Article pubs.acs.org/est
Lipophilic Contaminants Influence Cold Tolerance of Invertebrates through Changes in Cell Membrane Fluidity Martin Holmstrup,*,† Hélène Bouvrais,‡ Peter Westh,§ Chunhua Wang,§ Stine Slotsbo,† Dorthe Waagner,† Kirsten Enggrob,† and John H. Ipsen‡ †
Department of Bioscience, Aarhus University, Vejlsøvej 25, DK-8600 Silkeborg, Denmark Department of Physics, Chemistry and Pharmacy, MEMPHYS − Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark § Research Unit for Functional Biomaterials, Roskilde University, Universitetsvej 1, Building 18.1, DK-4000 Roskilde, Denmark ‡
S Supporting Information *
ABSTRACT: Contaminants taken up by living organisms in the environment as a result of anthropogenic contamination can reduce the tolerance of natural stressors, e.g., low temperatures, but the physiological mechanisms behind these interactions of effects are poorly understood. The tolerance to low temperatures of organisms that cannot regulate their body temperature (ectotherms) depends on their ability to increase the fluidity of their cellular membranes at low temperatures. Our study shows that contaminants accumulating in lipids of organisms alter the physical state of their membranes simply by being present. Contaminants of varying chemical structures can alter the membrane fluidity in either direction and correspondingly modulate the cold tolerance of intact animals.
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INTRODUCTION Adaptive changes in the composition of membrane phospholipids constitute a common feature of ectothermic organisms and are thought to preserve appropriate fluidity of the cell membrane under changing temperatures.1−3 A fully functioning cell membrane is in the liquid crystalline state, but when the temperature is lowered, the membrane is gradually ordered and stiffened and may ultimately undergo transition to a gel phase, which is incompatible with membrane functionality.4 Such a phase transition is a particular challenge to cold-tolerance because it can lead to a loss of intracellular metabolites and ions and a damaging uptake of sodium and calcium.1 Membrane malfunction may also depend on more subtle changes in the properties of the lipid matrix. For example, it has been suggested that solute-induced changes in the lateral pressures along the membrane may couple to the function of membrane proteins.5 The capacity of environmental contaminants to modify the membrane is reflected in the mechanical and thermodynamic properties, which can be characterized by biophysical techniques. In this work, we have chosen to measure the membrane bending rigidity and main transition temperature (Tm) of simple model membranes in the presence of contaminants. The mechanical properties of biomembranes are linked to a multitude of biological processes, e.g., membrane fusion, fission and membrane-pore formation. The membrane mechanics can be characterized by a few parameters, in particular, the © XXXX American Chemical Society
membrane bending rigidity, which reflects the stiffness of the membrane with respect to bending deformations. The bending rigidity, κ, can be estimated by vesicle fluctuation analysis,6 which has the advantages of being a noninvasive technique and allowing changes in the membrane mechanical properties to be investigated under precise and variable conditions. Recent improvements in this technique (both technical and theoretical7) have led to a gain in the precision of the estimated parameters that allow this tool to investigate complex systems. Furthermore, the bending rigidity has proved to be highly sensitive to the effects of membrane inclusions such as peptides.8 Membrane interactions can also be assessed from solute-induced changes in the phase behavior, which are readily measured by techniques such as scanning calorimetry (DSC). In particular, shifts in Tm show whether the solute favors the disordered fluid phase or the ordered gel phase. Our first approach was to use simple membranes (composed of a single lipid) to evaluate the impacts of contaminants: this minimalist approach enabled us to focus on the contaminant molecule itself. Recently, there has been an increased focus in cell biology on the role of lateral domain structures for the functioning of biological membranes, which is a manifestation of the phase separation in multicomponent membranes.9 The Received: May 5, 2014 Revised: July 21, 2014 Accepted: July 22, 2014
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spiked soil was thoroughly mixed and left overnight under a fume hood to allow the acetone to evaporate. Subsequently, the soil−water content was adjusted to about 50% of the water holding capacity (220 mL kg−1 dry soil). Five adult E. albidus were added to 10 mL plastic vials containing 5 g of moist soil. Thirty adult F. candida were added to 200 mL plastic beakers containing 60 g of moist soil. The containers were closed with perforated lids, allowing ventilation. Cold Tolerance Assays. Vials with E. albidus exposed to contaminated soil were kept at 2 °C for 8 days, and subsequently at −1.5 °C (±0.1 °C). After 24 h, a small ice crystal was added to the soil surface, which initiated freezing of the moist soil and subsequent freezing of the enchytraeids.19 After another 24 h at −1.5 °C, the vials were shifted to a programmable cooling cabinet, which lowered the temperature from −2 °C by 3 °C d−1 until the target temperatures of −5 °C (NP exposed worms) or −8 °C (PHE exposed worms), respectively, were reached. The vials were thawed at 5 °C for 24 h until the assessment of survival.19 The controls were kept at 2 °C throughout the experiments. Beakers with F. candida exposed to contaminated soil were kept at 20 °C for 8 days. Tap water was then added to the beakers and the collembolans were rapidly extracted from soil by flotation and transferred to Petri dishes with dry plaster of Paris. The collembolans were transferred with the aid of an aspirator to 1.5 mL Eppendorf vials (10 specimens vial−1) and then exposed to cold shock at various temperatures and durations, e.g., −5 °C, 2 h, in a temperature-controlled bath with a precision of ±0.05 °C (Lauda Eco RE 1050, VWR Bie and Berntsen A/S, Herlev, Denmark). After this cold shock treatment, the collembolans were allowed to recover on moistened plaster of Paris/charcoal Petri dishes at 20 °C (±1 °C) for 24 h before their survival was assessed as the ability to walk in a coordinated manner.20 Determination of PHE and NP in Animal Tissue. The animals were sampled for analysis immediately before cold exposure, i.e., after exposure for 8 days in soil. The E. albidus were rinsed in demineralized water and gently blotted with filter paper. The fresh weight of 30 individual samples transferred to Eppendorf vials was then determined using a Sartorius Micro SC 2 balance accurate to ±1 μg (Sartorius AG, Goettingen, Germany). The samples were frozen at −80 °C until analysis. The F. candida were sampled from the Petri dishes immediately after being collected by flotation, placed in preweighed Eppendorf vials, weighed and frozen. Further samples were used for the gravimetric determination of their water content after freeze-drying and total lipid content determination as previously described.21 Samples for analysis of PHE were transferred to 1.5 mL brown glass vials and 500 μL of acetonitrile was added. The samples were placed on ice, sonicated for 90 min and then kept at room temperature for 24 h, frozen at −18 °C for 24 h and finally kept at room temperature for 24 h. The samples were again sonicated for 90 min on ice and were then transferred to Eppendorf vials. After brief centrifugation (3 min; 2620g), the supernatant was transferred to autosampler vials ready for analysis. PHE standards were run in parallel and subjected to the same extraction procedure. A Shimadzu GCMS-QP2010 with an autosampler was used to perform the analysis (see the Supporting Information for further details). Samples of animals for analysis of NP were homogenized in 1.5 mL of 70% ethanol with a steel ball using a Tissuelyser II (Qiagen Gmbh, Hilden, Germany). The homogenate was
lateral lipid domains, which are characterized by differing compositions and lipid acyl-chain ordering, can be modified by environmental contaminants. For instance, numerical simulations have predicted that small lipophilic contaminants can alter the lateral domain structures dramatically,10 but very little experimental evidence to substantiate this hypothesis exists. Thus, it was also necessary to investigate the effects of the contaminants on “real membranes” to confirm, or not, the results we obtained from a single-component membrane. To understand the effects of contaminants on membrane properties, a molecular description of their interactions is necessary. However, the combination of a hydrophobic core and an interfacial zone with extreme dipolar field strengths, all within a few nm, make membrane−solute interactions quite complex and highly dependent on the chemical structure of the solute. Amphiphilic molecules, for example, generally partition into the membrane interface and cause lateral expansion and disordering of the membrane core.11,12 This effect is particularly strong for charged amphiphiles such as fatty acids.13 Membrane partitioning for more polar compounds is controversial,14−16 but even molecules that are repelled from the interface affect the membrane properties indirectly through osmotic gradients close to the surface.17,18 Here, we provide evidence that the membrane partitioning of two lipophilic compounds, namely phenanthrene (representing the family of aromatic hydrocarbons) and 4-nonylphenol (an amphiphilic, yet hydrophobic, organic compound, which in our case is a mixture of branched isomers of 4-nonylphenol), translates into oppositely directed changes of model membrane fluidity and bending rigidity, and that this phenomenon is congruent with the cold tolerance of intact invertebrates exposed to these contaminants. Partitioning of sublethal concentrations of phenanthrene (PHE) causes membranes to become more fluid in vitro, and this effect was congruent with an increased cold tolerance of the freeze-tolerant oligochaete, Enchytraeus albidus, and the chill-sensitive collembolan, Folsomia candida. In contrast to this, membrane partitioning of 4-nonylphenol (NP) reduced the fluidity of model membranes in vitro and also reduced the cold tolerance of these two animals. Our study links the partitioning of contaminants into membranes with phenotypic responses to cold, and hence fundamentally improves our understanding of combined exposure to contaminants and natural stressors.
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METHODS Animals. The Enchytraeus albidus (Oligochaeta, Enchytraeidae) were kept in moistened soil at 5 °C and fed rolled oats mixed with dried and crushed macroalgae as previously described.19 The Enchytraeids were adult and had a fresh weight of 2−3 mg when used for the experiments. The Folsomia candida (Collembola, Isotomidae) used in this study were from a laboratory culture kept at 20 °C (±1 °C) and in a 12:12 h light:dark cycle on Petri dishes with water-saturated plaster of Paris mixed with charcoal. The collembolans were fed dried baker’s yeast and were 6−8 weeks old with a fresh weight in the range from 150 to 200 μg when used for the experiments. Exposure to NP and PHE. The animals were exposed via soil using a well-defined natural soil (LUFA 2.2; Speyer, Germany). Briefly, the soil was spiked with NP (Aldrich, CAS # 84852-15-3, 100% pure) or PHE (Aldrich, CAS # 85-01-8, 98% purity) dissolved in acetone to obtain the desired concentrations (mg kg−1 dry soil). Chemical structures of NP and PHE are shown in Figure S1 (Supporting Information). The B
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of 2 mg mL−1 and multilamellar vesicles were produced by taking the samples through 10 temperature cycles (15−40 °C with 5 min of stirring at each temperature). The vesicles were loaded into a Nano differential scanning calorimeter (TA Instruments New Castle, DE, USA) and heated from 10 to 35 °C at a rate of 0.5 °C min−1 using pure water as a reference. The transition temperature (Tm) was identified as the apex of the transition. Multilamellar vesicles were chosen as they provide sharp peaks in Differential Scanning Calorimetry (DSC) and hence the best resolution for solute induced changes in Tm. Generally, Tm for multilamellar vesicles is indistinguishable from Tm of unilamellar vesicles of a size comparable to a living cell.22 Bending rigidity Measurements. Giant unilamellar vesicles (GUVs) were produced using either POPC lipids (1,2-palmitoyloleyl-sn-glycero-3-phosphocholine; Avanti Polar Lipids; Alabater, AL, USA) or whole-body membrane lipid extracts obtained as described above. GUVs of diameters in the range of 10−50 μm were formed from submicrometerliposomes (SUVs) using a modified electroswelling technique.23 These SUVs were composed either of pure POPC or were reconstituted from lipid extracts of the studied invertebrates. The GUVs were observed in a phase-contrast microscope at 20 °C and recorded at 25 frames per second with a video integration time of 4 ms in order to visualize the thermal fluctuations of the membranes. Their 2D contours were then extracted by homemade image processing software. This flickering was finally analyzed by statistical methods involving Fourier decompositions of the angular correlation function.7 For each set of conditions, a number of independent liposomes, 8−12, was analyzed and the same procedures were applied (detailed information on GUV technique is found in the Supporting Information). Statistics. The effect of contaminants on the survival rates was tested by using contingency tables (two-tailed Fisher’s exact test). The effect of contaminants on the bending rigidity of the GUVs was subjected to ANOVA followed by Tukey’s pairwise comparisons. The effect of contaminants on the relative phospholipid fatty acid composition and unsaturation indices was subjected to ANOVA and corrected for multiple testing (Bonferroni).
transferred to a glass tube, where 10 μL of 6.4% NaOH, 250 μL of 0.2 M K2CO3 and 20 μL of acetic anhydride (Ac2O) was added. After whirly mixing for 10 min, the homogenate was left for 3 h in the dark to allow glycation. This procedure was followed by solid phase extraction, during which NP was isolated and eluted on LiChrolut columns (bottom EN 100 mg, top RP-18 200 mg) (Merck KGaA, Darmstadt, Germany). After the columns were washed with 2 mL of Elga water followed by 2 mL of 96% ethanol and 2 mL of 70% ethanol, the homogenate was added to the columns. The retaining NP was then eluted with 1 mL of acetonitrile (super gradient for HPLC). The remaining water in the NP extract was removed by adding a few milligrams of sodium sulfate (Na2SO4). Extracts were centrifuged for 3 min at 3000g, and the supernatant was transferred to autosampler vials and analyzed by gas chromatography−mass spectrometry (GC−MS) (see the Supporting Information for further details). NP standards were run in parallel and subjected to the same extraction procedure. Analysis of Membrane Lipids. Whole-body sample tissue (3−5 mg dry weight) was crushed in 0.5 mL phosphate buffer using a TissueLyser II (Qiagen, Copenhagen, Denmark) at 30 Hz for 2 × 15 s. Crude extraction of all lipids was conducted by adding 1 mL of phosphate buffer, 3 mL of methanol and 1.5 mL of chloroform to the samples in 12 mL glass centrifuge tubes, which were then whirly mixed for 1 min and incubated for 2 h at room temperature. Another 1.5 mL of chloroform and 1.5 mL of phosphate buffer were added to each sample, which was again whirly mixed for 1 min and left at room temperature overnight. After centrifugation at 1600g for 10 min, the watery phase was discarded and chloroform was evaporated from the organic phase under nitrogen flow. To separate neutral lipids and lipids of medium polarity, e.g., cholesterol, from polar lipids, the samples were redissolved in 900 μL of chloroform and then slowly vacuum-filtered through solid-phase silica columns (100 mg; Bond Elute, Agilent Technologies, Santa Clara, CA, USA), which were preconditioned with 1.5 mL of chloroform. The neutral and mediumpolarity lipids were eluted with 1.5 mL of chloroform and 6 mL of acetone, respectively, and then discarded. Polar lipids (mainly phospholipids) were eluted with 1.5 mL of methanol, which was afterward evaporated under gentle nitrogen flow and then transesterified by mild alkaline methanolysis.20 After transmethylation, fatty acid methyl esters (FAMEs) were redissolved in 1 mL of heptane for gas chromatographic analysis coupled with mass spectrometry as previously described.20 The fatty acids were designated as X:Y, where X is the number of carbon atoms and Y is the number of double bonds. The degree of unsaturation (unsaturation index on a molar basis, UI) was calculated as Σ (% monoenes + 2 × % dienes + 3× % trienes...)/100. The molar ratio between unsaturated and saturated fatty acids was calculated (UFA/ SFA). Calorimetric Measurements. Stock solutions of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine; Avanti Polar Lipids; Alabater, AL, USA), NP and PHE were prepared gravimetrically in glass vials using a 1:1 chloroform - methanol mixture as the solvent. Stocks were mixed to obtain contaminant-to-lipid mole ratios of between 0 and 0.1, and all samples were dried, first under a stream of nitrogen until apparent dryness and then overnight at reduced pressure to runoff any traces of solvent. The dry lipid/contaminant mixtures were hydrated with Milli-Q water to a solid content
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RESULTS AND DISCUSSION NP and PHE Modulate Cold Tolerance in Opposite Directions. Initial experiments showed that acute lethality, i.e., without cold exposure, of NP and PHE in E. albidus was not observed at nominal concentrations below 80 and 200 mg kg−1 dry soil, respectively (Figure S2A,B, Supporting Information). In F. candida, no lethality was observed at nominal concentrations below 70 mg NP kg−1 and 50 mg PHE kg−1 dry soil (Figure S2C,D, Supporting Information). Previous studies reported that LC50 (3 week test) for PHE in E. albidus is 135 mg kg−1 dry soil,24 and that LC50 (8 d test) for NP in F. candida is 175 mg kg−1 dry soil.25 Where comparisons are possible, the present results are thus in good agreement with previous reports using the same species. When we tested the cold tolerance of these two invertebrate species, we found that sublethal nominal concentrations of NP (75−80 mg kg−1 dry soil) significantly reduced the survival rate after freezing at −6 °C from 80 to 33% (E. albidus), and survival of cold shock from 93 to 81% (120 min at −5 °C; F. candida) (Figures 1 and S1, Supporting Information). In contrast, sublethal nominal concentrations of PHE significantly increased survival after C
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Figure 1. Experiments showing that 4-nonylphenol (NP) reduced cold tolerance in Enchytraeus albidus (freezing at −6 °C; mean ± sem; N = 6 vials each containing 5 specimens) and Folsomia candida (cold shock at −5 °C for 120 min; mean ± sem; N = 5 vials each containing 20 specimens), whereas phenanthrene (PHE) improved cold tolerance in E. albidus (freezing at −8 °C; mean ± sem; N = 5 vials each containing 5 specimens) and in F. candida (cold shock at −5 °C for 180 min; mean ± sem; N = 10 vials each containing 10 specimens). All treatments were significantly different from the respective controls (two-tailed Chi-square tests (Fisher’s exact test); P < 0.05).
Figure 2. Effects of 4-nonylphenol (NP) and phenanthrene (PHE) on the phase transition temperature (Tm) of DMPC multilamellar vesicles using differential scanning calorimetry. Each point represents one measurement. Linear regressions are shown (NP: y = 0.049x + 0.067, R2 = 0.98, p = 0.002. PHE: y = −0.198x + 0.13, R2 = 0.99, p = 0.0001).
freezing at −8 °C from 75 to 100% (90 mg kg−1 dry soil; E. albidus), and after cold shock from 54 to 73% (50 mg kg−1 dry soil; 180 min at −5 °C; F. candida) (Figures 1 and S2, Supporting Information). NP and PHE Change the Fluidity of Model Membranes. Given the crucial importance of the physical membrane properties for functioning and survival at low temperatures, 1 we investigated the influence of these contaminants on the membrane scale. To understand the opposite effects of NP and PHE on cold tolerance, we measured their impacts on artificial and seminatural biological membranes through two different parameters, i.e., the phase transition temperature and the bending rigidity. This gives us indications regarding the physical state of the lipid bilayer. We first conducted measurements on one-component lipid bilayers in a reductionist approach in order to underline the effects of the contaminants on simple membranes. The interaction of PHE and NP with artificial multilamellar membranes consisting of DMPC (1,2-dimyristoyl-sn-glycero-3phosphocholine) had significant effects on the membrane phase transition temperatures. Concentrations of PHE equivalent to 2 mol % or higher in DMPC vesicles caused the phase transition temperature to decrease, indicating that the interactions with PHE molecules stabilize the fluid phase (Figure 2). In contrast to this, NP at the same concentrations increased the phase transition temperature. These effects on the phase behavior suggest that PHE promotes the fluidity of DMPC membranes while NP has the opposite effect. The calorimetric data were well aligned with studies on the bending rigidity of 1,2palmitoyloleyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUV). Here, 0.5 mol % of PHE (or a higher concentration) reduced the bending rigidity, which is also a signature of an increase in fluidity, and in contrast, NP made GUVs more rigid (Figure 3). Interestingly, it should be underlined that measurements of the membrane phase transition temperature and membrane bending rigidity seem quantitatively consistent. A comparatively larger molar concentration of NP than of PHE is required to achieve
Figure 3. Effects of 4-nonylphenol (NP) and phenanthrene (PHE) on mean (±sem; N = 4−8) bending rigidity of POPC giant unilamellar vesicles. Mol% indicates the molar ratio of the toxicant: POPC. Different letters above the bars indicate significant differences in pairwise comparisons (ANOVA, Tukey’s test, P < 0.05).
numerically similar changes of both phase transition temperature and bending rigidity respectively (Figures 2 and 3). NP and PHE Change the Fluidity of Reconstituted Membranes from Two Species of Soil Invertebrates. To increase the relevance of the in vitro membrane measurements for the in vivo results of cold-tolerance, and thus the relevance for our conclusions, we performed measurements of bending rigidity on GUVs made from reconstituted phospholipid extracts of both model species. In doing so, we found that both types of GUVs behaved qualitatively exactly like POPC GUVs when NP and PHE were added to the reconstituted phospholipid mixtures used to form the GUVs. Accordingly, NP made membranes more rigid, whereas PHE decreased bending rigidity and softened the membranes when occurring in a 10 mol % mixture with the phospholipids of these two species (Figure 4). Moreover, the survival rates of these model organisms exposed to freezing or cold shock in combination to either of these two chemicals are in perfect agreement with the observations we made for the in vitro effects of NP and PHE on membrane fluidity and rigidity. It is also interesting to note that the mixture of NP and PHE (5:5 mol %) in both E. albidus and F. candida GUVs led to a bending rigidity similar to that of the control, i.e., the effects of the two contaminants may cancel out D
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compounds in the membranes of the model species is difficult because the estimated lipid content includes not only neutral lipids and membrane lipids, but also cuticular lipids and waxes in which NP and PHE will also partition, but the results indicate that they are below or close to the levels where (lethal) narcotic effects (40−160 mmol kg−1 lipid) usually start to occur.35,36 Are Effects of NP and PHE on Cold Tolerance Due to Lipid Peroxidation Processes? Previous investigations have shown that effects of natural stressors, e.g., low temperature, combined with chemical stressors may result in synergistic or antagonistic interactions.37 Two hypotheses for such interactions have been proposed: contaminants may cause changes in the composition of phospholipids via oxidative stress and lipid peroxidation processes, thus altering the physical properties of the membranes,38 or merely change the physical properties of cellular membranes by their insertion in and interactions with the lipid bilayers.10,39,40 Both phenomena may potentially compromise membrane functionality with respect to challenging thermal conditions. Earlier studies have shown that heavy metals like copper (Cu2+) reduce the tolerance to freezing temperatures, possibly because of Cu-induced changes in the phospholipid fatty acid (PLFA) composition of the cell membranes in the freezetolerant earthworm, Dendrobaena octaedra.38 Lipid peroxidation increased in worms exposed to copper, and this metal ion had a significant negative effect on the polyunsaturated PLFA, linoleic acid (18:2ω6,9), which has previously been reported to correlate positively with freeze-tolerance in D. octaedra.38,41 We therefore analyzed the PLFA profiles of E. albidus and F. candida exposed to the two contaminants but found no significant effects of PHE or NP causing a decrease of PLFA unsaturation in either of these two species (Figures S3 and S4, Supporting Information). Although both PHE and NP are likely to create oxidative stress in toxic concentrations, which could damage cellular membranes, it seems that the effects on cold tolerance are not related to lipid peroxidation processes in this case. Instead, we suggest that the primary effect of these contaminants on cold tolerance is the result of a perturbation of the physical properties of the cell membrane. Specifically, cold tolerance was improved by PHE and reduced by NP in two species having fundamentally different cold-tolerance strategies, which intrigued us. Although animals relying on a supercooling strategy (such as F. candida) should maintain their membranes hydrated, functional and, probably, optimally fluid during winter, those relying on a freeze-tolerance strategy (such as E. albidus) would experience dehydration of their membranes and do not keep them functional, but rather protect their physical integrity during volumetric changes of the cell. Apparently, these two diverse cold-tolerance strategies resulting in very different membrane destinies require the same adaptive solutions. Several previous studies have demonstrated convincing correlations between membrane fluidity and cold tolerance during cold acclimation processes, but other adaptive processes than alterations of membrane lipid composition are likely to be involved in cold hardening.3,42 In the present study, we have replaced the often used temporal cold acclimation process with the action of environmental contaminants and provide clear indications that membrane fluidity is indeed the primary target for the changes in cold tolerance. Perspectives. The present study has shown interesting effects of common environmental contaminants on the physical
Figure 4. Influence of 4-nonylphenol (NP) and phenanthrene (PHE) on the mean (±sem; N = 8−12) bending rigidity of giant unilamellar vesicles made from reconstituted phospholipid extracts of E. albidus (left panel) and F. candida (right panel). Mol% indicates the molar ratio of the toxicant: POPC; “mix” indicates an equimolar mixture of NP and PHE (5:5 mol %). Different letters above the bars indicate significant differences in pairwise comparisons (ANOVA, Tukey’s test, P < 0.05).
each other when occurring in equi-molar concentrations. In fact, this additivity of effects also applies to the effects of the pollutant and temperature on membrane fluidity. NP and PHE Interact with Phospholipid Molecules. The effect of NP on the bending rigidity of POPC GUVs found here resembles that previously reported for cholesterol (POPC with 10 mol % cholesterol showed a bending rigidity of 54.4 kBT26). Indeed, cholesterol present in the membrane tends to make the phospholipid molecules more ordered, which results in a higher bending rigidity of GUVs. Besides cholesterol and other sterols, NP may be the only other compound that has been shown so far to increase the bending rigidity of lipid bilayers. Due to its hydrophobic nature, we expect that NP is located in the hydrophobic core of the lipid bilayer and might play a role in the packaging of the lipid tails. PHE is a hydrophobic molecule with a maximum solubility in water of 4.5 mg L−1.27 For comparison, the solubility of NP in water is 5.4 mg L−1.28 Accumulation of hydrophobic compounds in the essentially water-free core of lipid bilayers may seem intuitive, and has been demonstrated for example for small alkanes.29,30 A similar mode of interaction could be envisioned for PHE, but earlier studies have suggested that aromatic compounds tend to partition at the membrane-water interface31,32 or near the lipid head groups,33 and an interfacial mode of interaction could also be relevant for PHE. PHE and NP Accumulate in Invertebrate Membranes. NP and PHE are relatively lipophilic compounds,34 and are readily taken up by soil invertebrates from contaminated soil and accumulated mainly in body lipids (Table S1, Supporting Information). We estimated that lipids (in a broad sense) of E. albidus and F. candida amounted to 0.066 ± 0.005 and 0.088 ± 0.01 mg mg−1 fresh mass, respectively. On the basis of measurements of internal concentrations, we estimated that the molar percentage of contaminants in phospholipids was in the range from 0.2 to 7 mol % (Table S1, Supporting Information), which is below the concentrations used in bending rigidity tests with GUVs made from phospholipid extracts of the two test species, but comparable to the molar percentages used in tests with POPC and DMPC vesicles. PHE and NP will partition well into phospholipid membranes as into neutral lipids (“fat”).34 An exact estimation of the concentrations of these E
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properties of membranes, even at sublethal concentrations of these compounds. Having said this, we must recall that the concentrations of PHE and NP used in the present study were relatively high and probably above relevant environmental concentrations. However, our study improves the basis for mechanistic understanding that may guide future work in this area. Apart from the ecotoxicological aspects, these kinds of “perturbation experiments” have the potential to provide new and direct evidence for the importance of “homeoviscous adaptation” in the thermal biology of ectothermic organisms. The interaction between the effects of hazardous chemicals in the environment and nonchemical stressors such as adverse climatic conditions has received increasing attention in the past decade.43−45 The rationale behind this concern is that the presence of contaminants in the environment may alter climatic tolerance limits and hence an organism’s potential geographical distribution in future climatic scenarios, but very little is currently known of the underlying physiological mechanisms.37 Because low temperature is one of the most important environmental constraints for ectothermic animals of the temperate and Arctic regions, an understanding of these mechanisms may to some extend refine the development of a sound risk assessment of hazardous chemicals in cold environments.43 Moreover, our study suggests that the environmental effects of chemical compounds may be partly evaluated from simple biophysical studies of their effect on model membranes. Through our interdisciplinary approach, drawing on complementary fields such as ecophysiology, ecotoxicology, membrane biochemistry and membrane biophysics, we have been able to explain the synergism/ antagonism between the effects of physical and chemical stress in ectothermic organisms, which is now beginning to provide a mechanistic understanding of these phenomena. In conclusion, we suggest that interdisciplinary competences are necessary to study these complex phenomena in order to break down barriers.
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ASSOCIATED CONTENT
Chemical structures of focus compounds (Figure S1), results from more detailed dose−response trials (Figure S2), concentration of NP and PHE in tissues of the study species (Table S1), phospholipid fatty acid compositions (Figures S3 and S4) and detailed descriptions of methodologies. This material is available free of charge via the Internet at http:// pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*M. Holmstrup. E-mail:
[email protected]. Address: Department of Bioscience, Aarhus University, Vejlsøvej 25, DK-8600 Silkeborg, Denmark. Phone: +45 3018 3152. Fax: +45 8715 8901. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
This study was supported by The Danish Council for Independent Research (contract no. 10-084579). The authors thank N. Cedergreen for her statistical advice and J. Jacobsen for her graphical assistance. F
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