100 μm.

First condense domains in TMCL monolayer in the applied experimental conditions appear at ca. 4 mN/m. They have the irregular dendritic or flower-like shape and are quite large with the diameter of approx. 50 μm. Upon further compression the domains grow and approach eachother. The fusion of the domains can be observed from the surface pressure value of ca. 10 mN/m; however, even at 35 mN/m some crevices in the solid homogeneous monolayer can be 16 ACS Paragon Plus Environment

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observed being the remnants of the previous domain boundaries. It should be underlined that till the monolayer collapse (over 50 mN/m) no multilayer aggregates can be observed in the photos. In the presence of endrin at X(END) = 0.1 first condensed domains appear at ca. 7 mN/m and the dark regions typical to the LE state of the monolayer occupy greater areas that at comparable surface pressures for pure TMCL monolayer, which explains the increased compressibility noticed after END addition in the CS-1-π curves. First 3D aggregates appear at ca. 20 mN/m; however, they are small, with their dimensions (2-5 μm) close to the microscope resolution. The number of the aggregates is limited and even at high surface pressure they are not numerous. The images of the monolayer were very similar at X(END) = 0,3, whereas at X(END) = 0.7 3D aggregates were visible even at 0 mN/m. The TMCL domains in these conditions appear at a comparable surface pressure as at smaller X(END), have the same flower-like shape and dimensions as previously described. The multilayer aggregates are numerous and larger than at smaller endrin proportion; thus, it is obvious that most of the END molecules remains phase separated. Regarding the systems with mirex, this compound exhibits much higher tendency to separate from the binary system; thus, the images at X(MX) = 0.1 resemble the photos taken for the TMCL/END system at X(END) = 0.7. At higher mirex proportions the aggregates were numerous and larger disabling the observation of the evolution of the phospholipid domains.



Fig. 3. BAM images for the binary systems containing TMCL and HCH or HCB. Row A) system TMCL/HCH at X(HCH) = 0.1, row B) system TMCL/HCH at X(HCH) = 0.7, row C) system TMCL/HCB at X(HCB) = 0.7. The numbers in the photos denote surface pressure in mN/m. The scale bar is 100 μm.

The evolution of the TMCL texture in the presence of HCH is very similar to the described for one-component TMCL monolayer. The dendritic domains have narrower arms, but similar dimensions. It is important that even at very high surface pressure values (over 50 mN/m) no 3D aggregates can be observed and the monolayer is completely homogeneous. In Fig. 3 we show the BAM photos for X(HCH) = 0.1; however for X(HCH) = 0.3 and 0.5 the results were practically identical. Only at X(HCH) = 0.7 (row B) the shape of the domains changed, as they lose their dendritic morphology acquiring the oval or kidney-like shapes. It is important that even at high surface pressure and the large HCH proportion there are practically no 3D 18 ACS Paragon Plus Environment

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aggregates in the monolayer. HCB behaves very similarly to HCH and till X(HCB) = 0.5 the morphology of the film at a given surface pressure value resembles the pure TMCL monolayer. At X(HCB) = 0.7 the situation is also similar to HCH, that is the shape of TMCL condensed domains is changed from dendritic to oval. It can be also observed in the BAM photos taken at 35 mN/m that at X(PP) = 0.7 HCB is more prompt to form 3D aggregates than HCH. The case of the single-ring polychlorinated pesticides: HCH and HCB is especially interesting. The presence of these molecules neither affects the location and course of the π-A isotherm nor 3D aggregates can be observed in the BAM images. On the other hand, the presence of HCH and HCB even at X = 0.1 significantly lowers the values of compression modulus, which proves the presence of these molecules at the interface. HCH and HCB are hydrophobic non-polar molecules completely insoluble in water. Their volatility in the experimental conditions is also negligible, so once deposited at the interfacial region they cannot leave it. Thus, there are two options of their organization: 1) HCH and HCB form multiple small 3D aggregates much lower than the resolution of the microscope, which are invisible in the BAM images and 2) the molecules form the so-called add-layer on top of the phospholipid monolayer. Such add-layers were discovered for some perfluorinated molecules interacting with phospholipid Langmuir monolayers.

46-48

Generally, in such an organization

mode the terminal parts of the hydrocarbon chains of the phospholipid molecules interact with the molecules of the xenobiotic. Although the layers can be undulated and the terminal parts of the hydrocarbon chains can be interdigitated with the halogenated molecules, they are not built deep between the phospholipid molecules, so finally the mean molecular area is not increased and the π-A isotherms in the presence of HCH or HCB are identical with the π-A curve measured for the one-component TMCL monolayer. The add-layer is thin as compared with the phospholipid monolayer and the refraction index of perhalogenated molecules is comparable with water, 49 so the presence of the add-layer is not observed in BAM images.



Page 20 of 54

BAM is a method of optical microscopy and with its resolution of 2 µm it can provide important information about the monolayer texture but is unable to give the insight into the organization of the phospholipid molecules at the molecular scale. Thus, to gain the information regarding the molecular packing in a Langmuir monolayer the methods of the diffraction of synchrotron X-ray radiation (Grazing Incidence X-ray Diffraction, GIXD) should be applied. TMCL as well as other saturated membrane phospholipids including DMPE and DMPG form Langmuir monolayers 50-52 which are 2D crystalline which means that the monolayers diffract synchrotron radiation. Indeed, these phospholipids were many times investigated with GIXD and the measurements provided valuable information about the interactions between phospholipid molecules in the model membranes or about the effects of xenobiotics exerted on the monolayer structure. 53-55 In this studies we applied GIXD for studying the effects exerted by water insoluble PP and their water soluble metabolites on the 2D crystal structure of the condensed domains formed by anionic phospholipids TMCL and DMPG, typical to Gram positive bacteria, and DMPE – the main component of the inner membrane of Gram negative bacteriaThe measurements were performed at constant surface pressure of 25 mN/m at which the packing of the phospholipid hydrocarbon chain is comparable with their organization in real cellular membranes.

56

At 25 mN/m the investigated phospholipids form solid Langmuir

monolayers in which the molecules are arranged in the hexagonal lattice with their hydrophobic chains oriented perpendicular to the air/water interface.

50-52

These monolayers were

investigated previously by GIXD and the results were published by us and other authors 50-53,55 so we show the intensity maps and their interpretations for one-component phospholipid monolayers in the Supporting materials. In The GIXD experiments we focused mainly on endrin as on the basis of the π-A isotherms and BAM images it can be supposed that this cyclodiene pesticide can be incorporated in some extent into the TMCL monolayers. Additionally the GIXD measurements were performed for the mixture of TMCL with HCB at


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its highest proportion of 0.7. HCH and HCB were postulated on the basis of the BAM images to form the add-layers on top of the phospholipid monolayers, so it was interesting to check if such an upper layer can influence the crystalline packing of the phospholipid monolayer below. It turned out that END and HCB do not change the packing mode of TMCL molecules in the crystalline domains within the limits of experimental error. In all the cases only one diffraction signal with its intensity maximum at Qz = 0 was observed, which is typical to the 2D hexagonal lattice. Thus, the intensity maps, Bragg peaks and Bragg rods for these experiments are presented in Supporting materials, whereas the calculated lattice parameters are gathered in Table. 1. The comparison of the GIXD results and the conclusions drawn from the π-A isotherms leads to the notion that the results are mutually contradictory. However, this contradiction is only apparent. It can be supposed that the mixed regions of the monolayer remain in the liquid-expanded state, which does not diffract the X-ray beam. So, if the condensed phospholipid domains does not include PP molecules the organization of the phospholipid molecules is unchanged and GIXD data are identical as for the pure phospholipid monolayer. On the other hand, the increased presence of the LE phase in the monolayer is proved by the lower CS-1 values as well as by the dark regions visible in BAM images. Anionic phospholipids are the main components of the cellular membranes of multiple species of Gram positive soil bacteria. 32,33 Typically, both cardiolipins and phosphatidylglycerols are present and these organisms can considerably variate the CL/PG ratio reacting on the external stimuli. 57 The external stimulus is very often the presence of a xenobiotic in the vicinity of the biofilm. Therefore, it was reasonable to study also the effects of the investigated PP on the model membranes formed of DMPG. The resultant GIXD data are presented in Fig. 4, whereas the calculated lattice parameters are gathered in Table1.



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Table1. Parameters calculated for the investigated monolayers from the GIXD data a, b, γ lattice parameters, A – crystallographic area per one hydrocarbon chain, τ – tilt angle, Lxy – the range of crystallinity, Lz – the length of the scattering moiety

a, b,  (Å, Å, A (Å2)

system

τ (deg)

Lxy (Å)

Lz (Å)

deg) DMPE

4.949; 120

21.21

0

384

22.7

DMPG

4.942; 120

21.15

0

395

21.8

TMCL

4.922; 120

20.98

0

636

26.6

TMCL/END

4.909; 120

20.87

0

636

23.8

4.912; 120

20.90

0

757

23.3

4.919; 120

20.95

0

608

23.8

4.959; 120

21.30

0

294

16.3

13.5

X(END)=0.1 TMCL/END X(END)=0.7 TMCL/HCB X(HCB)=0.7 DMPG/END X(END)=0.1 DMPG/END

5.045; 8.586; 21.69

X(END)=0.3

90

DMPG/END

5.071; 8.596; 21.80

X(END)=0.5

90

DMPE CHA DMPG CHA

15.8

on 5.000; 8.566; 21.47

13.4

90

on 5.003; 8.572; 21.44

11.3

90


= 369 = 240

= 425

-

= 149

-

= 503 = 263 = 498 = 325

= 14.9 = 15.4

= 18.5 = 16.9 = 14.4 = 15.8

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TMCL

on 4.925; 120

21,01

0

650

20,6

CHA

Fig. 4. GIXD data: intensity maps I(Qxy,Qz), Bragg peaks I(Qxy) and Bragg rods I(Qz) for the mixtures of DMPG with endrin. a,b) X(END) = 0.1; c,d) X(END) = 0.3, e,f) X(END) = 0.5. The solid lines in the Bragg peak plots are Lorentz curve fits to the experimental data.

The limited addition of endrin to the DMPG monolayer at X(END) = 0.1 do not change the way of the DMPG molecules packing. Still one diffraction signal with its intensity maximum at Qz= 0 was registered and the lattice parameters and the range of crystallinity were very similar to those collected for one-component DMPG monolayer. The increase of X(END) to 0.3 changes the situation. The diffraction signal in the intensity map is smeared to larger Qz values



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and the interpretation of the Bragg peak leads to the conclusion that the observed signal is a superposition of two separate signals. One of them indexed as has its intensity maximum at Qz = 0, whereas the second signal indexed as is shifted from the horizon and has its intensity maximum at Qz = 0.3. The integrated intensity of the signal is roughly two times greater than that of . Such a combination of the diffraction peaks leads to the conclusion that at X(END) = 0.3 the hexagonal lattice of TMCL was deformed and the arrangement of the phospholipid molecules can now be described by the rectangular centered lattice. The deformation of the lattice was caused by the tilt of the phospholipid hydrocarbon chains from the monolayer normal. It can be inferred from the diffraction peaks sequence that the azimuth of the tilt is toward the nearest neighbor (NN) and the tilt angle τ = 13.5o. The further increase of X(END) to 0.5 leads to a significant weakening of the diffraction signal which is the effect of the monolayer amorphization and molecular disorder caused by the introduction of the additional END molecules to the monolayer. The sequence of the weak diffraction signals is similar to the previous case and the 2D crystal lattice can be described as rectangular centered with NN azimuth. The tilt angle τ was increased to 15.8o. The results are quite interesting because they underline the crucial role of the four-chain cardiolipin for the survival of soil bacteria in the environment polluted by highly hydrophobic substances. The dense, highly organized palisade of cardiolipin tails is much more difficult to cross than the membrane formed by the two-chained PG. Thus, the studies performed on the simplified model systems lead to the same conclusions as these drawn by Tsai and co-authors

57

that in harsh

condition cardiolipin is inevitably necessary for the survival of Gram positive soil bacteria.


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III.2. The effects of soluble PP metabolites

To study the effects of the presence of CHA or PCP in the aqueous subphase the monolayers of the studied phospholipids were spread on 10-4 M aqueous solutions of CHA or PCP and compressed in such condition. The resultant π-A isotherms compared with the π-A curves measured on pure water are gathered in Fig. 5. together with the compression modulus – surface pressure dependences.

Fig. 5. π-A isotherms and CS-1 – π dependences for the phospholipid monolayers spread on pure water and 10-4 M CHA and PCP solutions: a) DMPE, b) DMPC, c) DMPG, d) TMCL

The presence of CHA and PCP in the aqueous subphase affects all the investigated phospholipid monolayers. The most pronounced effects are observed for DMPE as the lift-off area of the π-



A isotherm is shifted ca. 20 Å2/mol. to higher mean molecular areas. The increase of surface pressure upon compression is moderate, what is typical to the monolayers in the LE state. The plateau typical to the LE-LC transition is shifted from 7 mN/m on water via 22 mN/m on CHA to 30 mN/m on PCP. At very high surface pressure values between 40 mN/m and the monolayer collapse all three isotherms overlap, which means that in such conditions the xenobiotic molecules were eliminated from the phospholipid monolayer. The presence of CHA and PCP increases also the compressibility of the DMPE monolayers which is reflected in the lowered values of compression modulus. According to the CS-1 criterion at high surface pressures the DMPE monolayer achieves the organization typical to the solid state on the aqueous subphase, but only the liquid condensed state is observed on the metabolite solutions. In the case of DMPC in the presence of the chlorinated metabolites the whole π-A isotherms are shifted toward greater mean molecular areas and the shift is greater for PCP than CHA. The isotherms remain separated and do not overlap even at high surface pressure values. The course of the curves is comparable which is also reflected in the overlapping CS-1-π plots. The monolayers of DMPC are in the LE state on water and the presence of CHA or PCP do not lead to noticeable changes of the monolayer condensation. In the case of DMPG the π-A isotherms on CHA and PCP solutions are shifted ca. 10 Å2 toward greater molecular areas. Also for this phospholipid the mean surface pressure characteristic to the LE-LC transition plateau is raised from 15 mN/m on water via 22 mN/m on the 10-4 M CHA solution to 37 mN/m noticed on the 10-4 M PCP solution.. At very high surface pressures all three isotherms practically overlap; however, their inclination is different which is reflected in the values of compression modulus. On water CS-1 achieves the maximal value of 270 mN/m typical to the solid state, on CHA 245, whereas on PCP only 135 mN/m which is typical to the monolayer in the LC state. In the case of TMCL the lift-off area of ca. 160 Å2/mol. is identical for the three isotherms and they practically overlap at the earlier stage of the monolayer compression. The plateau connected with the LE-


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LC transition is observed on water at ca. 5 mN/m, on CHA at ca. 8 mN/m, whereas on PCP is shifted to 20 mN/m. At higher surface pressure values all three isotherms overlap which indicates that the xenobiotic molecules are eliminated in such conditions from TMCL monolayers. The Langmuir experiments prove that both the water soluble metabolites of the investigated PP have profound effect on the organization of the model membranes. CHA induced especially significant changes into the organization of the DMPE monolayer being a simplified model of the inner membrane of Gram negative bacteria. PCP seems to be more membrane active than CHA and disturbs the organization of both zwitterionic and anionic phospholipids, as important changes in the π-A isotherms were observed both for DMPE as for TMCL. At higher surface pressures over the LE-LC transition the PP metabolites seems to be eliminated from the monolayers as the π-A isotherms practically overlap with each other. To have a deeper insight into the effects exerted by PP metabolites on the organization of the phospholipid molecules in the model membranes the GIXD method was applied. The presence of PCP in the subphase elevated the surface pressure of the LE-LC transition in the investigated monolayers to very high values. This leads to the amorphization of the monolayers at 25 mN/m and the lack of diffraction signal. Even at 35 mN/m the GIXD signal was weak and it was difficult to stabilize the monolayers for GIXD experiments. Therefore, the GIXD experiments were performed successfully only for the CHA containing subphases. The GIXD data collected for these monolayers spread on 10-4 M CHA solution and compressed to 25 mN/m are presented in Fig. 6 whereas the lattice parameters calculated from these data are gathered in Table1.



Fig. 6. GIXD data: intensity maps I(Qxy,Qz), Bragg peaks I(Qxy) and Bragg rods I(Qz) collected for the monolayers of: a,b) DMPE, c,d) DMPG, e,f) TMCL spread on 10-4 M CHA and compressed to 25 mN/m. Solid lines on the Bragg peak plots are fits of the Lorentz curves to the experimental data.

As it was discussed in the earlier chapter of this article regarding the effect off PP on the organization of the model membranes DMPE, DMPG and TMCL form on pure water hexagonal phases with upright molecules. It turned out that the CHA molecules present in the aqueous subphase interact with the DMPE and DMPG molecules which leads to the appearance of the tilt of the hydrocarbon chains of the phospholipid molecule from the monolayer normal. The tilt angle τ is 13.4 o for DMPE and 11.3 o for DMPG. The occurrence of the molecular tilt leads to the deformation of the hexagonal 2D crystal lattice and its transformation to the rectangular centered with the NN tilt azimuth. The Lxy parameter in the direction is greater in the 28 ACS Paragon Plus Environment

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presence of CHA than on pure water which means that the molecules of CHA, which is a dicarboxylic acid, can join the adjacent phospholipid headgroups via the net of hydrogen bonds and by this lead to the cross-linking of the adjacent molecules which results in the increase of the range of 2D crystallinity. On the other hand, the presence of CHA in the aqueous subphase does not affect the packing of TMCL molecules in the 2D crystalline domains. In this case only one diffraction signal with its intensity maximum at Qz = 0 was observed, defining the hexagonal crystal lattice. The location of the signal and the FWHM of Bragg peak and Brag rod were identical as on pure water leading to the same crystallographic parameters. The discussed here results again indicate the special durability of the model membranes formed by cardiolipin. In the previous chapter regarding the water insoluble PP it was discussed that it was connected with the dense packing of the upright oriented four hydrocarbon chains of the cardiolipin molecules. Here when the effect of the dicarboxylic acid CHA is concerned the discussion should focus on its interactions with the polar head-group of TMCL molecule. Cardiolipin can be treated as a dimeric form of phosphatidylglycerol and the dimerization is achieved by the junction of two adjacent polar head-groups. Therefore, the CL head-group is especially large as compared with other phospholipids, highly hydrated and cross-linked with other CL headgroups by the net of hydrogen bonds formed mainly by the hydroxyl group located at the second carbon atom of the middle glycerol moiety.

58

CHA interacting with the head-groups can

slightly modify their organization at low surface pressures, which was proved by the π-A isotherms, but the GIXD results prove that at 25 mN/m, when TMCL molecules are tightly packed the presence of CHA has no effect on their organization.



III.2.1. Penetration tests

In the experiments discussed in the previous subchapter the phospholipids were spread from their chloroform solutions at the 10-4 M polychlorinated metabolite solution/air interface. These experiments provided important information about the pesticide-phospholipid interactions; however, in real environmental conditions the cellular membrane has its specific composition and organization and to such a highly organized membrane the pesticide migrates. Therefore, in further experiments we decided to perform the penetration tests which are often applied in the studies of model membranes. 59, 60 The TMCL and DMPC monolayers were spread on pure water, condensed to the required surface pressure values and left for stabilization after which 500 μl of a concentrated 0.05 M ethanol solution of CHA or PCP was injected via the injection port to the deposition well of the Langmuir trough, deep below the monolayer. After the injection temporal evolution of surface pressure was monitored. The resultant Δπ-t curves for the investigated systems are gathered in Fig. 7.


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Fig. 7. Tests of the penetration of the water soluble metabolites to the phospholipid monolayers: a) CHA to TMCL, b) PCP to TMCL, c) CHA to DMPC, d) PCP to DMPC.

As it is visible in Fig. 7 after the injection of CHA and PCP below the monolayers three regions in the Δπ-t curves can be discern: 1) fast surface pressure increase in the first minutes of the experiment, 2) decrease of surface pressure in the next 10 to 20 minutes, 3) stabilization of surface pressure. In the case of CHA injection below TMCL monolayer the highest Δπ is observed for the monolayer compressed to 5 mN/m. 30 minutes after injection the Δπ stabilizes at the level of 6 mN/m. The injection of CHA below the TMCL monolayer compressed to 10 mN/m leads to the stabilization of Δπ at the level of 2 mN/m, whereas after the injection below the monolayer compressed to 20 and 30 mN;/m the Δπ stabilizes at the negative value of -5 and -11 mN/m, respectively. The negative values indicate that the appearance of CHA in the aqueous subphase can induce the nucleation of 3D domains in TMCL monolayers. The presence of such aggregates was proved by BAM. The injection of PCP leads to much higher maximal Δπ values. Δπ stabilizes at the level of ca. 15 mN/m regardless the surface pressure to which the monolayer was compressed. Only for the monolayer compressed to 30 mN/m Δπ stabilizes at 0 mN/m. For DMPC the injection of CHA and PCP leads also to the significant increase of surface pressure as Δπ stabilizes at ca. 17 mN/m for CHA and 12 mN/m for PCP. The Δπ-t curves are comparable for the monolayers compressed to 5 and 10 mN/m for CHA and 5, 10 and 20 mN/m for PCP. At higher surface pressures: 20 mN/m for CHA and 30 mN/m for DMPC the injection of the metabolites leads to the quick fall of Δπ value after the initial peak and its stabilization at negative values. DMPC forms expanded monolayers on the aqueous subphase and upon their compression no aggregates were observed even in the collapse region. On the contrary, such aggregates were formed when CHA or PCP were injected below DMPC monolayers compressed to higher surface pressures.



It can be inferred from the course of the Δπ-t curves that after the injection CHA and PCP molecules come quickly in contact with the model membranes leading to their destabilization. However, after the fast adsorption step the desorption or reorganization of the chlorinated metabolites is observed which leads to the decrease of the Δπ values. Δπ stabilizes on much higher values for PCP indicating its superior membrane activity as compared with CHA which was independently proved by the GIXD experiments. The flat PCP molecules are better fit to the phospholipid chains than the bulky polycyclic CHA moieties; thus, they can migrate deep between the chains destroying their lateral ordering. CHA molecules remain rather closer to the haedgroups leading to their cross-linking and in consequence to the change in the 2D crystal structure. To shed much light onto the changes caused by the presence of the PP metabolites on the conformation of the polar headgroups of TMCL and DMPC within the monolayers PMIRRAS spectroscopy was applied. As we were interested in the reorganizations of the polar headgroup the spectra were baseline corrected and interpreted only in the fingerprint and carbonyl regions, that is from 950 to 1850 cm-1. The whole spectra are shown in the Supporting materials. Here in Fig. 8. we focus only on the characteristic of the very intense band originating from the asymmetric P=O vibration as only for this band significant changes were observed in the presence of the chlorinated metabolites.


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Fig. 8. PM-IRRAS spectra registered for TMCL and DMPC monolayers compressed to 10 or 30 mN/m before (black line) and after the injection of HCNA or PCP solutions below the monolayers: a) TMCL on CHA, b) DMPC on CHA, c) TMCL on PCP, d) DMPC on PCP

In PM-IRRAS spectra if the transition momentum vector lies in the monolayer plane the resulting bands are positive if it is perpendicular to the monolayer plane the bands are negative, whereas when the orientation is intermediate the bands are weak or can hardly be observed in the spectrum.

42

At 10 mN/m on water the P=Oas band is weak for both investigated here

phospholipids, which can indicate the intermediate orientation of the transition momentum vector, whereas at 30 mN/m the P=Oas band has high intensity and is positive which means the reorientation of the phosphate group and the change of the transition momentum to the parallel orientation. In the first 5 minutes after the injection of the chlorinated metabolites a significant increase of the intensity of the P=Oas band can be observed at 10 mN/m meaning the 33 ACS Paragon Plus Environment


reorientation of the phosphate group. The increase can also be observed at 30 mN/m but it is less pronounced. In the case of TMCL significant differences between CHA and PCP can be observed with time elapse. At 10 mN/m 15 min after CHA injection the signal disappears, whereas with time elapse it becomes negative. At 30 mN/m the signal is not negative but practically disappears with time. The PM-IRRAS spectra corroborate and complement the conclusions drawn from the penetration tests. Short after the injection the CHA and PCP molecules appear in the proximity of the polar-head-group. PCP is negatively charged whereas CHA as a dicarboxylic acid can also be at least partially ionized. The appearance of the negative charges in the proximity of the negatively charged phosphate group leads to its reorientation, as there is less space for it. This switches the transition momentum to the parallel orientation and results in the intense P=Oas band in the spectrum. This effect is not observed at 30 mN/m, as the close packing of the phospholipid molecules itself induces the reorientation of the phosphate group. In the first minutes after injection both CHA and PCP molecules migrate to the monolayer and are located between the mirystoyl chains. This stage is common both for TMCL and DMPC. Bicyclic CHA is less structurally fit to the hydrocarbon chain than the PCP molecule and disturbs the close packing of the chains of TMCL, so its presence therein is not favorable from the thermodynamic point of view. Thus, with the elapse of time part of the CHA molecules leaves the hydrophobic regions of the monolayer achieving the closer contact with the polar head-group. The presence of CHA molecules in the head-group sublayer triggers the reorientation of the phosphate group, which finally results with the appearance of the intense P=Oas negative band in the spectrum. Similar trend is not observed for PCP. As it was interpreted previously PCP molecules fit better between the hydrophobic chains of TMCL and are strongly bound therein, so no desorption of these molecules was observed with time. As far as the DMPC monolayer is concerned, because of its LE state there is more space for CHA and PCP incorporation in the hydrophobic region, so the desorption of CHA molecules was here


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not observed. The P=Oas band was positive and had practically constant intensity with time elapse after the PP metabolites injection.

IV.

Conclusions

In this studies the effects of hydrophobic polychlorinated pesticides and their water soluble metabolites on the organization of model decomposer membranes were tested. The performed experiments proved that the cyclodiene PP as endrin and mirex exhibit limited membrane activity and can be incorporated into the phospholipid matrix. On the contrary, the representatives of monocyclic PP hexachlorocyclohexane and hexachlorobenzene turned out to be membrane inactive, as their incorporation into the model membranes was not observed. On the basis of BAM images and the changes in monolayer compressibility caused by the presence of HCH and HCB it was postulated that the molecules can form an add-layer on top of the phospholipid monolayer. The incorporation of the cyclodiene pesticides occurs at lower surface pressure values for all the tested phospholipids, whereas at high surface pressures, more relevant to the conditions in real membranes, PP incorporation is closely related with the phospholipid structure. In the membranes of Gram positive soil bacteria dominate the anionic phospholipids belonging to two classes: cardiolipins and phosphatidylglycerols. Our studies performed on the model systems proved that cardiolipins form closely packed, solid membranes exhibiting high resistance to cyclodiene PP incorporation. On the contrary, the incorporation of these pesticides to the monolayers of phosphatidylglycerol turned out to be possible even at high surface pressures. It was proved by multiple studies performed on soil bacteria that the proper cardiolipin to phosphatidylglycerol ratio is crucial for their survival and that the bacteria living in harsh conditions exhibit especially high CL level. Our studies performed on model systems also corroborate this observation. PP when released to the environment can be oxidized 35 ACS Paragon Plus Environment


to water soluble metabolites. The metabolites are often common to different pesticide classes. Cyclodiene pesticides are often oxidized to chlorendic acid, whereas polychlorinated cyclohexanes and polychlorinated benzenes to pentachlorophenol. Therefore, in a set of experiments overwhelming the Langmuir technique, penetration tests, GIXD and PM-IRRAS the effects of CHA and PCP on the model membranes were tested. It turned out that the monolayers formed by anionic phospholipids are less susceptible to CHA and PCP incorporation than the model membranes formed by the zwitterionic phospholipids. Regarding the anionic phospholipids, the GIXD experiments proved that the structure of the cardiolipin crystalline domains was not affected by the presence of CHA, whereas this metabolite induced significant changes of the phospholipid molecules organization in the DMPG monolayers. The first part of the studies regarding the water insoluble PP indicated that the tested cyclodiene PP were incorporated to the model membranes profoundly affecting their properties, whereas HCH and HCB were rather inactive. On the contrary, PCP – the common metabolite of HCH and HCB turned out to be much more active than CHA. PCP destroyed the 2D crystalline structure of TMCL, DMPG and DMPE, which proved that it was incorporated into the phospholipid domains even at high surface pressures. PCP was the only compound which severely affected the organization of TMCL. The species of microorganisms able to decompose polychlorinated substances both in sewage treatment plants and for the remediation of the polluted arable lands are highly required. Our studies performed on model systems prove that Gram positive bacteria with their membranes rich in cardiolipin should be especially suitable for this purpose. The studies indicated also with accordance with previously published results that PCP is a very dangerous environmental pollutant and that the effectiveness of the degradation of PP can be severely lowered in the presence of this substance. Therefore, the studies regarding the biodegradation of PCP should be intensified and the microorganisms applied for PP biodegradation should be applied in consortia containing PCP degrading bacteria.


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Supporting Information π-A isotherms and CS-1- π dependences for the binary systems with DMPC. Detailed BAM characterization of the investigated monolayers, GIXD results for the one-component phospholipid monolayers and for the binary monolayers containing TMCL. PM-IRRAS spectra in the whole fingerprint and carbonyl region for the studies of the interaction of water soluble PP metabolites with TMCL and DMPC.

Acknowledgements

This project was financed by the National Science Centre (No 2016/21/B/ST5/00245). We gratefully acknowledge SOLEIL for provision of synchrotron radiation facilities and we would like to thank Dr. Philippe Fontaine for assistance in using SIRIUS beamline.

V.

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Graphical abstract 254x190mm (96 x 96 DPI)

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Structural formulas of the investigated pesticides and their metabolites 254x190mm (96 x 96 DPI)


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π-A isotherms and CS-1 – π curves (insets) for the investigated binary systems: a) TMCL/END, b) TMCL/MX, c) TMCL/HCH, d) TMCL/HCB 272x208mm (300 x 300 DPI)



Selected BAM images for the binary monolayers containing TMCL and the cyclodiene pesticides: row A) onecomponent TMCL monolayer, row B) system TMCL/END, X(END) = 0.1, row C) system TMCL/END, X(END) = 0.7, row D) system TMCL/MX, X(MX) = 0.1. The numbers in the photos denote surface pressure in mN/m, the scale bar is 100 μm. 254x190mm (96 x 96 DPI)


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BAM images for the binary systems containing TMCL and HCH or HCB. Row A) system TMCL/HCH at X(HCH) = 0.1, row B) system TMCL/HCH at X(HCH) = 0.7, row C) system TMCL/HCB at X(HCB) = 0.7. The numbers in the photos denote surface pressure in mN/m. The scale bar is 100 μm. 254x190mm (96 x 96 DPI)



GIXD data: intensity maps I(Qxy,Qz), Bragg peaks I(Qxy) and Bragg rods I(Qz) for the mixtures of DMPG with endrin. a,b) X(END) = 0.1; c,d) X(END) = 0.3, e,f) X(END) = 0.5. The solid lines in the Bragg peak plots are Lorentz curve fits to the experimental data. 254x190mm (96 x 96 DPI)


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. π-A isotherms and CS-1 – π dependences for the phospholipid monolayers spread on pure water and 10-4 M CHA and PCP solutions: a) DMPE, b) DMPC, c) DMPG, d) TMCL 272x208mm (300 x 300 DPI)



GIXD data: intensity maps I(Qxy,Qz), Bragg peaks I(Qxy) and Bragg rods I(Qz) collected for the monolayers of: a,b) DMPE, c,d) DMPG, e,f) TMCL spread on 10-4 M CHA and compressed to 25 mN/m. Solid lines on the Bragg peak plots are fits of the Lorentz curves to the experimental data. 254x190mm (96 x 96 DPI)


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Tests of the penetration of the water soluble metabolites to the phospholipid monolayers: a) CHA to TMCL, b) PCP to TMCL, c) CHA to DMPC, d) PCP to DMPC. 272x208mm (300 x 300 DPI)



PM-IRRAS spectra registered for TMCL and DMPC monolayers compressed to 10 or 30 mN/m before (black line) and after the injection of HCNA or PCP solutions below the monolayers: a) TMCL on HCNA, b) DMPC on HCNA, c) TMCL on PCP, d) DMPC on PCP 254x190mm (96 x 96 DPI)


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Effects of Polychlorinated Pesticides and Their Metabolites on

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