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Effects and Location of Coplanar and Non-Coplanar PCB in a Lipid Bilayer: A Solid-State NMR Study Christian Totland, Willy Nerdal, and Signe Steinkopf Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01723 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016
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Effects and Location of Coplanar and NonCoplanar PCB in a Lipid Bilayer: A Solid-State NMR Study Christian Totland†*, Willy Nerdal†, Signe Steinkopf‡ †University of Bergen, Norway, Department of Chemistry, Allégaten 41, N-5007 Bergen, Norway ‡Bergen University College, Department of Biomedical Laboratory, Sciences and Chemical Engineering
*Corresponding Author: Email:
[email protected] Phone: +47 55588233
KEYWORDS. Solid-State NMR; model bilayer; polychlorinated biphenyls; PCB; POPC
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ABSTRACT
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Coplanar and non-coplanar polychlorinated biphenyls (PCBs) are known to have different
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routes and degree of toxicity. Here, the effects of non-coplanar PCB 52 and coplanar PCB 77
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present at 2 mol% in a model system consisting of POPC liposomes (50 % hydrated) are
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investigated by solid-state
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the outer part of the bilayer, near the segments of the acyl chain close to the glycerol group.
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Despite similar membrane locations, the coplanar PCB 77 shows little effect on the bilayer
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properties overall, except for the four nearest neighboring lipids, while the effect of PCB 52 is
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more dramatic. The first ~2 layers of lipids around each PCB 52 in the bilayer form a high
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fluidity lamellar phase, whereas lipids beyond these layers form a lamellar phase with slight
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increase in fluidity compared to a bilayer without PCB 52. Further, a third high mobility
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domain is observed. The explanation for this is the interference of several high fluidity
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lamellar phases caused by interactions of PCB 52 molecules in different leaflets of the model
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bilayer. This causes formation of high curvature toroidal region in the bilayer, and might
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induce formation of channels.
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C and
31
P NMR at 298K. Both PCBs intercalate horizontally in
PCB 77 PCB 52
TOC graphic
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INTRODUCTION
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The use of polychlorinated biphenyls (PCBs) has been highly restricted or banned in several
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countries since the 1970s and 80s, although a worldwide ban was not in place until 2001.1
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However, the PCBs low biodegradability and high lipophilicity cause these compounds to
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accumulate in the adipose tissue of mammals as well as other creatures such as birds and
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fish.2,
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locations such as Antarctica.4 Due to the high environmental persistence and high resistance
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to decomposition in living organisms,5, 6 PCBs accumulate up the food chain, causing human
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exposure through both aquatic and terrestrial food sources. The possible effects of prolonged
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human exposure have therefore received considerable attention.7
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Despite slow degradation, PCBs are still found in the atmosphere, even in remote
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There are as much as 209 possible configurations of PCB, where the degree and route of
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toxicity of various congeners varies significantly.7 From studies of structure-activity
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relationships it is suggested that coplanar PCBs without chlorine substitution in ortho position
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have biological properties similar to dioxin (2,3,7,8-tetrachlorobibenzo-p-dioxin), showing
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carcinogenic effects by acting on the aryl hydrocarbon receptor.7, 8 The non-coplanar PCBs on
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the other hand, show different and more complex routes of toxicity, with for example
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estrogenic and neurotoxic activity.9-11 Additionally, non-coplanar PCBs have shown to
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increase the fluidity of cellular membranes,12-14 which may affect the activity of multiple
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membrane proteins.15 Consequently, the physiological effects of non-coplanar PCBs may be
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far-reaching compared to coplanar PCBs.
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Most previous literature focuses on effects of PCBs on cellular membranes, which
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illuminates effects of PCBs but makes it difficult to investigate the mechanisms of action. In
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order to access the core mechanisms it is helpful to utilize a model lipid membrane. PCB 77
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and PCB 52 have been used in several earlier studies,14, 16, 17 representing coplanar and non-
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coplanar PCB, respectively. Tan et al. showed that PCB 52 causes rapid cell death in
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thymocytes and cerebellar granule cell neurons, altering multiple membrane components,
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while PCB 77 showed none of these effects.14,
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polarization measurements that PCB 52 increases membrane fluidity, which was linked to the
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stereochemistry of the more bulky PCB 52 compared to PCB 77, causing a greater impact on
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membrane properties when intercalated in the bilayer structure.16 Campbell et al. investigated
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interaction of these PCBs with a model 1,2-dimyristoyl-sn-glycero-3-phosphocholine
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(DMPC) lipid membrane using atomic force microscopy (ATM) and differential scanning
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calorimetry (DSC).17 It was observed that bilayers with PCB 52 had a lower gel-to-liquid
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phase transition temperature. Additionally, fluorescence correlation spectroscopy of PCB in
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1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) fluid bilayers showed that PCB 52
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diffuses more slowly than PCB 77 in the bilayer.17 It was further concluded that, contrary to
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PCB 52, PCB 77 does not intercalate into the lipid bilayers. This latter conclusion was later
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questioned by Jonker and van der Heijden.18
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It was further shown from fluorescent
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In order to fully evaluate the effect and location of different PCBs in a lipid bilayer, atomic-
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level investigation of the bilayer is required. A technique capable of this is nuclear magnetic
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resonance (NMR) spectroscopy.19-22 The amount of PCB interacting with a membrane
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relevant to human exposure is relatively small, and remains below the detection limit of 13C
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NMR. Still, high-resolution magic angle spinning (MAS) 13C NMR can give detailed insight
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into the lipid-PCB interaction by monitoring changes in lipid
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NMR gives direct and precise information on bilayer morphology and fluidity.23 These
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techniques are applied here on a system comprising of uni-lamellar liposomes of POPC – a
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biologically abundant lipid – with and without PCB 52 or PCB 77.
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C spectra. Additionally,
31
P
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The purpose of this work is to establish how non-coplanar and coplanar PCBs interact with
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a lipid bilayer and what possible consequences such interactions have to the bilayer
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properties.
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MATERIALS AND METHODS
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Materials. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was obtained from
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Avanti Polar Lipids (Birmingham, AL). PCB 52 and PCB 77 where obtained from Sigma
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Aldrich.
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Liposome Preparation. The amount of PCB in the samples is 2.0 mol%, which is above
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the limit where nonspecific toxicity occurs for non-coplanar PCB.24 The lipid samples were
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dissolved in chloroform, which was then evaporated and the samples where lyophilized
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overnight. The dry samples were suspended in degassed, argon bubbled water and left on a
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water bath for 1 h at 40°C. In order to obtain mostly uni-lamellar liposomes the sample was
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freeze-thawed seven times using liquid N2.22 The samples were pH-adjusted to 7.4 by adding
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a small amount of 0.05M NaOH with freezing and thawing between each adjustment. The
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electrolyte concentration as a result of the pH adjustment was in the order of 10-4 M. Samples
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were subjected to 24 h of lyophilization giving partially hydrated liposomes with a hydration
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level of ~12 water molecules per lipid molecule.21, 22 Thus, samples with about 50 wt% water
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(fully hydrated lipids) could be obtained by adding degassed argon-bubbled water.25, 26 The
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samples were then equilibrated at 40°C for 48 h and packed in NMR rotors in an argon-
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atmosphere and stored in the freezer.
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NMR. The NMR data was obtained on a Bruker AV III HD 500 instrument. The instrument
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is equipped with magic angle spinning (MAS) hardware for 4 mm rotors (ZrO2). Experiments
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were carried out at a sample temperature of 298 K.
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spinning rate of 6 kHz with high-power proton decoupling during acquisition. The
13
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experiments were acquired with a relaxation delay of 5s and 15000 scans. For the static
31
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spectra 5000 scans were collected with a relaxation delay of 3s between transients. MAS 31P
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experiments were carried out with a rotor spinning speed of 2.5 kHz. Simulations of static 31P
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NMR spectra were carried out with the Topspin software (version 3.5).
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C spectra were obtained at a sample C P
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B
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C *
POPC/ PCB52
POPC/ PCB77
*
POPC
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50
0
- 50 50
0
ppm
0
-2
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Figure 1. Recorded (A) and simulated (B) static 31P NMR spectra of POPC samples (50wt.%
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hydration) with and without PCB 52 or 77. The central peak of corresponding MAS spectra
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with 2 kHz spinning are shown in C: An asterisk marks an isotropic high mobility phase
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(does not give spinning side bands), while the remaining peaks represent lamellar phases. The
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amount of PCB in the samples is 2 mol% relative to the lipid.
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RESULTS AND DISCUSSION
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31
P NMR. Figure 1 shows the static (A), simulated (B) and MAS (C) 31P NMR spectra of
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the three samples containing POPC, POPC + PCB 77 or POPC + PCB 52 with 50w%
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hydration. The spectrum of pure POPC shows as expected a single lamellar phase with
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chemical shift anisotropy (CSA) at 43 ppm. The static spectrum of the POPC+PCB 77 sample
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(Figure 1 A) shows the same lamellar phase, but with a small fraction of a higher mobility
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isotropic phase. As seen from the simulated spectrum (Figure 1 B), this isotropic phase
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consists of a negligible fraction of POPC in the sample. It is not clear whether this isotropic
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phase is related to interaction with PCB 77, as a small fraction of isotropic POPC may occur
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during sample preparation regardless of PCB. Although not visible in the static spectrum, the 6 ACS Paragon Plus Environment
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corresponding MAS spectrum of the POPC + PCB 77 sample (Figure 1 C) shows presence of
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a small additional lamellar phase, corresponding to 8.5 % of the sample lipids. This lamellar
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phase is not observed in the static spectrum due to low abundance. If this lamellar phase is
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related to lipids interacting with PCB 77, it corresponds to about four affected lipids per PCB
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77 molecule.
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However, changes in POPC morphology in the presence of PCB 52 are clearly seen in the
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31
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spectrum (Figure 1 A&B), which is typical for a micellar-like phase. This phase corresponds
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to 18% of sample lipids. In addition, two lamellar phases with greater fluidity than that
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observed for the POPC and POPC+PCB 77 samples. The CSAs of the two lamellar phases
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with 2 mol% PCB 52 present are 31 and 17 ppm, representing 50 % and 32 % of sample
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lipids, respectively. The CSA value is inversely proportional to both the axial rotation of the
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lipids and the degree of lipid oscillation. Hence, higher lipid mobility or larger angle of the
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oscillation will induce higher bilayer fluidity and cause lower CSA values. This indicates
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increased fluidity compared to the lower mobility lamellar phase observed in the POPC and
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POPC+PCB 77 samples (CSA = 43 ppm).
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P spectra (Figure 1), where a high-mobility phase is indicated by a sharp peak in the static
The
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P NMR data confirm results from earlier studies on both cellular and model lipid
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bilayer samples where the presence of PCB 52 is found to increase bilayer fluidity.14,
17, 26
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Similar to previous studies, PCB 77 do not significantly affect the membrane fluidity;
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however, 4 lipids per PCB 77 adopt a lamellar phase with slightly different properties. The
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nature of the lipid morphologies are discussed in a separate section.
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sn-1 O
O P
O
C B A
O
O
α β H3C
γ
O
+ CH γ3
N
CH3
2’ O
1
16’
1’
CH3
2
9
CH3
10
18
O
sn-2
γ
B 0.25
PCB 77 PCB 52
∆δ (ppm)
0.15 0.05
γ
β
1 2 3 α C B A 1’ 2’ 3’ 4
6’ 11/8’ 7 -9’
16 17 9 10 14’ 15’
-0.05 -0.15
130
-0.25
131
Figure 2. A: Molecular structure of POPC. B: Differences in 13C chemical shifts (∆δ) of the
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POPC lamellar phase in the presence of PCB 77 () and PCB 52 (□), compared to a sample
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of POPC only. A positive value of ∆δ means that the peak in question shifts to higher ppm
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values in the presence of PCB (∆δ = δw/PCB – δPOPC). The molecular structure of POPC is
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shown with labelling of the carbons according to the assignment in the plot and in Figure 3
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and Figure S1 and S2 of the Supporting Information.
137 138
13
C NMR. There have been controversies regarding the localizations of PCB 52 and 77
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with respect to the bilayer. It has previously been proposed that PCB 77 does not intercalate
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between the lipid molecules and only interacts with the head group,17 something that was later
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argued as unlikely due to the lipophilic nature of PCB.18 In this section the locations of PCB
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52 and 77 are determined by 13C NMR.
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Figures 2 and 3 show the effect of PCB intercalation in the POPC bilayer. Compared to the 13
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reference sample with only POPC, the
C chemical shifts of lamellar POPC are altered in
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samples containing PCB 77 or PCB 52 (Figure 2 B). From the plot of chemical shift
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differences in Figure 2, two main observations are made. First, the pattern of chemical shift
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1 9
A
10
B
POPC
POPC/ PCB77
* POPC/ PCB52
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178
176
174
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[ppm]
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Figure 3. 13C MAS NMR spectra of the carbonyl (A) and C=C (B) regions of the POPC (top),
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POPC + PCB 77 (middle) and POPC + PCB 52 (bottom) samples at 298 K with 50 wt.%
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hydration. Peak labels are in accordance with the molecular structure given in Figure 2. The
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amount of PCB in the sample is 2 mol% relative to the lipid. The peak labelled with an
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asterisk is interpreted as POPC in a different phase. Assignments are based on ref. 27
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differences is similar for both the PCB 52 and PCB 77 sample. However, the changes in
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chemical shift are slightly larger for the PCB 52 sample. Second, the changes in chemical
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shift are more pronounced for the carbonyl carbons (1’, 1) and subsequent acyl chain carbons.
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This indicates both that PCB 52 and 77 intercalate at the same position in the POPC bilayer,
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and that this position is below the POPC carbonyl where POPC carbons in general experience
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a more crowded chemical environment, as evident from the decrease in chemical shift.
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The displacement of the carbonyl resonance to higher chemical shift values (Figure 3 A)
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reflects a conformational change where the angle between the acyl chain and head group
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increases upon intercalation of PCB. The increased angle reduces stearic interactions between
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the carbonyl carbon and surrounding nuclei which in turn reduces the chemical shift. An
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accuracy of
