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The pesticide picloram affects biomembrane models made with Langmuir monolayers
Colloids and Surfaces B: Biointerfaces 181 (2019) 953–958
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The pesticide picloram affects biomembrane models made with Langmuir monolayers
T
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Tibebe Lemmaa, , Gilia Cristine Marques Ruiza, Osvaldo N. Oliveira Jr.b, Carlos J.L. Constantinoa a b
Faculdade de Ciências e Tecnologia (FCT)-Universidade Estadual Paulista (UNESP)-Presidente Prudente, SP, 19060-900, Brazil São Carlos Institute of Physics, University of Sao Paulo, CP 369, São Carlos, SP, 13560-970, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: DOPC/SM DOPC/SM/Chol GUV Picloram Monolayer π-A isotherm
Cell membrane models are useful to obtain molecular-level information on the interaction of biologically-relevant molecules such as pesticides whose activity is believed to depend on its effects on the membrane. In this study, we investigated the interaction between the widely used pesticide picloram with Langmuir monolayers of binary and ternary mixtures comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM) and cholesterol (Chol), which could be taken as representative of ocular membranes in humans. Picloram expanded the molecular area of DOPC/SM and DOPC/SM/Chol monolayers as the pesticide penetrated the hydrophobic region of the mixtures. A clear correlation was also found between the compressibility modulus (Cs−1) and the presence of cholesterol in the ternary monolayer. Data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) confirmed that picloram interacts with both the acyl chains and headgroups. Spectral shifts and band broadening were induced by picloram, particularly for the phosphate and choline groups, probably owing to its H-bonding ability. The effects reported here on the lipid monolayers may be evidence of the possible activity of picloram on mammalian cell membranes, which highlights the importance of strict control of the level of exposure of humans dealing with pesticides.
1. Introduction Pesticides are toxic substances useful in food production for protecting agricultural products by controlling or repelling pests such as insects, weeds, and organisms responsible for plant diseases (microbes). The term ‘pesticide’ encompasses various groups including, but not limited to, herbicides, insecticides, fungicides, bactericides, and rodenticides. The several classes of pesticides include organophosphate pesticides (OPPs), organochlorine pesticides (OCPs) [1,2], carbamates [3], synthetic pyrethroids [4], biopesticides [5], and microbial pesticides [6]. Though essential in modern high-production agriculture, pesticides continue to pose danger to human health (especially farm workers), animals, and the environment due to extensive usage and improper handling. The type and severity of the problems depend on the pesticide`s toxicity, chemistry and the mode of action on specific targets. Some are very toxic to a degree of specificity (targeting certain organs or accumulating) and may cause serious injury to humans and non-target organisms such as bees, birds, wild life and plants in or near the target site. Exposure of humans to these pesticides can occur in several ways, through oral exposure, dermal absorption, inhalation, and contaminated drinking water and food. The most serious exposure
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occurs through the eyes and skin during handling of pesticides and spraying or mixing of materials [7]. In this study, we investigate effects from picloram (3,5,6-trichloro4-amino-2-pyridinecarboxylic acid) (Fig. S1), used as a broad-spectrum herbicide to control weeds in sugar cane, wheat, pasture, pineapple, sorghum, and other woody plant species [8,9]. Picloram is absorbed by plant roots and foliage and tends to translocate throughout the plant with the tendency to accumulate at its upper portion. It belongs to the chlorobenzoic acid family, being the most persistent and mobile of herbicides in the soil with a half-life in the field from 20 to 300 days [10]. It has been used in small as well as in large-scale agricultural crops throughout the industrial world and developing nations. As elsewhere, many pesticides are used in Brazil and the country recently has become a major global consumer of agrotoxins, consuming one billion liters of pesticides in the 2009/10 seasons [11]. The widespread and largely unregulated use of chemical pesticides, however, has led to a harmful effect on both human and the environment. Studies have been conducted to link occupational exposure (spraying, mixing and handling of sprayed plants) to pesticides with an increased risk factor for chronic diseases, particularly among those who lack proper training and use less protective clothing. Harm to public health associated with
Corresponding author. E-mail address: [email protected] (T. Lemma).
https://doi.org/10.1016/j.colsurfb.2019.06.060 Received 16 April 2019; Received in revised form 18 June 2019; Accepted 25 June 2019 Available online 26 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 181 (2019) 953–958
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corresponds to the liquid-expanded (LE) phase, from 100 to 250 mN/m it refers to the liquid-condensed (LC) monolayer and the solid phase of monolayers has even higher modulus [18]. The PM-IRRAS spectra at the air/water interface with and without picloram were recorded using a KSV PMI 550 spectrometer (Helsinki, Finland) connected with an external reflection setup. A detailed experimental method and the optical configurations used have been described elsewhere [19]. Briefly, the main optical setup consists of an IR source, a Michelson interferometer from a Bruker IFS66 (Bruker, Karlsruhe, Germany) spectrometer, a photoelastic modulator (PEM, Hinds Instruments, PEM-90, ZnSe modulator optical head) and a reflection unit. The beam has its polarization modulated between p (on the plane of incidence) and polarization perpendicular to this plane (s) with a frequency of 50 kHz. For each spectrum, 300 scans and acquisition time of 10 min were taken for both the background and sample, using 8 cm−1 per modulation center. Band fitting and baseline correction were performed on Origin 8.0 software. In order to compare the vibrational spectroscopic properties, cast films were obtained from DOPC/SM and DOPC/SM/Chol, which also included picloram. The solvents used for casting were methanol:chloroform in a 7:3 ratio, with the exception of picloram for which ethanol was used. Smooth films were obtained by casting the solution on germanium substrates, and left to evaporate at 40 °C. The FTIR spectra of the cast films in Figs. S2 and S3 of the Supporting Information were acquired at room temperature in the transmission mode with a Bruker Tensor 27 spectrometer equipped with a mercury cadmium telluride (MCT) detector. The spectra were recorded at a resolution of 4 cm−1 for 256 scans within the wavenumber region of 600–4000 cm−1. The manipulation and evaluation of the spectra, in particular, the baseline correction and smoothing, were carried out using the Bruker OPUS software. Table S1 in the Supporting Information shows the band assignments for the spectra in Figs. S2 and S3.
these pesticides includes interference with cellular function, damage to the reproductive, endocrine, immune, or respiratory system, especially of agriculture workers that come to contact with pesticides with inhalation of sprayed pesticide or by ingestion of pesticide in foods or by skin contacts [12]. The potential role of these pesticides exposure has been studied on numerous pesticides-induced biological interactions including tissue, cell, enzymes [13,14] in the human body. Indeed, there is a need to assess the long and short-term effect of these pesticides and evaluate the routes of exposure and toxicity, especially for those humans chronically exposed. The action mechanism of pesticides, as it occurs for pharmaceutical drugs, depends on their interaction with cell membranes. Investigation of such interactions to obtain molecular-level information in living cells is not straightforward, which is why cell membrane models are employed. Lipid monolayers, for instance, provide an excellent biomimetic model for studying phenomena such as ligand-membrane interactions. Typically, the cell membrane is represented by a lipid-based model which consists of one or two lipid bilayers from artificial phospholipids. Binding or insertion of ligand molecules into the lipid monolayer can be inferred through a change in the surface pressure-mean molecular area (π-A) isotherm or compressibility modulus. In Langmuir monolayers, lipids are compressed to the lateral pressure corresponding to a cell membrane (˜30 mN/m) [15]), and the composition of the lipids and subphase can be varied. Moreover, the temperature can be set to mimic a biological condition so that the data obtained from Langmuir monolayers may be useful in predicting interactions with cell membranes [16]. In our study, we have used mixtures of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) and egg sphingomyelin (SM), and DOPC, SM and cholesterol (Chol) to investigate the effect of picloram, including an attempt to obtain molecular-level information using in-situ polarizationmodulated infrared reflection absorption spectroscopy (PM-IRRAS). 2. Materials and methods
3. Results and discussion All chemicals were of reagent or analytical grade except where stated otherwise. 3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid (Picloram), glucose, sucrose, and cholesterol were purchased from Sigma–Aldrich (Brazil). DOPC, SM and Chol were all obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Ultrapure water was obtained from an ultrapure water system (Direct-Q Water Purification System, Brazil). Phosphate buffer (pH 7.4, 50 mM, 50 mM NaCl) was prepared with ultrapure water (18 MΩ cm). All the buffer solutions were refrigerated until use. The π-A isotherms were measured with a Langmuir trough (KSV NIMA, model KN 2002) at 23 °C. The monolayers of the ternary and binary mixture of DOPC/SM/Chol and DOPC/SM, with or without the pesticide, were obtained by spreading the solution of lipids in methanol/chloroform (7:3) ratio with a 50 μL microsyringe. About 35–40 μL of the lipid mixture were spread homogeneously on the subphase containing picloram and the solvent was allowed to evaporate for 15 min before compression proceeded. The compression speed was carefully selected (10 mm min–1) to avoid nucleation of three-dimensional phase and quasi-static state, which do not represent the true phase of the system. The data were also checked for the value of the liftoff point to detect loss of materials during subsequent compression. The curves were subjected to deconvolution using OriginPro (version 10). The compressibility modulus (Cs−1) was calculated from the surface pressure isotherms using:
Cs−1 = −
3.1. Surface pressure isotherms 3.1.1. DOPC/SM + picloram Cholesterol is believed to play a role in regulating membrane trafficking and the permeation of molecular species across the lipid bilayer [20]. To study this putative role of cholesterol in the interactions with picloram, monolayers were first obtained with the zwitterionic DOPC/ SM mixture without cholesterol. The π-A isotherms of the binary mixtures from DOPC and SM in Fig. 1 show that incorporation of picloram causes the liftoff area per molecule to increase from 97 to 108 Å2, thus indicating migration of picloram to the air/water interface even in the gas phase. The areas per molecule at five surface pressures are compared for monolayers containing (or not) picloram in Tables S2 and S3 in the Supporting Information, which indicates that picloram is expected to remain at the interface even at high surface pressures. In addition, picloram had little effect on the collapse pressure, which changed from 47 mN/m for DOPC/SM to 45 mN/m in the presence of picloram. The inset in Fig. 1 shows no significant changes in Cs−1 between the DOPC/SM monolayer and that containing picloram, with both monolayers being in the LC phase at 30 mN/m, the pressure believed to correspond to the lipid packing in a cell membrane [21]. Therefore, we can conclude that although picloram has no significant effect on the film elasticity, it penetrates into the hydrocarbon region of the lipid monolayer.
1 ⎛ ∂A ⎞ A ⎝ ∂π ⎠
3.1.2. DOPC/SM/Chol + picloram Incorporation of cholesterol into the lipid composition (DOPC/SM) to form DOPC/SM/Chol monolayers increases the packing density (condensation effect) and the rigidity of the acyl chains [22]. This is noted by comparing the results in Figs. 1 and 2, with the maximum Cs−1 changing from 115 mN/m for DOPC/SM to 180 mN/m for DOPC/
Where, A is the mean molecular area (MMA) in Å2 and π is the corresponding surface pressure. According to Davies and Rideal [17] the compressibility modulus values can characterize the phase state of a monolayer. For instance, in the gaseous phase the modulus ranges between 0 and 20 mN/m, whereas a modulus from 12.5 to 50 mN/m 954
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However, the area expansion is smaller than for DOPC/SM, with an increase of 4 to 6 Ų at 30 mN/m. Thus, the effect of picloram appears to be somewhat hindered by cholesterol. The modulating effect of cholesterol in bilayers has been investigated extensively, including condensing, permeability and lateral diffusion of molecules [27–31]. A steep increase in pressure was observed from 5 to 40 mN/m, and the monolayer collapse occurred at around 43 mN/m. Although the DOPC/ SM/Chol-picloram π-A isotherm approached that of DOPC/SM/Chol close to collapse, picloram molecules were not squeezed out of the mixed film. The Cs−1 vs π curves in the inset in Fig. 2 indicate the formation of condensed phases with small effects from picloram, especially at π = 30 mN/m. 3.2. PM-IRRAS spectra for DOPC/SM and DOPC/SM/Chol monolayers 3.2.1. DOPC/SM vs DOPC/SM/Chol in the hydrocarbon tail region The in situ PM-IRRAS spectra in the region (2700–3000 cm−1) in Fig. 3 provides information about the acyl moiety in the lipid molecules, with CeH vibrations at 2915–2940 and 2840–2870 cm−1 assigned to asymmetric (CH2) and symmetric (CH2) stretching modes of the methylene, respectively. Changes in CH2 scissoring (1473 and 1468 cm−1), symmetric bending (1370–1380 cm−1), wagging (1200–1400 cm−1) and CH3 asymmetric vibration modes, known to be sensitive to perturbation of the acyl group, can also be used to assess the effects [32,33]. The DOPC/SM spectrum in Fig. 3A shows two broad bands at 2916 and 2846 cm−1, assigned to the asymmetric and symmetric stretching mode of CH2, respectively. Both bands have spectral overlapping, which should be expected owing to the mixture of different compounds. The shoulder at 2941 cm−1 is due to the asymmetric CH3 stretching mode. There is also another broad band at 2767 cm−1, which is accompanied by a band at 2720 cm−1 from the co-contribution of NH(NH3+) and CH3 stretching, respectively [34]. As seen in Fig. 3B, incorporation of picloram caused the CH2 asymmetric stretching mode at 2916 cm−1 to shift to 2921 cm−1, while the CH2 symmetric stretching at 2846 cm−1 split into two unresolved bands at 2838 and 2806 cm−1, with broadening. The broad band at 2767 cm−1 remained nearly in the same position and the weaker feature at 2720 cm−1 split into two very weak bands at 2709 and 2736 cm−1. For the DOPC/SM/ Chol mixture, Fig. 3C shows the band due to CH2 asymmetric stretching mode in the same position as for the DOPC/SM monolayer, while the band assigned to the symmetric stretching mode of CH2 is blue shifted
Fig. 1. Surface pressure-mean molecular area (π-A) isotherms for (A) mixture of DOPC/SM on air/water subphase and (B) on 1 mM aqueous solution of picloram at 23⁰C. The inset shows the data for compressibility modulus as a function of the surface pressure. All the isotherm performed in triplicate but we use the result of only one isotherm for each mixture.
SM/Chol. It is postulated that under normal physiological conditions, diffusion of molecular species through a lipid monolayer strongly depends on the composition and packing of the lipids [23,24]. It is also documented that the packing effect occurs through cholesterol interaction with the lipids, preferably with SM [25,26]. The π-A isotherm for the monolayer of DOPC/SM/Chol at 23 °C in Fig. 2A features a typical behavior of stable lipid monolayers at the air/water interface. On ultrapure water, the π-A curve of the lipid mixture shows three distinct regions: the initial slope from 0 mN/m (160 Å2/molecule) corresponds to the two-dimensional gas (G) phase. This is followed by the first limb of increasing surface pressure at ˜2 mN/m for both cases signaling the emergence of the liquid-expanded (LE) phase. Upon further compression, a transition to the liquid-condensed (LC) phase is observed at ˜5 mN/m and the surface pressure rapidly increases up to monolayer collapse (˜43 mN/m), forming a condensed monolayer. An expansion in the ternary monolayer is induced by picloram with the liftoff area increasing from 78 to 90 Å2/molecule (Fig. 2B), probably because it penetrates into the tail region without affecting the monolayer elasticity.
Fig. 2. Surface pressure-mean molecular area (π-A) isotherm plot of monolayers at a compression rate of 10 mm/min (A) mixture of DOPC/SM/Chol and DOPC/SM monolayers spread on air/water subphase and (B) on 1 mM picloram of aqueous solution at 22 °C. The inset show the compressibility modulus calculated for the isotherms as a function of the surface pressure. All the isotherm performed in triplicate but we use the result of only one isotherm for each mixture. 955
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Fig. 3. PM-IRRAS spectra in C–H stretching region for (A) DOPC/SM; (B) DOPC/SM-picloram; (C) DOPC/SM/Chol; (D) DOPC/SM/Chol-picloram. All the spectra were measured at 30 mN/m.
to 2850 cm−1, suggesting the preferential orientation of the symmetric bond in the presence of cholesterol. The broad bands at 2767 and 2720 cm−1 shift to 2760 and 2715 cm−1 respectively, along with an intensity increase. This apparent change is strong evidence that cholesterol causes perturbation of the acyl group as a result of the H-bond interactions with the lipid, preferentially with sphingomyelin in the mixture. [35,36] Incorporation of picloram affected the acyl chains, according to Fig. 3D. The band due to CH2 symmetric stretching mode is blue-shifted from 2916 in Fig. 3C to 2924 cm−1, while the mode ascribed to asymmetric CH2 is red-shifted to 2838 cm−1, which appears at 2850 cm−1 in Fig. 3C. The band at 2760 cm−1 also changed, with the broad band splitting into two shoulders (2770 and 2743 cm−1). Thus, the combined evidence from the spectral shift, intensity and band broadening indicates that picloram penetrates the hydrophobic core and induces conformational changes in the acyl moiety of both DOPC/ SM and DOPC/SM/Chol monolayers.
attached group on the SM (Fig. S3 in the Supporting Information) [34]). This is rather broad compared with the solid-state spectra in Fig. S3 in the Supporting Information, therefore leading to the expectation that the two carbonyls may undergo H-bonding or overlap with other modes. The structural differences between the two carbonyl groups manifest in their broad and medium intense absorption spectra in Fig. 4B. Interestingly, the band at 1659 cm−1 broadens and splits into two modes at 1657 and 1685 cbm−1, while that at 1745 cm−1 does not shift significantly at the air/picloram interface in Fig. 4. This may indicate that the picloram molecules have preferential interaction with SM (amide group) than with DOPC of the ternary mixture, thereby explaining the strong broadening effects and spectral shift for the 1659 cm−1 mode. On the other hand, in the spectrum of DOPC/SM, the two modes shift in opposite direction to 1616 and 1735 cm−1 compared with the ternary mixture, and the relative intensity of both modes decreases significantly (Fig. 4C). This spectral shift and broadening may point to intermolecular H-bonding near the head group (both PO4 and N+(CH3)3) or self-association of head groups. The spectrum also shows two weak bands at 1767 and 1690 cm−1 owing to the interaction of the two head groups. A similar behavior has been observed in the acyl group spectrum, although the effect was much less pronounced. Fig. 4D shows that upon inserting picloram in the DOPC/SM monolayer, the negative band near 1616 cm−1 shifts slightly to 1621 cm−1, which
3.2.2. DOPC/SM and DOPC/SM/Chol in the headgroup region The PM-IRRAS spectrum of DOPC/SM/Chol at air/water interface in Fig. 4A is dominated by two types of carbonyl (C]O) stretching modes, at 1659 cm−1 and near 1745 cm−1. Generally, the ester carbonyl groups of lipids give rise to the characteristic band at 1745 cm−1, while the carbonyl mode at 1659 cm−1 probably originates from the amide
Fig. 4. PM-IRRAS spectra in 1600-1800 cm−1 region for: (A) DOPC/SM/Chol, (B) DOPC/SM/ Chol-picloram, (C) DOPC/SM and (D) DOPC/ SM-picloram. All the spectra measured at 30 mN/m. The spectra demonstrate that the addition of cholesterol to the binary system causes significant broadening and peak shift at the same time. The strong positive absorption band indicates that the IR transition dipole moments are parallel to the interface.
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present at the headgroup rather than distributed throughout the lipid mixture. This may also explain why the binary system is more susceptible to such a perturbation due to the absence of cholesterol in the mixture. The in-situ PM-IRRAS spectrum of the ternary complex at the air/ water interface is given in Fig. S5C. There are overlapping bands arising from the contribution of cholesterol in the mixture. The ternary spectrum exhibits two weak bands at 1326 and 1370 cm−1 ascribed to the wagging mode of CH2 and the bending mode of the CH3 group. The broad and medium intense band at 1406 cm−1 is the CH symmetrical bending mode in N+(CH3)3 group. The other intense band at 1462 cm−1 is due to the CH3 bending mode, while the last two bands at 1526 and 1575 cm−1 can be assigned to NeH bending modes, respectively. For the DOPC/SM/Chol mixture at the air/picloram interface, the PM-IRRAS spectrum exhibits a significant spectral variation in comparison with the results obtained from the ternary system at the air/water interface. The absorption bands at 1326 and 1370 cm−1 blueshifted to 1350 and 1391 cm−1 and tending to merge into one unresolved broad band. The same applies to the two bands at 1406 and 1462 cm−1. The two intense and broad bands merge into one single and merge into one very broad band centered at 1516 cm−1. The broad peak at 1516 cm−1 split into two bands at 1506 and 1535 cm−1, while the band at 1575 cm−1 become a broad shoulder on the low-wavenumber side of the band at 1557 cm−1 (Fig. S5D). This is consistent with a strong interaction at the N+(CH3)3, which influences the neighboring phosphate and ester group on the headgroup.
indicates that picloram disturbed the headgroup packing. In addition, the band intensity decreased sharply and a weak shoulder appeared near 1657 cm−1 which confirms the presence of other modes. An even more pronounced downshift (1690 cm−1) was observed for the mode at 1735 cm−1, which confirms the strong interaction between picloram and the headgroup. The vibrational modes in the spectral region 1000 to 1300 cm−1 result from the symmetric and antisymmetric stretching of the phosphate group and CeOePeOeC and COeOeC stretching modes [34]. The PM-IRRAS spectra of both DOPC/SM and DOPC/SM/chol monolayers in Fig. S4 show spectral shift and band broadening compared with those of the spectra collected from the cast film. This suggests that the headgroup may be involved in inter- and/or intramolecular Hbonding. For the DOPC/SM mixture in Fig. S4A, there is an intense, broad band at 1216 cm−1 assigned to asymmetric PO2− stretching mode with a weak shoulder near 1243 cm−1. This broad band split into a triplet at 1271, 1231 and 1203 cm−1 with weak intensity. The asymmetric (COeOeC) stretching mode also shifts from 1156 cm−1 to 1161 cm−1 in Fig. S4B. The symmetric (PO2−) stretching mode at 1082 cm−1 remains in the same position but its intensity increases owing to the presence of picloram. This behavior may be due to intermolecular H-bonding between the lipid headgroup with the picloram molecule. The two bands from the stretching mode of the COePeOeC group at 1045 and 1020 cm−1 merge into a single broad peak and redshifted to 1041 cm−1, along with the intensity increase. With picloram in the subphase, the in-situ PM-IRRAS spectrum of the DOPC/SM/Chol monolayer exhibited broad bands, with decreased band intensity and band shifts compared to the same monolayer without picloram. The asymmetric band at 1211 cm−1 split into two weak bands (not well resolved) at 1196 and 1218 cm−1, which is attributed to the perturbation induced by picloram in the headgroup environment, such as with hydrogen bonds. Therefore, the results confirm that the headgroup are affected by picloram, mostly owing to changes in H-bonding. The spectral region for DOPC/SM and DOPC/SM/chol monolayers most affected by picloram is the choline region between 1300–1600 cm−1, as shown in the spectra in Fig. S5 in the Supporting Information. The choline moiety is the outermost fragment of the binary (ternary) system with quaternary nitrogen based cationic functional group and plays an important role in both chemical stability and structural conformation at the interface. The choline groups are highly hydrated and the most susceptible fragments to alterations in the presence of active molecules at the interface. This leads to a conformation change and rearrangement of the other lipid components on the headgroup, which may even affect the hydrocarbon chains [34]. The PM-IRRAS spectrum of DOPC/SM mixture at the air/water interface is shown in Fig. S5A, featuring seven broad bands at 1333, 1373, 1410, 1448, 1500, 1531 and 1566 cm−1 assigned as follows: 1333 and 1337 cm−1 to wagging modes of the CH2 groups and to the symmetric bending of modes of the CH2, the band at 1410 cm−1 is the CH asymmetric bending mode in N+(CH3)3 The broad band at 1448 cm−1 involves the asymmetric bending mode of CH3 and the last two distinctly unresolved modes at 1500 and 1531 cm−1 are associated with the overlapping asymmetric bend of CH in N+(CH3)3 group and deformation modes of (CeH) and (NeH) groups, respectively [34]. When picloram is incorporated in the monolayer, nearly all the vibrational modes undergo spectra distortion, broadening and intensity decrease, in particular in the DOPC/SM system in Fig. S5B. The bands at 1410 and 1448 cm−1 are blue-shifted to 1443 and 1466 cm−1 and almost completely merged into one broad band. The band at 1500 cm−1 shifted to 1507 cm−1 and a weak shoulder appears below the main band. Furthermore, the band at 1566 cm−1 broadens and shifted to 1580 cm−1, while the band at 1333 cm−1 is red shifted, respectively. A similar pattern was observed for the band at 1373 cm−1, which is blueshifted and split into a doublet at 1367 and 1389 cm−1. The changes in relative intensities and band positions of several modes at the air/picloram interface suggests that considerable amount of picloram may be
4. Conclusions In this work, we investigated the interaction of picloram with Langmuir monolayers of binary and ternary mixtures made with DOPC/ SM and DOPC/SM/Chol, which were characterized with surface pressure isotherms and PM-IRRAS. The results from π-A isotherms pointed to picloram affecting the packing and intermolecular interactions of the membrane-forming lipid molecules, with a small but non-negligible area expansion, indicating that picloram occupies an additional area at the interface. This shows that the picloram penetrates into the hydrophobic region of the mixtures and perturbs the mechanical properties and film packing. From PM-IRRAS data, we observed that picloram affects both the headgroups and acyl chains of DOPC/SM and DOPC/ SM/Chol monolayers, with stronger effects on the headgroups probably due to its H-bonding ability. It is hoped that the effects of picloram on Langmuir monolayers representing simple models of cell membranes may be correlated with its activity. Acknowledgments The experimental work summarized in this paper was supported by FAPESP (2016/06424-5, 2013/14262-7). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.060. References [1] B. Junhong, L. Qiongqiong, Z. Qingqing, W. Junjing, G. Zhaoqin, Z. Guangliang, Organochlorine pesticides (OCPs) in wetland soils under different land uses along a 100-year chronosequence of reclamation in a Chinese estuary, Sci. Rep. 5 (2015) 17624. [2] K. Jones, P. de Voogt, Persistent organic pollutants (POPs): state of the science, Environ Pollut 100 (1999) 209–221. [3] A. Vale, M. Lotti, Organophosphorus and carbamate insecticide poisoning, Handb. Clin. Neurol. 131 (2015) 149–168. [4] S.J. Maund, P.J. Campbell, J.M. Giddings, M.J. Hamer, K. Henry, E.D. Pilling, J.S. Warinton, J.R. Wheeler, Ecotoxicology of synthetic pyrethroids, Top. Curr. Chem. 314 (2012) 137–165.
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The pesticide picloram affects biomembrane models made with Langmuir monolayers
T
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Tibebe Lemmaa, , Gilia Cristine Marques Ruiza, Osvaldo N. Oliveira Jr.b, Carlos J.L. Constantinoa a b
Faculdade de Ciências e Tecnologia (FCT)-Universidade Estadual Paulista (UNESP)-Presidente Prudente, SP, 19060-900, Brazil São Carlos Institute of Physics, University of Sao Paulo, CP 369, São Carlos, SP, 13560-970, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: DOPC/SM DOPC/SM/Chol GUV Picloram Monolayer π-A isotherm
Cell membrane models are useful to obtain molecular-level information on the interaction of biologically-relevant molecules such as pesticides whose activity is believed to depend on its effects on the membrane. In this study, we investigated the interaction between the widely used pesticide picloram with Langmuir monolayers of binary and ternary mixtures comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM) and cholesterol (Chol), which could be taken as representative of ocular membranes in humans. Picloram expanded the molecular area of DOPC/SM and DOPC/SM/Chol monolayers as the pesticide penetrated the hydrophobic region of the mixtures. A clear correlation was also found between the compressibility modulus (Cs−1) and the presence of cholesterol in the ternary monolayer. Data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) confirmed that picloram interacts with both the acyl chains and headgroups. Spectral shifts and band broadening were induced by picloram, particularly for the phosphate and choline groups, probably owing to its H-bonding ability. The effects reported here on the lipid monolayers may be evidence of the possible activity of picloram on mammalian cell membranes, which highlights the importance of strict control of the level of exposure of humans dealing with pesticides.
1. Introduction Pesticides are toxic substances useful in food production for protecting agricultural products by controlling or repelling pests such as insects, weeds, and organisms responsible for plant diseases (microbes). The term ‘pesticide’ encompasses various groups including, but not limited to, herbicides, insecticides, fungicides, bactericides, and rodenticides. The several classes of pesticides include organophosphate pesticides (OPPs), organochlorine pesticides (OCPs) [1,2], carbamates [3], synthetic pyrethroids [4], biopesticides [5], and microbial pesticides [6]. Though essential in modern high-production agriculture, pesticides continue to pose danger to human health (especially farm workers), animals, and the environment due to extensive usage and improper handling. The type and severity of the problems depend on the pesticide`s toxicity, chemistry and the mode of action on specific targets. Some are very toxic to a degree of specificity (targeting certain organs or accumulating) and may cause serious injury to humans and non-target organisms such as bees, birds, wild life and plants in or near the target site. Exposure of humans to these pesticides can occur in several ways, through oral exposure, dermal absorption, inhalation, and contaminated drinking water and food. The most serious exposure
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occurs through the eyes and skin during handling of pesticides and spraying or mixing of materials [7]. In this study, we investigate effects from picloram (3,5,6-trichloro4-amino-2-pyridinecarboxylic acid) (Fig. S1), used as a broad-spectrum herbicide to control weeds in sugar cane, wheat, pasture, pineapple, sorghum, and other woody plant species [8,9]. Picloram is absorbed by plant roots and foliage and tends to translocate throughout the plant with the tendency to accumulate at its upper portion. It belongs to the chlorobenzoic acid family, being the most persistent and mobile of herbicides in the soil with a half-life in the field from 20 to 300 days [10]. It has been used in small as well as in large-scale agricultural crops throughout the industrial world and developing nations. As elsewhere, many pesticides are used in Brazil and the country recently has become a major global consumer of agrotoxins, consuming one billion liters of pesticides in the 2009/10 seasons [11]. The widespread and largely unregulated use of chemical pesticides, however, has led to a harmful effect on both human and the environment. Studies have been conducted to link occupational exposure (spraying, mixing and handling of sprayed plants) to pesticides with an increased risk factor for chronic diseases, particularly among those who lack proper training and use less protective clothing. Harm to public health associated with
Corresponding author. E-mail address: [email protected] (T. Lemma).
https://doi.org/10.1016/j.colsurfb.2019.06.060 Received 16 April 2019; Received in revised form 18 June 2019; Accepted 25 June 2019 Available online 26 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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corresponds to the liquid-expanded (LE) phase, from 100 to 250 mN/m it refers to the liquid-condensed (LC) monolayer and the solid phase of monolayers has even higher modulus [18]. The PM-IRRAS spectra at the air/water interface with and without picloram were recorded using a KSV PMI 550 spectrometer (Helsinki, Finland) connected with an external reflection setup. A detailed experimental method and the optical configurations used have been described elsewhere [19]. Briefly, the main optical setup consists of an IR source, a Michelson interferometer from a Bruker IFS66 (Bruker, Karlsruhe, Germany) spectrometer, a photoelastic modulator (PEM, Hinds Instruments, PEM-90, ZnSe modulator optical head) and a reflection unit. The beam has its polarization modulated between p (on the plane of incidence) and polarization perpendicular to this plane (s) with a frequency of 50 kHz. For each spectrum, 300 scans and acquisition time of 10 min were taken for both the background and sample, using 8 cm−1 per modulation center. Band fitting and baseline correction were performed on Origin 8.0 software. In order to compare the vibrational spectroscopic properties, cast films were obtained from DOPC/SM and DOPC/SM/Chol, which also included picloram. The solvents used for casting were methanol:chloroform in a 7:3 ratio, with the exception of picloram for which ethanol was used. Smooth films were obtained by casting the solution on germanium substrates, and left to evaporate at 40 °C. The FTIR spectra of the cast films in Figs. S2 and S3 of the Supporting Information were acquired at room temperature in the transmission mode with a Bruker Tensor 27 spectrometer equipped with a mercury cadmium telluride (MCT) detector. The spectra were recorded at a resolution of 4 cm−1 for 256 scans within the wavenumber region of 600–4000 cm−1. The manipulation and evaluation of the spectra, in particular, the baseline correction and smoothing, were carried out using the Bruker OPUS software. Table S1 in the Supporting Information shows the band assignments for the spectra in Figs. S2 and S3.
these pesticides includes interference with cellular function, damage to the reproductive, endocrine, immune, or respiratory system, especially of agriculture workers that come to contact with pesticides with inhalation of sprayed pesticide or by ingestion of pesticide in foods or by skin contacts [12]. The potential role of these pesticides exposure has been studied on numerous pesticides-induced biological interactions including tissue, cell, enzymes [13,14] in the human body. Indeed, there is a need to assess the long and short-term effect of these pesticides and evaluate the routes of exposure and toxicity, especially for those humans chronically exposed. The action mechanism of pesticides, as it occurs for pharmaceutical drugs, depends on their interaction with cell membranes. Investigation of such interactions to obtain molecular-level information in living cells is not straightforward, which is why cell membrane models are employed. Lipid monolayers, for instance, provide an excellent biomimetic model for studying phenomena such as ligand-membrane interactions. Typically, the cell membrane is represented by a lipid-based model which consists of one or two lipid bilayers from artificial phospholipids. Binding or insertion of ligand molecules into the lipid monolayer can be inferred through a change in the surface pressure-mean molecular area (π-A) isotherm or compressibility modulus. In Langmuir monolayers, lipids are compressed to the lateral pressure corresponding to a cell membrane (˜30 mN/m) [15]), and the composition of the lipids and subphase can be varied. Moreover, the temperature can be set to mimic a biological condition so that the data obtained from Langmuir monolayers may be useful in predicting interactions with cell membranes [16]. In our study, we have used mixtures of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) and egg sphingomyelin (SM), and DOPC, SM and cholesterol (Chol) to investigate the effect of picloram, including an attempt to obtain molecular-level information using in-situ polarizationmodulated infrared reflection absorption spectroscopy (PM-IRRAS). 2. Materials and methods
3. Results and discussion All chemicals were of reagent or analytical grade except where stated otherwise. 3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid (Picloram), glucose, sucrose, and cholesterol were purchased from Sigma–Aldrich (Brazil). DOPC, SM and Chol were all obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Ultrapure water was obtained from an ultrapure water system (Direct-Q Water Purification System, Brazil). Phosphate buffer (pH 7.4, 50 mM, 50 mM NaCl) was prepared with ultrapure water (18 MΩ cm). All the buffer solutions were refrigerated until use. The π-A isotherms were measured with a Langmuir trough (KSV NIMA, model KN 2002) at 23 °C. The monolayers of the ternary and binary mixture of DOPC/SM/Chol and DOPC/SM, with or without the pesticide, were obtained by spreading the solution of lipids in methanol/chloroform (7:3) ratio with a 50 μL microsyringe. About 35–40 μL of the lipid mixture were spread homogeneously on the subphase containing picloram and the solvent was allowed to evaporate for 15 min before compression proceeded. The compression speed was carefully selected (10 mm min–1) to avoid nucleation of three-dimensional phase and quasi-static state, which do not represent the true phase of the system. The data were also checked for the value of the liftoff point to detect loss of materials during subsequent compression. The curves were subjected to deconvolution using OriginPro (version 10). The compressibility modulus (Cs−1) was calculated from the surface pressure isotherms using:
Cs−1 = −
3.1. Surface pressure isotherms 3.1.1. DOPC/SM + picloram Cholesterol is believed to play a role in regulating membrane trafficking and the permeation of molecular species across the lipid bilayer [20]. To study this putative role of cholesterol in the interactions with picloram, monolayers were first obtained with the zwitterionic DOPC/ SM mixture without cholesterol. The π-A isotherms of the binary mixtures from DOPC and SM in Fig. 1 show that incorporation of picloram causes the liftoff area per molecule to increase from 97 to 108 Å2, thus indicating migration of picloram to the air/water interface even in the gas phase. The areas per molecule at five surface pressures are compared for monolayers containing (or not) picloram in Tables S2 and S3 in the Supporting Information, which indicates that picloram is expected to remain at the interface even at high surface pressures. In addition, picloram had little effect on the collapse pressure, which changed from 47 mN/m for DOPC/SM to 45 mN/m in the presence of picloram. The inset in Fig. 1 shows no significant changes in Cs−1 between the DOPC/SM monolayer and that containing picloram, with both monolayers being in the LC phase at 30 mN/m, the pressure believed to correspond to the lipid packing in a cell membrane [21]. Therefore, we can conclude that although picloram has no significant effect on the film elasticity, it penetrates into the hydrocarbon region of the lipid monolayer.
1 ⎛ ∂A ⎞ A ⎝ ∂π ⎠
3.1.2. DOPC/SM/Chol + picloram Incorporation of cholesterol into the lipid composition (DOPC/SM) to form DOPC/SM/Chol monolayers increases the packing density (condensation effect) and the rigidity of the acyl chains [22]. This is noted by comparing the results in Figs. 1 and 2, with the maximum Cs−1 changing from 115 mN/m for DOPC/SM to 180 mN/m for DOPC/
Where, A is the mean molecular area (MMA) in Å2 and π is the corresponding surface pressure. According to Davies and Rideal [17] the compressibility modulus values can characterize the phase state of a monolayer. For instance, in the gaseous phase the modulus ranges between 0 and 20 mN/m, whereas a modulus from 12.5 to 50 mN/m 954
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However, the area expansion is smaller than for DOPC/SM, with an increase of 4 to 6 Ų at 30 mN/m. Thus, the effect of picloram appears to be somewhat hindered by cholesterol. The modulating effect of cholesterol in bilayers has been investigated extensively, including condensing, permeability and lateral diffusion of molecules [27–31]. A steep increase in pressure was observed from 5 to 40 mN/m, and the monolayer collapse occurred at around 43 mN/m. Although the DOPC/ SM/Chol-picloram π-A isotherm approached that of DOPC/SM/Chol close to collapse, picloram molecules were not squeezed out of the mixed film. The Cs−1 vs π curves in the inset in Fig. 2 indicate the formation of condensed phases with small effects from picloram, especially at π = 30 mN/m. 3.2. PM-IRRAS spectra for DOPC/SM and DOPC/SM/Chol monolayers 3.2.1. DOPC/SM vs DOPC/SM/Chol in the hydrocarbon tail region The in situ PM-IRRAS spectra in the region (2700–3000 cm−1) in Fig. 3 provides information about the acyl moiety in the lipid molecules, with CeH vibrations at 2915–2940 and 2840–2870 cm−1 assigned to asymmetric (CH2) and symmetric (CH2) stretching modes of the methylene, respectively. Changes in CH2 scissoring (1473 and 1468 cm−1), symmetric bending (1370–1380 cm−1), wagging (1200–1400 cm−1) and CH3 asymmetric vibration modes, known to be sensitive to perturbation of the acyl group, can also be used to assess the effects [32,33]. The DOPC/SM spectrum in Fig. 3A shows two broad bands at 2916 and 2846 cm−1, assigned to the asymmetric and symmetric stretching mode of CH2, respectively. Both bands have spectral overlapping, which should be expected owing to the mixture of different compounds. The shoulder at 2941 cm−1 is due to the asymmetric CH3 stretching mode. There is also another broad band at 2767 cm−1, which is accompanied by a band at 2720 cm−1 from the co-contribution of NH(NH3+) and CH3 stretching, respectively [34]. As seen in Fig. 3B, incorporation of picloram caused the CH2 asymmetric stretching mode at 2916 cm−1 to shift to 2921 cm−1, while the CH2 symmetric stretching at 2846 cm−1 split into two unresolved bands at 2838 and 2806 cm−1, with broadening. The broad band at 2767 cm−1 remained nearly in the same position and the weaker feature at 2720 cm−1 split into two very weak bands at 2709 and 2736 cm−1. For the DOPC/SM/ Chol mixture, Fig. 3C shows the band due to CH2 asymmetric stretching mode in the same position as for the DOPC/SM monolayer, while the band assigned to the symmetric stretching mode of CH2 is blue shifted
Fig. 1. Surface pressure-mean molecular area (π-A) isotherms for (A) mixture of DOPC/SM on air/water subphase and (B) on 1 mM aqueous solution of picloram at 23⁰C. The inset shows the data for compressibility modulus as a function of the surface pressure. All the isotherm performed in triplicate but we use the result of only one isotherm for each mixture.
SM/Chol. It is postulated that under normal physiological conditions, diffusion of molecular species through a lipid monolayer strongly depends on the composition and packing of the lipids [23,24]. It is also documented that the packing effect occurs through cholesterol interaction with the lipids, preferably with SM [25,26]. The π-A isotherm for the monolayer of DOPC/SM/Chol at 23 °C in Fig. 2A features a typical behavior of stable lipid monolayers at the air/water interface. On ultrapure water, the π-A curve of the lipid mixture shows three distinct regions: the initial slope from 0 mN/m (160 Å2/molecule) corresponds to the two-dimensional gas (G) phase. This is followed by the first limb of increasing surface pressure at ˜2 mN/m for both cases signaling the emergence of the liquid-expanded (LE) phase. Upon further compression, a transition to the liquid-condensed (LC) phase is observed at ˜5 mN/m and the surface pressure rapidly increases up to monolayer collapse (˜43 mN/m), forming a condensed monolayer. An expansion in the ternary monolayer is induced by picloram with the liftoff area increasing from 78 to 90 Å2/molecule (Fig. 2B), probably because it penetrates into the tail region without affecting the monolayer elasticity.
Fig. 2. Surface pressure-mean molecular area (π-A) isotherm plot of monolayers at a compression rate of 10 mm/min (A) mixture of DOPC/SM/Chol and DOPC/SM monolayers spread on air/water subphase and (B) on 1 mM picloram of aqueous solution at 22 °C. The inset show the compressibility modulus calculated for the isotherms as a function of the surface pressure. All the isotherm performed in triplicate but we use the result of only one isotherm for each mixture. 955
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Fig. 3. PM-IRRAS spectra in C–H stretching region for (A) DOPC/SM; (B) DOPC/SM-picloram; (C) DOPC/SM/Chol; (D) DOPC/SM/Chol-picloram. All the spectra were measured at 30 mN/m.
to 2850 cm−1, suggesting the preferential orientation of the symmetric bond in the presence of cholesterol. The broad bands at 2767 and 2720 cm−1 shift to 2760 and 2715 cm−1 respectively, along with an intensity increase. This apparent change is strong evidence that cholesterol causes perturbation of the acyl group as a result of the H-bond interactions with the lipid, preferentially with sphingomyelin in the mixture. [35,36] Incorporation of picloram affected the acyl chains, according to Fig. 3D. The band due to CH2 symmetric stretching mode is blue-shifted from 2916 in Fig. 3C to 2924 cm−1, while the mode ascribed to asymmetric CH2 is red-shifted to 2838 cm−1, which appears at 2850 cm−1 in Fig. 3C. The band at 2760 cm−1 also changed, with the broad band splitting into two shoulders (2770 and 2743 cm−1). Thus, the combined evidence from the spectral shift, intensity and band broadening indicates that picloram penetrates the hydrophobic core and induces conformational changes in the acyl moiety of both DOPC/ SM and DOPC/SM/Chol monolayers.
attached group on the SM (Fig. S3 in the Supporting Information) [34]). This is rather broad compared with the solid-state spectra in Fig. S3 in the Supporting Information, therefore leading to the expectation that the two carbonyls may undergo H-bonding or overlap with other modes. The structural differences between the two carbonyl groups manifest in their broad and medium intense absorption spectra in Fig. 4B. Interestingly, the band at 1659 cm−1 broadens and splits into two modes at 1657 and 1685 cbm−1, while that at 1745 cm−1 does not shift significantly at the air/picloram interface in Fig. 4. This may indicate that the picloram molecules have preferential interaction with SM (amide group) than with DOPC of the ternary mixture, thereby explaining the strong broadening effects and spectral shift for the 1659 cm−1 mode. On the other hand, in the spectrum of DOPC/SM, the two modes shift in opposite direction to 1616 and 1735 cm−1 compared with the ternary mixture, and the relative intensity of both modes decreases significantly (Fig. 4C). This spectral shift and broadening may point to intermolecular H-bonding near the head group (both PO4 and N+(CH3)3) or self-association of head groups. The spectrum also shows two weak bands at 1767 and 1690 cm−1 owing to the interaction of the two head groups. A similar behavior has been observed in the acyl group spectrum, although the effect was much less pronounced. Fig. 4D shows that upon inserting picloram in the DOPC/SM monolayer, the negative band near 1616 cm−1 shifts slightly to 1621 cm−1, which
3.2.2. DOPC/SM and DOPC/SM/Chol in the headgroup region The PM-IRRAS spectrum of DOPC/SM/Chol at air/water interface in Fig. 4A is dominated by two types of carbonyl (C]O) stretching modes, at 1659 cm−1 and near 1745 cm−1. Generally, the ester carbonyl groups of lipids give rise to the characteristic band at 1745 cm−1, while the carbonyl mode at 1659 cm−1 probably originates from the amide
Fig. 4. PM-IRRAS spectra in 1600-1800 cm−1 region for: (A) DOPC/SM/Chol, (B) DOPC/SM/ Chol-picloram, (C) DOPC/SM and (D) DOPC/ SM-picloram. All the spectra measured at 30 mN/m. The spectra demonstrate that the addition of cholesterol to the binary system causes significant broadening and peak shift at the same time. The strong positive absorption band indicates that the IR transition dipole moments are parallel to the interface.
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present at the headgroup rather than distributed throughout the lipid mixture. This may also explain why the binary system is more susceptible to such a perturbation due to the absence of cholesterol in the mixture. The in-situ PM-IRRAS spectrum of the ternary complex at the air/ water interface is given in Fig. S5C. There are overlapping bands arising from the contribution of cholesterol in the mixture. The ternary spectrum exhibits two weak bands at 1326 and 1370 cm−1 ascribed to the wagging mode of CH2 and the bending mode of the CH3 group. The broad and medium intense band at 1406 cm−1 is the CH symmetrical bending mode in N+(CH3)3 group. The other intense band at 1462 cm−1 is due to the CH3 bending mode, while the last two bands at 1526 and 1575 cm−1 can be assigned to NeH bending modes, respectively. For the DOPC/SM/Chol mixture at the air/picloram interface, the PM-IRRAS spectrum exhibits a significant spectral variation in comparison with the results obtained from the ternary system at the air/water interface. The absorption bands at 1326 and 1370 cm−1 blueshifted to 1350 and 1391 cm−1 and tending to merge into one unresolved broad band. The same applies to the two bands at 1406 and 1462 cm−1. The two intense and broad bands merge into one single and merge into one very broad band centered at 1516 cm−1. The broad peak at 1516 cm−1 split into two bands at 1506 and 1535 cm−1, while the band at 1575 cm−1 become a broad shoulder on the low-wavenumber side of the band at 1557 cm−1 (Fig. S5D). This is consistent with a strong interaction at the N+(CH3)3, which influences the neighboring phosphate and ester group on the headgroup.
indicates that picloram disturbed the headgroup packing. In addition, the band intensity decreased sharply and a weak shoulder appeared near 1657 cm−1 which confirms the presence of other modes. An even more pronounced downshift (1690 cm−1) was observed for the mode at 1735 cm−1, which confirms the strong interaction between picloram and the headgroup. The vibrational modes in the spectral region 1000 to 1300 cm−1 result from the symmetric and antisymmetric stretching of the phosphate group and CeOePeOeC and COeOeC stretching modes [34]. The PM-IRRAS spectra of both DOPC/SM and DOPC/SM/chol monolayers in Fig. S4 show spectral shift and band broadening compared with those of the spectra collected from the cast film. This suggests that the headgroup may be involved in inter- and/or intramolecular Hbonding. For the DOPC/SM mixture in Fig. S4A, there is an intense, broad band at 1216 cm−1 assigned to asymmetric PO2− stretching mode with a weak shoulder near 1243 cm−1. This broad band split into a triplet at 1271, 1231 and 1203 cm−1 with weak intensity. The asymmetric (COeOeC) stretching mode also shifts from 1156 cm−1 to 1161 cm−1 in Fig. S4B. The symmetric (PO2−) stretching mode at 1082 cm−1 remains in the same position but its intensity increases owing to the presence of picloram. This behavior may be due to intermolecular H-bonding between the lipid headgroup with the picloram molecule. The two bands from the stretching mode of the COePeOeC group at 1045 and 1020 cm−1 merge into a single broad peak and redshifted to 1041 cm−1, along with the intensity increase. With picloram in the subphase, the in-situ PM-IRRAS spectrum of the DOPC/SM/Chol monolayer exhibited broad bands, with decreased band intensity and band shifts compared to the same monolayer without picloram. The asymmetric band at 1211 cm−1 split into two weak bands (not well resolved) at 1196 and 1218 cm−1, which is attributed to the perturbation induced by picloram in the headgroup environment, such as with hydrogen bonds. Therefore, the results confirm that the headgroup are affected by picloram, mostly owing to changes in H-bonding. The spectral region for DOPC/SM and DOPC/SM/chol monolayers most affected by picloram is the choline region between 1300–1600 cm−1, as shown in the spectra in Fig. S5 in the Supporting Information. The choline moiety is the outermost fragment of the binary (ternary) system with quaternary nitrogen based cationic functional group and plays an important role in both chemical stability and structural conformation at the interface. The choline groups are highly hydrated and the most susceptible fragments to alterations in the presence of active molecules at the interface. This leads to a conformation change and rearrangement of the other lipid components on the headgroup, which may even affect the hydrocarbon chains [34]. The PM-IRRAS spectrum of DOPC/SM mixture at the air/water interface is shown in Fig. S5A, featuring seven broad bands at 1333, 1373, 1410, 1448, 1500, 1531 and 1566 cm−1 assigned as follows: 1333 and 1337 cm−1 to wagging modes of the CH2 groups and to the symmetric bending of modes of the CH2, the band at 1410 cm−1 is the CH asymmetric bending mode in N+(CH3)3 The broad band at 1448 cm−1 involves the asymmetric bending mode of CH3 and the last two distinctly unresolved modes at 1500 and 1531 cm−1 are associated with the overlapping asymmetric bend of CH in N+(CH3)3 group and deformation modes of (CeH) and (NeH) groups, respectively [34]. When picloram is incorporated in the monolayer, nearly all the vibrational modes undergo spectra distortion, broadening and intensity decrease, in particular in the DOPC/SM system in Fig. S5B. The bands at 1410 and 1448 cm−1 are blue-shifted to 1443 and 1466 cm−1 and almost completely merged into one broad band. The band at 1500 cm−1 shifted to 1507 cm−1 and a weak shoulder appears below the main band. Furthermore, the band at 1566 cm−1 broadens and shifted to 1580 cm−1, while the band at 1333 cm−1 is red shifted, respectively. A similar pattern was observed for the band at 1373 cm−1, which is blueshifted and split into a doublet at 1367 and 1389 cm−1. The changes in relative intensities and band positions of several modes at the air/picloram interface suggests that considerable amount of picloram may be
4. Conclusions In this work, we investigated the interaction of picloram with Langmuir monolayers of binary and ternary mixtures made with DOPC/ SM and DOPC/SM/Chol, which were characterized with surface pressure isotherms and PM-IRRAS. The results from π-A isotherms pointed to picloram affecting the packing and intermolecular interactions of the membrane-forming lipid molecules, with a small but non-negligible area expansion, indicating that picloram occupies an additional area at the interface. This shows that the picloram penetrates into the hydrophobic region of the mixtures and perturbs the mechanical properties and film packing. From PM-IRRAS data, we observed that picloram affects both the headgroups and acyl chains of DOPC/SM and DOPC/ SM/Chol monolayers, with stronger effects on the headgroups probably due to its H-bonding ability. It is hoped that the effects of picloram on Langmuir monolayers representing simple models of cell membranes may be correlated with its activity. Acknowledgments The experimental work summarized in this paper was supported by FAPESP (2016/06424-5, 2013/14262-7). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.060. References [1] B. Junhong, L. Qiongqiong, Z. Qingqing, W. Junjing, G. Zhaoqin, Z. Guangliang, Organochlorine pesticides (OCPs) in wetland soils under different land uses along a 100-year chronosequence of reclamation in a Chinese estuary, Sci. Rep. 5 (2015) 17624. [2] K. Jones, P. de Voogt, Persistent organic pollutants (POPs): state of the science, Environ Pollut 100 (1999) 209–221. [3] A. Vale, M. Lotti, Organophosphorus and carbamate insecticide poisoning, Handb. Clin. Neurol. 131 (2015) 149–168. [4] S.J. Maund, P.J. Campbell, J.M. Giddings, M.J. Hamer, K. Henry, E.D. Pilling, J.S. Warinton, J.R. Wheeler, Ecotoxicology of synthetic pyrethroids, Top. Curr. Chem. 314 (2012) 137–165.
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