Environ. Sci. Technol. 2004, 38, 1139-1147
Partitioning of Selected Estrogenic Compounds between Synthetic Membrane Vesicles and Water: Effects of Lipid Components HIROSHI YAMAMOTO* AND HOWARD M. LILJESTRAND Department of Civil Engineering, The University of Texas at Austin, 1 University Station C1786, Austin, Texas, 78712-0273
Partition coefficients of the steroid estrogens 17βestradiol, estriol, estrone, and 17R-ethynylestradiol and the industrial estrogenic compounds p-nonylphenol, p-(tert)octylphenol, bisphenol A, butylbenzylphthalate, and dibutylphthalate between liposome membrane vesicles and water (Klipw values) were determined using equilibrium dialysis. A moderate linear correlation with R2 values of as low as 0.679 were found for the relationship between log Klipw and log Kow for these compounds. Effects of lipid components used to prepare the membrane vesicles on the partitioning of 17β-estradiol and p-nonylphenol were also evaluated. For both, Klipw values were larger for the vesicles prepared from phospholipids composed of shorter acyl chains such as dilauroyl-phosphatidylcholine than those composed of longer acyl chains such as distearoylphosphatidylcholine. Partition coefficients were higher for the vesicles prepared from phospholipids including unsaturated acyl chains such as dioleoyl-phosphatidylcholine than those solely composed of saturated acyl chains such as distearoyl-phosphatidylcholine. Both shorter acyl chains and double bonds resulted in a more fluid conformation of the lipid bilayer with the liquid crystalline phase rather than the gel phase and greater partitioning. In contrast, higher cholesterol contents reduced the partitioning coefficient. The presence of cholesterol in the void of the lipid bilayer possibly led to the stabilization of the bilayer and the decrease in the partitioning of 17βestradiol or p-nonylphenol molecules. These results suggest that phase transition is of critical importance in selecting lipid components to evaluate the bioconcentration for these compounds.
Introduction The spread of steroid estrogens and estrogenic compounds throughout the biosphere has been suspected to link with reproductive disorders reported in humans and wildlife (e.g., ref 1). While steroid estrogen concentrations as high as 200 ng/L have been found in treated municipal wastewaters and receiving water bodies (e.g., refs 2-5), industrial estrogenic * Corresponding author phone: +81-29-850-2855; fax: +81-29850-2880; e-mail:
[email protected]. Present address: National Institute for Environmental Studies, Endocrine Disruptors and Dioxin Research Project, 16-2 Onogawa, Tsukuba, Ibaraki 3058506, Japan. 10.1021/es034311w CCC: $27.50 Published on Web 01/15/2004
2004 American Chemical Society
compounds with moderate hydrophobicity, such as alkylphenols and phthalates, have been detected in effluents and surface waters as high as 40 µg/L and in sediments and food products in concentrations as high as 10 mg/kg (2, 4, 6-9). Some of these industrial compounds have production rates as high as 1000 metric tons annually (e.g., ref 10) and could impact organisms through multiple pathways. Despite the growing concern about these endocrine disrupting compounds, little knowledge has been accumulated about their fate and behavior in the environment. Especially, the bioconcentration of steroid estrogens and other moderately hydrophobic estrogenic compounds has not been successfully determined except for alkylphenols (e.g., ref 11) due to their reproductive effects at relatively low concentrations and their moderate or weak accumulation (12). Actual biological uptake of micropollutants is of critical importance to evaluate the fate and behavior of these compounds both in the environment and in biological treatment processes. Furthermore, the uptake could be the rate-limiting step in metabolism and/or exerting estrogenic effects. For hydrophobic organic species, octanol-water partitioning has had considerable success as a simple model system to evaluate bioavailability and toxicity of chemical compounds through linear free energy relationships and quantitative structure-activity relationships (13, 14). Alternatively, the bioconcentration factor measurements using various kinds of living organisms have also widely used due to the similarity of the system with the actual pathway of the biological uptake. However, the octanol-water partitioning system has some thermodynamic differences from the actual uptake by biota (15) due to the use of the organic solvent, 1-octanol, as the model biological phase, while the bioconcentration factor measurement has limitations in cost, time, and reproducibility due to the use of fish, planktons, and other living organisms. To overcome these limitations, intermediate systems such as the micelle-water system (e.g., ref 16) and the membrane vesicle (liposome)-water system (17-19) have recently become available and been used both in pharmacology and environmental toxicology to evaluate the bioconcentration of chemical compounds. In this study, synthetic membrane vesicles (liposomes) were used as the model biological phase because of the similarity in chemical structure between the vesicle and the actual biological membrane, both of which consist of phospholipid bilayer. Additionally, diffusion through the lipid bilayer is believed to be the dominant pathway of moderately or highly hydrophobic chemical compounds through biological membranes (20, 21). Furthermore, the membrane vesicle suspension could be a model of a bioreactor with suspended growth materials such as the activated sludge and could estimate the bioavailability of the compounds of concern. Consequently, the partitioning of four steroid estrogens and five industrial estrogenic compounds, all of which are polar and moderately hydrophobic, between membrane vesicles and water was examined using equilibrium dialysis to evaluate the bioconcentration of these compounds. The correlation between the resulting partition coefficient (log Klipw value) and the octanol-water partition coefficient (log Kow) of each selected estrogenic compound was evaluated. Lipid components and fractions differ depending upon their animal or plant species source (20, 21). In general, mammalian membranes contain 20-40% of cholesterol in addition to phospholipids, while those of fish species contain a substantial amount of phospholipids with highly unsaturated acyl chains (e.g., docosahexanoic group) (22-24). However, differences in lipid components used to prepare VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Chemical structure, aqueous solubility limit, acid dissociation constant, and reported octanol-water partition coefficient of selected steroid estrogens and estrogenic compounds. a, from ref 61; b, from ref 37; c, from ref 38; d, estimated by using the Hammett and Taft equation (62); e, from ref 62; f, from ref 62; g, estimated from phenol using the fragment method (64); h, estimated from Kow using log Sw ) -1.016 log Kow + 0.515 for mixed organic compounds (65); i, estimated from Kow using log Sw ) -0.943 log Kow + 0.208 for phthalate (66); j, not defined; k, from ref 66. the membrane vesicles are suggested to change the conformations (22, 23) of the lipid bilayers and the partition coefficients of hydrophobic organic compounds (18, 25). Little information, however, is available on the effects of lipid components on the partitioning of moderately hydrophobic chemicals, such as steroid estrogens and alkylphenols, into membrane vesicles. In this study, the partition coefficients of 17β-estradiol (E2) and p-nonylphenol (NP) as model compounds were determined for membrane vesicle suspensions prepared from cholesterol and several phosphatidylcholines with unsaturated and saturated acyl chains including the major lipid components of animal cell membranes.
Experimental Section Materials. 17β-Estradiol (E2), one of the most common steroid estrogens, and p-nonylphenol (NP), a decomposition product of nonylphenol ethoxylate industrial detergents (26), were selected as primary target compounds in this study and used for the experiments to investigate the effects of lipid components. Estriol (E3) and estrone (E1), both metabolites of E2 and important pregnancy steroid estrogens, as well as 17R-ethynylestradiol (EE2), the main component of the contraceptive pill, were also chosen. p-(tert)Octylphenol (OP), bisphenol A (BPA), butylbenzylphthalate (BBP), and dibutylphthalate (DBP) were selected as synthetic estrogenic compounds because of their high industrial production and the lack of information about their bioconcentration. The chemical structure, aqueous solubility, the octanol-water partition coefficient (log Kow value), and the acid dissociation constant (pKa value) of these species are shown in Figure 1. E2, E3, NP, OP, BPA, and BBP were 1140
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purchased from Aldrich Chemical Co. (Milwaukee, WI). E1, EE2, and DBP were purchased from Sigma Chemical Co. (St. Louis, MO). Phospholipids and sterols are the main lipid components of most biological membranes (22-24). In this study, cholesterol was selected from the various sterols found in membranes because of its ubiquitous presence in mammalian cell membranes. For phospholipids, several phosphatidylcholines (PCs) were selected based on the consideration of their spontaneous formation of lamellar bilayers and ubiquitous existence in eukaryotic cell membrane (23, 24). The fatty acid component (acyl chain) of the phospholipids is highly dependent on the animal species, but palmitoyl (C16:0) followed by oleoyl (C18:1, 9-cis) are the most abundant groups (22-24) for most eukaryotes. In this study, dipalmitoyl-PC (DPPC, C16:0, 16:0) and palmitoyloleoyl-PC (POPC, C16:0, 18:1) were used as the primary phospholipid components, with dilauroyl-PC (DLPC, C12:0, 12:0), dimiristoyl-PC (DMPC, C14:0, 14:0), distearoyl-PC (DSPC, C18:0, 18:0), and diarachitoyl-PC (DAPC, C20:0, 20:0) as additional saturated acyl chains and dioleoyl-PC (DOPC, C18:1, 18:1) as another unsaturated acyl chain. Cholesterol was purchased from Sigma Chemical Co. (St. Louis, MO). DPPC, POPC, DLPC, DMPC, DSPC, DAPC, and DOPC were purchased from Avanti Polar Lipids (Alabaster, AL). Methods. The membrane vesicle suspensions were prepared using the thin film hydration technique developed by Mueller et al. (27). The resulting membrane vesicle suspensions were made more uniform in size by the rapid extrusion process developed by Hope and co-workers (e.g., ref 26) and as modified by Matsubara et al. (29). The phospholipid (and/
TABLE 1. Partition Coefficients (Klipw Values, L/kg-liposome) of Selected Estrogenic Compounds between Membrane Vesicles Prepared from POPC, DPPC, and Mixture of DPPC/Cholesterol (60:40 wt %) and Water estrogenic compounds
KlipwPOPC (( standard deviation)
KlipwDPPC (( standard deviation)
KlipwDPPC/cholesterol (( standard deviation)
E2 EE2 E1 E3 NP OP BPA BBP DBP
6.15 ((0.88) × 103 1.57 ((0.03) × 104 8.36 ((0.60) × 103 9.09 ((4.91) × 10 3.18 ((0.62) × 105 4.04 ((0.35) × 105 2.91 ((0.30) × 104 4.75 ((1.06) × 104 1.55 ((0.43) × 104
2.94 ((0.24) × 102 5.62 ((1.31) × 102 4.13 ((0.32) × 102 1.99 ((1.59) × 10 6.87 ((0.92) × 104 3.48 ((1.29) × 105 1.79 ((0.99) × 103 5.74 ((1.18) × 102 9.29 ((4.05) × 103
1.94 ((0.50) × 102 1.65 ((0.69) × 102 2.82 ((2.31) × 102 1.47 ((0.89) × 10 6.88 ((3.91) × 103 1.38 ((0.39) × 105 1.78 ((1.23) × 102 1.25 ((0.94) × 102 1.03 ((0.45) × 102
or cholesterol) solution in chloroform was evaporated in a round-bottom flask, pure water was gently added, and the flask was left overnight. The resulting suspension was extruded through the 1.2 µm Millipore polycarbonate membrane 13 times at least 10 °C above the main phase transition temperature (Tm). The membrane was rinsed carefully with pure water prior to the extrusion process. Particle size distribution of the resulting membrane vesicles was determined using both quasi-elastic laser light scattering analyzer (QELS) and transmission electron microscopy (TEM), and the number standard mean diameter was found to be between 0.4 and 0.6 µm (cf., Supporting Information). Partition coefficients of the selected steroid estrogens and estrogenic compounds (Klipw) between membrane vesicles (liposomes) and water were determined using the equilibrium dialysis procedure developed by Escher and Schwarzenbach (18). The measurement cells were made of two 10 mL glass vials and connected to each other by a Teflon joint. Regenerated cellulose dialysis membrane (Por 7 MWCO 10,000, purchased from Spectrum Scientific Co.) was installed between two vials and supported by the Teflon joint. Slight removal of relatively hydrophobic compounds (e.g., as high as 7% for NP and 3% for E2) was observed but is much less than the removal by liposome (cf., Supporting Information). The pH and ionic strength of all solutions were set at 7 and 0.02 M, respectively, using potassium phosphate buffer and sodium chloride. At pH 7, ionic species of all the selected compounds are much smaller than nonionic species because of the relatively high pKa values (Figure 1). At least three replicates of the test and reference cells were prepared for a single measurement of the partition coefficient. Sodium azide (0.02%) was added to all cells to minimize microbial activity. Cells were mixed using a tumbler in the dark at room temperature (22 °C) for 7 days. The equilibrium of the system was apparently aqueous phase rate limiting and was attained in 7 days (cf., Supporting Information). The concentration of the selected steroid estrogens and estrogenic compounds was measured using an HPLC (Waters 2690) equipped with the PDA detector (Waters 996) for both sides of the reference cells (Cref, mg/L) as well as the side without liposome in the testing cell (Cw, mg/L). The partition coefficients of selected steroid estrogens and estrogenic compounds were calculated as described by Escher and Schwarzenbach (18) as follows
Klipw (L/kg-liposome) )
Clip Cw m
(1)
where Clip (mg/L) is the concentration of the selected compound in the side with liposome in the testing cell and m is the concentration of liposome (kg-liposome/L), as measured using the Dohrmann Apollo 9000 TOC analyzer.
The mass balance of the two cells is given by
Cref ) Cw + Clip
(2)
By substituting Clip in eq 1, Klipw can be expressed in terms of Cw, m, and Cref as follows:
Klipw (L/kg-liposome) )
Cref - Cw Cwm
(3)
Equation 3 was used to determine the Klipw from data from experiments where the initial concentration (Co) was set between 300 µg/L and 2 mg/L for all the selected steroid estrogens and estrogenic compounds. Preliminary experiments were conducted, and the concentration of the liposome was set to attain Cw of approximately 50% of Co. The liposome concentration ranged from 3.7 to 1200 mg-liposome/L depending upon the Klipw values of the selected compounds. For some combinations of estrogenic compounds (e.g., E3) and membrane suspensions, Cw was below 10% as a result of low Klipw values and the upper limit of the membrane concentration prepared.
Results and Discussion Relationship between Liposome-Water Partition Coefficients and Octanol-Water Partition Coefficients. Partition coefficients (Klipw values) of all nine selected compounds between water and membrane vesicles prepared from three different lipid compositions, DPPC, POPC, and DPPC/ cholesterol (60:40 wt %), were determined, as summarized in Table 1. Correlations between log Klipw and log Kow values are plotted in Figure 2. In Figure 2, regression lines obtained in this study and by other researchers are also shown. The highly hydrophobic alkylphenols, NP and OP, had greater Klipw values for all the membrane vesicles. The Klipw values of E3, the least hydrophobic of the selected species, were lower than those of the other steroid estrogens and estrogenic compounds. In addition for all of the selected compounds, the log Klipw values were greater for the liposome prepared from POPC than from DPPC, and the lowest Klipw values were obtained for DPPC/cholesterol (60:40 wt %). Although all the selected compounds had Klipw values in the order of POPC > DPPC > DPPC/cholesterol (60:40 wt %), the difference of Klipw values between each liposome was inconsistent. For example, Klipw values of the three steroid estrogens (E2, EE2, and E1) as well as the estrogenic bisphenol A and phthalates (BBP and DBP) for POPC liposome were at least 16 times as high as those for the DPPC liposome. In contrast, Klipw values of the two alkylphenols were only slightly greater for liposome made from POPC than from DPPC, i.e., by 4.6-fold and 2.3-fold for NP and OP, respectively. These larger differences of Klipw values for steroid estrogens, BPA, VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and phthalates were possibly a result of increased penetration of these relatively planar-structured (ringed-structured) compounds into the lipid bilayer due to the coarser distribution of POPC molecules in the bilayer, as discussed further below. The reported log Klipw value of EE2 for the liposome prepared from egg PC is 3.81 (30), which is between the log Klipw obtained in this study for liposome prepared from DPPC () 2.75) and POPC () 4.20). As illustrated in Figure 2(a) log Kow and log Klipw for the selected compounds showed a moderate or weak linear correlation, and all the linear regression slopes were near unity as follows:
log KlipwPOPC ) 0.859 log Kow + 0.604 R2 ) 0.785(at 22 °C) (4) log KlipwDPPC ) 1.028 log Kow - 1.225 R2 ) 0.753(at 22 °C) (5) log KlipwDPPC/cholesterol ) 0.873 log Kow - 1.106 R2 ) 0.679(at 22 °C) (6) These relationships are much less strong than those obtained by other researchers (e.g., refs 31-34) who obtained R2 values of 0.9 or greater. These moderate/weak relationships are possibly attributed to the diversity of chemical structures in the compounds selected in this study. BPA had a higher Klipw value compared to the linear regression lines for liposome from all three lipid components, whereas DBP and BBP were slightly below the regression lines. NP and OP were consistently relatively close to or slightly above the regression lines. The Klipw values for the three steroid estrogens, E2, E1, and EE2, showed the opposite trend for DPPC and POPC liposome, i.e., the higher log Klipw was found for that with lower reported log Kow. Consequently, significant limitations are suggested for log Kow values to predict the log Klipw or the bioconcentration, not only for structurally different compounds but also for structurally similar compounds with similar log Kow values, such as steroid estrogens. Further investigation using more steroid compounds is needed, because the reported and the estimated log Kow values for steroid estrogens were inconsistent (35-38). Gobas et al. (31) reviewed conventional studies such as the results by Diamond and Katz (39) and their results and found the correlation of log Klipw for DMPC liposome with log Kow for 25 nonpolar compounds with log Kow between 1 and 5.5 as follows
log KlipwDMPC ) 1.19 log Kow + 0.645 R2 ) 0.96 (at 25 °C) (7) where DMPC is in the liquid crystalline phase. This linear regression line is drawn in Figure 2b to compare with the Klipw values obtained in this study. This line lies far above those obtained in this study, which is possibly attributed to the polarity of the selected compounds. Vaes et al. (32) also examined the relationship between log Klipw for DMPC and log Kow for 19 polar and nonpolar chemical compounds at 35 °C, where the phospholipid bilayer again exists as the liquid crystalline phase. They found the relationship (Figure 2b) as follows:
log KlipwDMPC ) 0.997 log Kow + 0.085 R2 ) 0.92 (at 35 °C) (8) FIGURE 2. Relationship between partition coefficients and the reported octanol-water partition coefficients with (a) regression lines obtained in this study, (b) those obtained by other researchers, and (c) empirical third-order polynomial fit. 1142
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In contrast to eq 7, eq 8 nearly overlies the regression line obtained in this study and is similar to eq 4 for POPC. This agreement suggests that Klipw values for DMPC and POPC, both at the liquid crystalline phase, are similar to each other for polar compounds, which is discussed further in the next
section. Escher et al. (33) also found the relationship (Figure 2b) as follows:
log KlipwDPPC/DOPC ) 0.78 log Kow + 1.12 R2 ) 0.92 (at 20 °C) (9) This trend line again lies close to eq 4. At 20 °C, phospholipid bilayer prepared from the mixture of DPPC and DOPC exists as the liquid crystalline phase (40). Furthermore, Van Wezel et al. (34) examined the relationship between log Klipw with log Kow of seven chlorobenzenes for DPPC liposome at several different temperatures. The relationship below the transition temperature (gel phase) was
log KlipwDPPC ) 0.80 log Kow - 0.90
R2 ) 0.98 (at 23 °C) (10)
At the above the transition temperature (liquid crystalline phase), the regression line significantly changed and became
log KlipwDPPC ) 0.89 log Kow - 0.01
R2 ) 0.99 (at 43 °C) (11)
As can be seen from Figure 2b, eq 10 lies close to the log Klipw values obtained in this study for DPPC and DPPC/cholesterol, whereas eq 11 lies close to the log Klipw values for POPC. This suggests the phase transition (and/or temperature) is possibly of critical importance in the determination of log Klipw values. In contrast, Dulfer and Govers (17) used a second-order polynomial fit to relate log Klipw with log Kow and obtained a satisfactory nonlinear correlation for PCBs with DMPC, DPPC, DSPC, and DAPC. While no second-order trend was evident in this study, a third-order polynomial with a sigmoid curve fits the data empirically for all three liposomes (Figure 2c):
log KlipwPOPC ) 0.367 (log Kow)3 - 4.67 (log Kow)2 + 19.85 log Kow - 23.91
R2 ) 0.920 (12)
log KlipwDPPC ) 0.549 (log Kow)3 - 6.56 (log Kow)2 + 25.73 log Kow - 30.38
R2 ) 0.968 (13)
log KlipwDPPC/cholesterol ) 0.564 (log Kow)3 6.69 (log Kow)2 + 25.90 log Kow - 30.46
R2 ) 0.984 (14)
These better fits using the empirical third-order polynomial rather than the linear regression suggest that some mechanisms such as hydrogen bonding other than the hydrophobic interaction bonding may play important roles in the partitioning (41). However with the limited number of the chemicals (e.g., n ) 9), and with their diverse chemical structure, and with relatively large standard deviations for some data (as high as 80% relative error), it would be difficult to provide specific physical-chemical interpretations for the statistical results, especially given the inconsistencies in reported Kow values, which could create artifacts. Thus, higher order polynomial fits were not used in this study, and further investigation is necessary to better predict log Klipw values from log Kow values using methods such as the structural fragment methods (41) in addition to the examination of the mechanisms to cause the better fit for the third-order polynomial than the linear regression. Comparison of Liposome-Water Partition Coefficient and Bioconcentration Factor. As presented above, not many studies have been successful in the determination of bioconcentration factors (BCF) for steroid estrogens. Lai et al. attempted to determine the BCF value of natural and
synthetic steroid estrogens by Chlorella vulgaris, but they failed to quantify any except that for estrone (E1), which was 27 (12). This value is comparable to the Klipw value obtained in this study for DPPC and DPPC/cholesterol but is oneorder lower than that for POPC. Other researchers have attempted to determine the BCF values of alkylphenols (42). For example, Ahel et al. determined the BCF value of p-nonylphenol (NP) by several freshwater organisms (11). Relatively higher BCF of up to 10 000 were found for some macrophytic algae, but the BCF of fish tissue was much lower and was between 13 and 410. Additionally, the BCF of NP by fathead minnows ranged from 245 to 380 (43). Our Klipw values were comparable to that for the algae but much higher than those for fish tissues. Furthermore, the BCF value of 4-tert-octylphenol (OP) by rainbow trout was 1190 for lipid but was between 100 and 260 for other tissues (44). In contrast to alkylphenols, not much has been investigated for the bioconcentration of phthalic esters and bisphenol A. The BCF value of butylbenzyl phthalate (BBP) by bluegill sunfish ranged from 1.7 to 9.4 depending on tissues (45), while the BCF of bisphenol A (BPA) of freshwater clam Pisidium amnicum was 144 (46). Generally, these measured BCF values are comparable or slightly lower than our Klipw values for fat but significantly lower than our Klipw values for some other tissues. These lower BCF values were possibly attributable to the difference in lipid contents and metabolic pathway to reduce the chemicals in the living organisms. Effects of Lipid Components on the Partition Coefficients. As noted in the previous section, the partition coefficients of estrogenic compounds varied among the membrane vesicles prepared from three lipid components. The effects of lipid composition on partitioning were further examined using 17β-estradiol (E2) and p-nonylphenol (NP) as model compounds. Klipw values of E2 and NP were determined and compared for the liposome prepared from phosphatidylcholines (PCs) with different lengths of saturated acyl chains (or alkyl aliphatic carbon chains), for the membrane vesicles prepared from PCs with saturated and unsaturated acyl chains, and for those with different cholesterol contents. Effects of the Length of the Acyl Chain. Klipw values of E2 and NP for the liposome prepared from five phosphatidylcholines (PCs) with different lengths of saturated acyl chains, DLPC (C12:0, 12:0), DMPC (C14:0, 14:0), DPPC (C16:0, 16:0), DSPC (C18:0, 18:0), and DAPC (C20:0, 20:0), were examined. Table 2 shows these Klipw values, the pretransition temperature, main transition temperature (Tm), and the phase of the lipid bilayers at room temperature. As illustrated in Figure 3, both the pretransition and the main transition temperatures of three PCs with relatively longer acyl chains, DPPC, DSPC, and DAPC, are above room temperature (22 °C), and their lipid bilayers exist as a gel phase at room temperature, whereas the main transition temperature of DLPC is at less than room temperature, and its bilayers exist as the liquid crystalline phase. Room temperature is between the main transition and pretransition temperature for DMPC, and its bilayers exist as an intermediate phase called “ripple phase” (Figure 3). The obtained Klipw values of E2 and NP were larger for the PCs composed of shorter acyl chains, but the difference between Klipw values for the liposome prepared from DSPC and DAPC was not significant. The significantly greater Klipw values for the DLPC liposome were attributed to the lower hydrophobic interaction and coarse distribution of the phospholipid molecules in the bilayer (23), which results not only in the higher fluidity of the lipid molecules but also in better penetration of moderately or highly hydrophobic chemical compounds such as E2 and NP into the lipid bilayer. Conversely, the lower Klipw value for the liposome prepared VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Partition Coefficients of E2 and NP between Membrane Vesicles Prepared from Phosphatidylcholine with Different Acyl Chains and Water lipid
acyl chain compositiona
Tpb (°C)
Tmc (°C)
lipid bilayer phase at room temperature
Klipw value for E2 (L/kg-liposome)d
Klipw value for NP (L/kg-liposome)d
DLPC DMPC DPPC DSPC DAPC POPC DOPC
(12:0, 12:0) (14:0, 14:0) (16:0, 16:0) (18:0, 18:0) (20:0, 20:0) (16:0, 18:1) (18:1, 18:1)
NA 15 35 51 62 NA NA
-2 23 41 55 64 2 -22
liquid crystalline ripple gel gel gel liquid crystalline liquid crystalline
5.46 (( 0.16) × 103 2.01 (( 0.15) × 103 2.94 (( 0.24) × 102 1.72 (( 0.66) × 102 1.57 (( 0.74) × 102 6.15 (( 0.88) × 103 6.88 (( 0.55) × 103
4.48 (( 0.54) × 105 3.93 (( 0.17) × 105 6.87 (( 0.92) × 104 1.32 (( 0.33) × 104 1.33 (( 0.27) × 104 3.18 (( 0.62) × 105 3.32 (( 0.56) × 105
a Number of carbon: number of double bond, number of carbon, number of double bond. temperature. d (( standard deviation).
b
Pretransition temperature. c Main transition
FIGURE 3. Effects of temperature on the conformation and fluidity of lipid bilayer (ref 20, modified by authors). from three PCs of the gel phase was due to the greater hydrophobic interaction and more rigid conformation of phospholipid molecules in the bilayer. The Klipw values of E2 and NP for the DMPC liposome, which is in the ripple phase, were between Klipw values for DLPC and the three other PCs in the gel phase. The results obtained in this study agree with those of Antunes-Madeira and Madeira (e.g., ref 47), who found Klipw values of pesticides, parathion, lindane, DDT, and marathion in the order of DMPC > DPPC > DSPC at 10 °C below the main transition temperature of each phospholipid. Oguri et al. (48) also found Klipw values of highly hydrophobic PAHs in the order of DLPC > DMPC > DPPC > DSPC > DAPC at room temperature which agree with the results obtained in this study for E2 and NP. However, the results are not comparable with those obtained by Dulfer and Govers (17), who found partition coefficients of a wide variety of PCBs in the order of DPPC > DMPC > DSPC > DAPC at 37 °C, where DPPC was in the ripple phase and DMPC was in the liquid crystalline phase. These greater partition coefficients for the ripple phase (DPPC) than the liquid crystalline phase (DMPC) suggest a larger hydrophobic interaction between the very hydrophobic PCBs and a longer carbon chain of DPPC than DMPC. Effects of the Unsaturated Acyl Chain. The existence of an unsaturated acyl group in a phospholipid bilayer results in a bend in the carbon-chain and a larger distance between the phospholipid molecules (23), which leads to the lower hydrophobic interaction and lower transition temperature or higher fluidity (Figure 3). Table 2 shows the main transition temperature, the phase of lipid bilayer in room temperature, and Klipw values of E2 and NP for the liposome prepared from POPC and DOPC. As can be seen from Table 2, a lipid bilayer composed of unsaturated PCs, POPC, and DOPC, has a main transition temperature lower than room temperature and exists as a liquid crystalline phase, whereas that composed of the 1144
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saturated PCs such as DPPC and DSPC is in the gel phase. The Klipw values of E2 and NP for the liposome prepared from PCs with unsaturated acyl chains were significantly greater than the Klipw values of those prepared from PCs with saturated acyl chains and with a similar number of carbons (e.g., DPPC and DSPC). The larger Klipw values for the liposome prepared from unsaturated acyl chains in the liquid crystalline phase are analogous to the greater Klipw values for the DLPC liposome also in the liquid crystalline phase at room temperature. Thus, the greater fluidity of the bilayer could allow the E2 and/or NP molecules to penetrate into the hydrophobic region of the bilayer for the POPC and DOPC liposomes as with the DLPC liposome, as suggested in the previous section. These larger Klipw values of E2 and NP for the DOPC and POPC liposomes compared with those for DPPC agree with the results obtained by Lakowicz and co-workers (25, 49), who obtained four to five times as high Klipw values of lindane, methoxychlor, and hexachlorocyclohexane for the DOPC and POPC liposomes as for the DPPC liposome. Sarmento and co-workers (50) also obtained Klipw values for DOPC higher than those for DSPC using dopamine agonists. As noted above, the degree of saturation in the acyl chain of the phospholipids varies among biological species. The biggest difference among animal species is the significant fraction of the omega-3-polyunsaturated acyl chain such as the docosahexanoic group (22 carbons with six double bonds, C22:6) and the eicosapentaenoic group (C20:5) in fish (51). Since significantly larger Klipw values were found for membrane vesicles prepared from unsaturated phospholipids, even larger partition coefficients were suggested for membranes composed of even highly unsaturated acyl chains in fish compared with the other animal species. Nevertheless, further investigation is needed using the membrane vesicles prepared from phospholipids composed of these highly unsaturated acyl chains. In addition, the relationship between the number of double bonds and the partition coefficient
FIGURE 4. Effects of phase transition temperatures of lipid bilayers on the Klipw values. should also be investigated based on the consideration of the effects of the transition temperature of the phospholipid. Effects of the Transition Temperature. Figure 4 shows the relationship between log Klipw and the main transition temperature plotted for all the PCs composed of the saturated and unsaturated acyl chain used in this research. As can be seen from Figure 4, Klipw values of E2 and NP for the liposome prepared from the lipid components to form the liquid crystalline phase at room temperature, DLPC, DOPC, and POPC, were similar to each other. This similarity suggests that Klipw values of E2 and NP do not change significantly if the lipid component forms a liquid crystalline phase at the temperature, although the investigation using a lipid component with an even lower transition temperature (e.g., dicapryl-PC and diarachidonoyl-PC) is needed. Klipw values of both E2 and NP for the liposome prepared from the other PCs which form the gel phase at room temperature, those prepared from DPPC, DSPC, and DAPC, are less than that for the liposome prepared from DLPC, DOPC, and POPC. The log Klipw value for DMPC was between the log Klipw value for the liposome in the liquid crystalline phase and that for the liposome in the gel phase, but the log Klipw values were closer to the liquid crystalline phase for NP and closer to the gel phase for E2. This discrepancy was possibly attributed to the uncertainty in room temperature. As presented above, these significant effects of phase transition temperature were observed by van Wezel et al. (34) for seven chlorobenzenes using the DPPC liposome at a wide range of transition temperature. They found that the partition coefficients varied significantly near the transition temperature, and those at liquid crystalline phase were 2-3 orders of magnitude higher than those at the gel phase for all the selected compounds. Their observation suggests the importance of the phase transition temperature and agrees with ours for E2 and NP. Effects of Cholesterol. Effects of cholesterol content on the partition coefficients (Klipw values) were investigated using phosphatidylcholines (PCs) consisting of DSPC, DPPC, POPC, and DOPC. Cholesterol contents were set at 40% (PC: cholesterol ) 60:40 wt %), 20% (PC:cholesterol ) 80:20 wt %), and none (PC only), and Klipw values were determined for the four PCs. The log Klipw value for each PC and cholesterol content are illustrated in Figure 5. As in Figure 5(a), log Klipw values of E2 decreased with increasing cholesterol contents. The effects of decreasing log Klipw values by increasing cholesterol were more significant for the liposome prepared from an unsaturated acyl chain, POPC and DOPC, than that from a saturated acyl chain only, DPPC and DSPC. As with E2, log Klipw values of NP also decreased with increasing cholesterol contents, as shown in
FIGURE 5. Relationship between cholesterol contents and Klipw values for (a) 17β-estradiol (E2) and (b) nonylphenol (NP). Figure 5(b). Unlike E2 however, the effects of cholesterol on log Klipw values were similar to each other for all four PCs, such that Klipw for DPPC and POPC are approximately 10 times as high as DPPC/cholesterol (60:40 wt %) and POPC/ cholesterol (60:40 wt %), respectively. These smaller log Klipw values for the liposome prepared from higher cholesterol contents agree with other reported results. Korten et al. (52) found approximately a 25% lower Klipw value of thiopental for the liposome prepared from egg lecithin/cholesterol (85:15 wt %), approximately a 50% lower Klipw value for egg lecithin/cholesterol (70:30 wt %), and approximately a 75% lower Klipw value for egg lecithin/ cholesterol (50:50 wt %) as compared with Klipw for the liposome prepared from egg lecithin only. Lakowicz and Hogen (49) also obtained the partition coefficients of lindane for liposome prepared from DPPC only was approximately four times as high as that for DPPC/cholesterol (75:25 wt %). Additionally, Antunes-Madeira and Madeira (e.g., ref 47) found a significant decrease in Klipw values for four pesticides with the increase in cholesterol mole fraction in egg PC from 0 to 50%. More recently, Banachir et al. (53) found that the leakage of a fluorescence probe from the enclosed volume of the liposome prepared from POPC/cholesterol (70:30 wt %) was approximately one-tenth of that from the liposome prepared from POPC only. In contrast, Wang et al. (54) suggested that the significant change in the Klipw value of a polyene antibiotic Nystatin A1 with a minute change in ergosterol or cholestrol content on either side of the mole fraction (e.g., 25.0 mol %) was possibly attributed to the disappearance of an ordered phase in the phospholipid bilayer above 25% of the cholesterol contents in DPPC confirmed by dipyrene-PC excimer/monomer measurements (55). Huang et al. (56) also suggested that the presence of cholesterol in DPPC resulted in a broadened and higher main transition temperature, which might lead to lower Klipw values. VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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the effects of lipid components other than the bilayer conformation. In this study, the main focus has been on the effects of acyl chains and cholesterol contents on the lipid/water partitioning of moderately hydrophobic estrogenic compounds. As presented above, the partitioning of estrogenic compounds in liposome and water could be significantly affected by mechanisms other than the hydrophobic interactions since most of these compounds are polar. Among these other mechanisms, the hydrogen bonding possibly plays an important role (41). Furthermore, these polar interactions are possibly attributed to the interactions between polar estrogenic compounds and polar headgroups of the phospholipids (e.g., ref 60). Thus, further investigation for different headgroups such as phosphatidyl ethanolamines, phosphatidylglycerol, and sphingolipids is necessary to better understand the effects of lipid components. Interactions between headgroups and the selected compounds might be much more significant at pH higher than their pKa values, which also needs to be investigated in the future. FIGURE 6. Relationship between log Klipw values for POPC liposome and those for DPPC or DPPC/cholesterol liposome. The existence of cholesterol in the lipid bilayer decreases the interfacial surface area covered by lipid molecules, increases the rigidity, and decreases the fluidity (57, 58). The greater resistance of the lipid bilayer against the membranedisrupting surfactants was also observed for the lipid bilayer prepared from the larger cholesterol contents (59). Moreover, the decrease in the partition coefficient with the increase of cholesterol content suggests lower partition coefficients for E2, NP, and other moderately or highly hydrophobic compounds for biological membranes in mammalian species than those for other animal species at room temperature. However given that homeothermal mammalian and avian species have body temperatures as high as 37 °C, further investigation with model lipid components as well as temperature is needed. Implications of This Study. As presented above, the effects of lipid components such as the length of an acyl chain, the degree of saturation, and the cholesterol content significantly affected the liposome/water partition coefficients (Klipw values) of the selected estrogenic compounds. Thus, the lipid components used to prepare the membrane vesicles are of critical importance and should be selected carefully when used for the evaluation of the bioconcentration of these compounds. However, the results for the selected estrogenic compounds also suggest that the Klipw values are similar to each other for the liposome prepared from PCs in the liquid crystalline phase. Among these PCs, POPC is possibly a model of living organisms in an aquatic environment and in a bioreactor with suspended growth materials because of both the presence of abundant saturated (i.e., palmitoyl-group) and unsaturated (i.e., oleoyl-group) acyl chains in their structure and the sufficiently low transition temperature (-22 °C). On the other hand, the selection of temperature is an important factor because of the importance of the phase transition determined mostly by ambient temperature. However, the log Klipw values using DPPC liposome linearly correlate with log Klipw values using POPC liposome with the slope of nearly 1, while those using DPPC/cholesterol only weakly correlate (Figure 6). These results suggest that the DPPC liposome with its lipid bilayer in the gel phase could be a surrogate for that in the liposome liquid crystalline phase such as POPC. In contrast, the DPPC/cholesterol is not suitable for the surrogate due to the possible difference in the bilayer conformation. Further investigation for the liposome prepared from DPPC and DPPC/cholesterol at the above transition temperature is also necessary to confirm 1146
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Supporting Information Available Particle size analyses of the membrane vesicles, mean diameter of the prepared liposome after the extrusion process (Table S1), morphological analysis of membrane vesicles using TEM, electron micrograph of liposome prepared from DPPC (Figure S1), from DPPC/cholesterol (Figure S2), and from POPC (Figure S3), and results of preliminary experiments to determine the equilibration time (Figure S4). This material is available free of charge via the Internet at http:// pubs.acs.org.
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Received for review April 6, 2003. Revised manuscript received November 25, 2003. Accepted November 26, 2003. ES034311W
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