Article pubs.acs.org/est
Isomer-Specific Binding Affinity of Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) to Serum Proteins Sanjay Beesoon† and Jonathan W. Martin*,† †
Division of Analytical & Environmental Toxicology, Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3 S Supporting Information *
ABSTRACT: Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent contaminants in human serum, and these were historically manufactured as technical mixtures of linear and branched isomers. The isomers display unique pharmacokinetics in humans and in animal models, but molecular mechanisms underlying isomer-specific PFOS and PFOA disposition have not previously been studied. Here, ultrafiltration devices were used to examine (i) the dissociation constants (Kd) of individual PFOS and PFOA isomers with human serum albumin (HSA) and (ii) relative binding affinity of isomers in technical mixtures spiked to whole calf serum and human serum. Measurement of HSA Kd’s demonstrated that linear PFOS (Kd = 8(±4) × 10−8 M) was much more tightly bound than branched PFOS isomers (Kd range from 8(±1) × 10−5 M to 4(±2) × 10−4 M). Similarly, linear PFOA (Kd = 1(±0.9) × 10−4 M) was more strongly bound to HSA compared to branched PFOA isomers (Kd range from 4(±2) × 10−4 M to 3(±2) × 10−4 M). The higher binding affinities of linear PFOS and PFOA to total serum protein were confirmed when both calf serum and human serum were spiked with technical mixtures. Overall, these data provide a mechanistic explanation for the longer biological half-life of PFOS in humans, compared to PFOA, and for the higher transplacental transfer efficiencies and renal clearance of branched PFOS and PFOA isomers, compared to the respective linear isomer.
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INTRODUCTION As a result of their historical production and use in many consumer and industrial applications over the past 60 years,1,2 perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent xenobiotics detected in human blood today.3 Based on animal models, they are shown to have a range of toxic effects including neurotoxicity,4 developmental toxicity,5 hepatotoxicity,6 and possible disruption of the endocrine system.7,8 Human epidemiology studies of PFOS and/or PFOA have furthermore reported associations with biochemical or physiological end points including cholesterol metabolism and cardiovascular risk,9−12 immune system disorders,13,14 hyperuricemia,15−17 endocrine dysfunction,18−21 neurobehavioral disorders,22−24 reproductive system dysfunction,25−28 and low birth weight babies.29−32 The major historical manufacturer of PFOS and PFOA, the 3M Company, voluntarily phased out33 the production of these two compounds between years 2000 and 2002 after their wide human34 and environmental distributions35 were first reported. Prior to this, the 3M Company manufactured PFOS and PFOA for over 4 decades by an industrial process known as electrochemical fluorination (ECF). This process yielded a consistent mixture of branched and linear isomers in the resulting commercial products. For PFOS, the composition was approximately 70% linear and 30% branched, while for PFOA © 2015 American Chemical Society
the composition was approximately 80% linear and 20% branched.36 Two studies have compared the relative toxicities of linear and branched isomers of PFOS or PFOA. In the first, Loveless et al.37 fed the ammonium salt of PFOA (APFO) to rats and mice in one of three following formulas: (i) pure linear APFO, (ii) pure branched APFO, and (iii) a mixture of linear and branched APFO (ratio of 78:22). The authors concluded that branched isomers of APFO were less toxic than the linear isomer based on results that the pure branched APFO treatment had the least adverse effect on body weights, food intake and efficiency, hepatic peroxisomal β-oxidation, and organ weights. Whether the lower toxicity of branched isomers was related to their faster elimination (discussed below) or to their interaction with molecular receptors is unknown. In the second study, O’Brien et al.38 used microarray technology to compare the effects of technical PFOS (a mixture of linear (60−70%) and branched PFOS (30−40%)), versus pure linear PFOS, on the transcriptional profiles of chicken embryonic hepatocyte cultures. At equivalent 10 μM concentrations, Received: Revised: Accepted: Published: 5722
November 4, 2014 March 30, 2015 March 31, 2015 March 31, 2015 DOI: 10.1021/es505399w Environ. Sci. Technol. 2015, 49, 5722−5731
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serum albumin and reported that over 99% of PFOS (0.013 μM to 0.260 μM) and PFOA (0.023 μM to 0.320 μM) were noncovalently bound to albumin. However, to date, no studies have examined the isomer specific binding of PFOS or PFOA, even though in the earliest study Jones et al.55 suggested that the “consequences of dif ferential binding of straight-chain and branch-chain forms of PFOS to albumin require f urther investigation”. Ng and Hungerbuhler60 recently raised the question as to whether different bioaccumulation potentials of linear and branched isomers can be explained by their dissimilar binding affinities to albumin, while Greaves and Letcher43 suggested further research to understand how molecular size and geometries of linear and branched PFOS isomers can influence their protein binding affinities and excretion patterns. Here, we hypothesized that the binding affinities of linear PFOS and PFOA to serum proteins would be higher than any corresponding branched isomer. If true, then under physiological conditions this would leave a higher proportion of unbound branched isomers in the serum to cross the placental barrier, or to be filtered through the glomerular membrane and excreted in urine; both of which have been observed to occur in humans. To test this hypothesis, an ultrafiltration method was employed to estimate the dissociation constants (Kd) of linear and three individual branched isomers (3m, 4m, and 5m) of PFOS and PFOA with human serum albumin and to study the behavior of technical PFOS and PFOA mixtures spiked in whole calf serum and human serum.
technical PFOS upregulated 278 genes, compared to only 104 genes for pure linear PFOS, and downregulated 62 genes, compared to only 26 genes for pure linear PFOS. The very limited information available for PFOS must be cautiously interpreted, but this study suggests that branched isomers may induce different effects than linear PFOS, which could translate into different toxicities. PFOS and/or PFOA isomer specific disposition has been studied in fish,39 chicken eggs,40 rodents,41,42 polar bears,43 and humans.44 In rainbow trout and zebrafish,39 preferential bioaccumulation of linear PFOS was shown in fish tissues compared to all branched isomers. Chicken eggs injected (into the air cell) with technical PFOS had higher bioaccumulation of linear PFOS in the embryonic liver tissue compared to the branched isomers.40 In Sprague−Dawley rats41,42 exposed orally to technical mixtures of PFOS and PFOA, the proportion of branched isomers in urine (the primary mode of excretion) was higher than in the blood or in the corresponding technical mixture, also indicating a preference to accumulate the linear isomer over branched isomers. Greaves and Letcher43 analyzed PFOS isomers in polar bear tissues, and results suggested isomer-specific pharmacokinetics, which the authors postulated could be due to isomer-specific protein affinities. The only study to date on the isomer-specific elimination of PFOS or PFOA in humans also showed a consistent preferential urinary excretion of branched isomers.44 In this pharmacokinetic context, it is therefore interesting to note that human blood is often enriched in branched PFOS isomers (i.e., >30% branched PFOS) compared to historical ECF products,45,46 as this cannot be explained by the above experimental information whereby the linear isomer is consistently the most bioaccumulative. One possible explanation for enrichment of branched isomers in human serum is preferential metabolism of branched PFOS-precursors to branched PFOS in vivo.47,48 PFOS and PFOA isomers can cross the human placenta and enter into fetal circulation, and three studies have shown the preferential enrichment of branched PFOA and PFOS isomers in cord blood, relative to maternal blood.45,49,50 The higher transplacental transfer efficiencies of branched PFOS and PFOA isomers are surprising. First, because more hydrophobic molecules are generally more efficient at crossing the lipid bilayer of endothelial cells,51 and based on their elution orders in reversed phase chromatography,52 branched PFOS and PFOA isomers are less hydrophobic than the linear molecules. Second, as predicted by Sastry53,54 the more “rounded” a molecule is, the less efficient it will be at crossing the placental barrier. Thus, when the geometries of the branched isomers (higher cross sectional diameters) are compared to linear isomers, the transplacental movement of the branched isomers would be predicted to be lower. In a direct binding assay, conducted at a technical PFOS concentration (i.e., mixture of isomers) of 2.35 mg/mL with bovine serum albumin (1 mg/mL), and detection of the albumin-bound fraction of PFOS by quadrupole time-of-flight mass-spectrometry, Jones et al.55 reported that nearly all (>98%) PFOS was bound to bovine albumin. Using equilibrium dialysis, Zhang et al.56 later concluded that one molecule of serum albumin can bind up to 45 molecules of PFOS noncovalently. For PFOA, Han et al.57 used size exclusion chromatography and ligand binding methods to determine that over 90% of PFOA was bound to albumin in human or rat blood under normal physiological conditions. Bischel et al.58,59 used equilibrium dialysis with 200 μM bovine
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MATERIALS AND METHODS Chemical Standards and Reagents. The same nomenclature proposed by Benskin et al.52 was used for the individual branched PFOS and PFOA isomers, whose structures are shown in Figure S1. Linear PFOS (50 μg/mL), linear PFOA (50 μg/mL), and mass-labeled internal standards (MPFACMXA) were from Wellington Laboratories (Guelph, ON, Canada). Technical mixtures of PFOS (Br-PFOS) and PFOA (T-PFOA) were also from Wellington Laboratories. High concentration standards of individual isomers, 3m-PFOS (23.3 μg/mL), 3m-PFOA (44.4 μg/mL), 4m-PFOS (26.4 μg/mL), 4m-PFOA (58.0 μg/mL), 5m-PFOS (42.8 μg/mL), and 5mPFOA (84.0 μg/mL) were kindly donated by Wellington Laboratories on a one-time basis (Guelph, ON, Canada) and are not commercially available at this high concentration. The ECF-PFOS standard (80% linear and 20% branched) used here was donated from the 3M Company (Saint Paul, MN, United States of America). Fatty acid and globulin free human serum albumin (HSA; purity ≥99% by agarose gel electrophoresis) was purchased as a lyophilized powder from Sigma-Aldrich (Oakville, ON, Canada). All chemicals used in the preparation of organic and aqueous mobile phases (methanol, water, formic acid and ammonium hydroxide) for liquid chromatography were of HPLC grade from Fisher Scientific (Ottawa, ON, Canada). Incubation and Ultrafiltration of Pure Isomers with HSA. HSA (0.006 mM) was prepared in HPLC grade water, and 900−1000 μL aliquots were dispensed into 1.5 mL Eppendorf safe-lock polypropylene microcentrifuge tubes. This particular concentration of HSA was chosen such that it falls within the range of PFOS and PFOA (both linear and branched) concentrations used in this experiment, thus a 1:1 PFOS (or PFOA) molar ratio was achieved at a given point within the range of selected concentrations. The albumin solutions (pH 6.4) were then spiked with pure linear, or pure 5723
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representing multiple discrete points for the calibration range of 0 mM to 0.012 mM, were also diluted 100-fold prior to analysis. The ultrafiltrates were diluted 20-fold with methanol, and a portion of these were spiked with 5 μL of a 100 ng/mL mixture of mass-labeled internal standards (MPFAC-MXA). All samples were centrifuged at 14 000 rpm (Thermo IEC Micromax RF, Thermo Fisher Scientific, Ottawa, Canada) for 30 min, and supernatants were then transferred to HPLC vials for analysis. The method of Benskin et al.49 was adapted to identify and quantify the individual linear and branched isomers of PFOS and PFOA in all samples. More details are in the Supporting Information. Calculation of Isomer-Specific Dissociation Constants (Kd). For all samples (blanks, quality controls, ultrafiltrates, and uncentrifuged), the relative response (RR) of each PFOS or PFOA isomer was calculated relative to linear 13C-labeled PFOS or PFOA internal standard. The percentage of free PFOS or PFOA isomer in a sample was calculated by eq 1, using linear PFOS as the example:
branched, isomer standards such that the total volume reached 1000 μL in all tubes. The concentrations of individual PFOS and PFOA isomers ranged from 0.002 mM to 0.012 mM, and residual methanol concentration ranged from 2 to 10%. All tubes were mixed by gentle inversion, to avoid frothing and possible denaturing of the albumin61 and were incubated at 37 °C in a circulating water bath (BÜ CHI Labortechnik GmbH, Flawil, Switzerland) for 1 h, after which 700 μL was transferred by pipet to a Centrifree ultrafiltration (UF) device (Figure S2), taking care to avoid air bubbles. These devices can accommodate a maximum volume of 1 mL, and the molecular weight membrane cutoff was 30 000 Da. After loading the UF devices, they were centrifuged at 1500g at 37 °C for 30 min (Sorvall, ST 40 R, Thermo Fisher Scientific, Ottawa, Canada). The remaining 300 μL of incubated solution (not centrifugedthus representing total PFOS and PFOA concentrations) and the ultrafiltrates were kept at −20 °C pending sample preparation and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). A series of quality control samples (containing no protein) were similarly prepared with HPLC-grade water spiked with different concentrations of PFOS or PFOA to examine for any nonspecific binding of PFOS or PFOA to the UF device, or to the walls of the centrifuge tubes. Previous studies have shown that the Centrifree UF device is a quick, reliable, and cost-effective method for measuring the free fraction of drugs or hormones in blood. For example, Dow et al.62 suggested the Centrifree UF device as an excellent method for estimating the free fraction of pharmaceuticals in plasma, and elegantly derived the mathematical equation for calculating the percentage of plasma protein binding. In an attempt to estimate the bioavailability of triiodothyronine and thyroxine, Gu et al.63 used the Centrifree UF device to separate bound and free fractions of these hormones. More recently, Jensen et al.64 used the Centrifree UF device to quantify the bound and free enantiomers of warfarin in human plasma, and the authors highlighted the exceptional reliability of the method. Incubation and Ultrafiltration of Technical PFOS and PFOA Mixtures in Calf and Human Serum. Unlike the dissociation constant experiments described above, whereby pure individual isomers were spiked into pure HSA, in these secondary studies whole serum (containing all endogenous plasma proteins) was spiked with technical mixtures of linear and branched isomers. Next, 1 mL aliquots of whole calf serum (Lampire Biological Laboratories Inc., Pipersville, PA 18947, USA) were spiked with 100 ng/mL, 200 ng/mL, and 300 ng/ mL of Wellington technical PFOS (78.8% linear) and Wellington technical PFOA (79.0% linear), respectively. Then, 1 mL aliquots of diluted (1 in 10, with sterile physiological saline) pooled human serum (Lampire Biological Laboratories Inc., Pipersville, PA 18947, USA) were spiked in triplicate at mg/L concentrations with the historically relevant ECF-PFOS mixture from 3M, and Wellington technical PFOA. The main reason for diluting the human serum prior to spiking was to diminish the background level of PFOS and PFOA. Also, because the sample was a commercial one (Lampire Laboratories, Pipersville, PA, USA), it was most likely from nonfasting individuals and was visibly turbid. Sample Preparation and Analysis. Prior to instrumental analysis, the remaining uncentrifuged samples, representing total (free + bound) PFOS or PFOA, were diluted 100-fold with methanol. The individual pure standards (in methanol),
%Free linearPFOS =
RR UF × 100 RR UC
(1)
where RRUF represents the relative response of linear PFOS in the ultrafiltrate (i.e., free linear PFOS) and RRUC represents the relative response of linear PFOS in the uncentrifuged sample (i.e., free and albumin-bound linear PFOS). From eq 1 above, the concentration of free linear PFOS (CFreelinearPFOS) and bound linear PFOS (CBoundlinearPFOS) at a given spiked concentration of linear PFOS in 0.006 mM albumin can be calculated according to eqs 2 and 3, respectively. CFree linearPFOS =
%Free linearPFOS × TotalConclinearPFOS 100 (2)
CBoundlinearPFOS = TotalConclinearPFOS − CFree linearPFOS (3)
The various isomer free ligands (LF; i.e., linear and branched isomers of PFOS and PFOA) over a range of concentrations bind reversibly to the protein receptor (R) at a fixed concentration (i.e., 0.006 mM human serum albumin) to form a complex (LR), as in eq 4. K on
LF + R ⇐ ⇒ LR
(4)
K off
At equilibrium, the rate of the forward reaction (Vf), where Vf = kon[L][R] is equal to the reverse reaction (Vb), where Vb = koff[LR] (eq 5), which can be rearranged to solve for the dissociation constant (Kd; eq 6).
kon[L F][R] = koff [LR]
(5)
k K [L F][R] [L ][R] = off = Kd F = off = Kd [LR] kon [LR] Kon
(6)
Thus, Kd is a measure of the relative binding affinity between L and R, with high values of Kd indicating low binding affinities, and low values of Kd indicating high binding affinities. To experimentally derive an estimate for the binding between PFOA and rat or human serum proteins, Han et al.57 formerly proposed the following variant of Scatchard equation linking LF to Kd: 5724
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Figure 1. Scatchard plots for calculation of the dissociation constants (Kd) and number of binding sites (n) for linear and three individual branched isomers of PFOS. The p value for regression was 3m- > 4m- > 5m- > iso- > linearPFOS. With the only exception being 1m-PFOS, the current 5727
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Figure 4. Box-plots (5th, 25th, 50th, 75th, 95th percentiles) showing percentage of individual (A) PFOS and (B) PFOA isomers in ultrafiltrates of spiked human serum (serum diluted 10-fold prior to spiking). Higher values indicate less serum protein binding.
Significance and Limitations of the Findings. The higher binding affinities of linear PFOS and PFOA to HSA and other serum protein, relative to branched isomers, can help to explain various human and rodent pharmacokinetic data. For example, the higher transplacental transfer efficiencies of branched isomers of PFOS and PFOA in humans45,49,50 could be explained based on the fact that linear PFOS and PFOA are adsorbed most strongly to serum proteins and thus are less bioavailable to cross the placental barrier. Moreover, the higher rates of renal clearance for branched PFOS and PFOA isomers (relative to linear) in humans44 and rodents41,42 are also consistent with the current data because, as xenobiotics bind to serum albumin, or other large serum proteins, they are not available to be filtered and eliminated in urine by the kidney. D’eon et al.70 noted the exceptional binding of PFOA to HSA by nuclear magnetic resonance (NMR) and postulated that the interactions of perfluorinated substances with albumin must have a direct bearing on elimination kinetics of these compounds from the human body; the current data support this proposition. Using a computer simulation for the interaction between HSA and PFOS or PFOA, Salvalaglio et al.65 made a number of key observations that are relevant to the current discussion, even though they did not discriminate between linear and branched isomers in their modeling. First, based on the free binding energies of PFOS-HSA and PFOA-HSA complexes at thermodynamic equilibrium, they reported a maximum number of nine PFOA and 11 PFOS molecules that can be adsorbed on HSA at the critical micelle concentration, which the authors suggested as one possible explanation for the higher bioaccumulation potential of PFOS compared to PFOA. We calculated only two binding sites for linear PFOS and linear PFOA by Scatchard analysis; thus like Salvalaglio et al.65 we did not find a significant difference in this parameter for linear PFOS and linear PFOA with HSA. However, we suggest that both Kd and the number of binding sites must be considered together for bioaccumulation potential modeling. Salvalaglio et al.65 also suggested that the hydrophobic nature of the perfluoroalkyl chain plays a major role in the PFAA-HSA complex formation and that this association should favor bioaccumulation. Our findings agree with this, taking for example that the branched isomers are less hydrophobic (e.g.,
PFOS isomer binding to total human serum proteins (relative to linear PFOS), of which HSA is known to be the major component. The findings in spiked serum generally support the results from Kd experimentation, but direct comparison of the results is difficult and beyond the scope of the current work, mainly because of the complex mixture of serum proteins present here. It is also not warranted to directly compare the results for PFOS isomers versus PFOA isomers, which were spiked at much different concentrations, for reasons of detection limits in the ultrafiltrate. Isomer specific PFOS analysis of the spiked human serum samples and ultrafiltrates (Figure 4A) showed no obvious overall structure−activity relationship, but an interesting result was that 1m-PFOS (which is a minor isomer in technical PFOS) was more tightly bound to serum proteins than any isomer, including linear PFOS. Analysis of variance (2-way ANOVA, IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.) indicated a statistically significant isomer effect (p < 0.001), and posthoc comparisons indicated that the only statistically significant differences were for 1mPFOS, whereby its percentage in the ultrafiltrate was significantly lower than for any other isomer. This phenomenon could explain the exceptionally high biological half-life of 1mPFOS in men (geometric mean of 55 years) estimated by Zhang et al.44 from paired serum and urine concentrations, which is much longer than for any other PFOS isomer. With this exceptional ability to bind to human serum proteins, only a very small fraction of 1m-PFOS would pass into the glomerular filtrate and be excreted through urine. For PFOA, higher proportions of branched isomers, compared to linear PFOA, were also consistently detected in the ultrafiltrates of female human serum at all six different spiking concentrations (0.5, 1.0. 1.5, 2.0, 2.5, and 3.0 mg/L). There was no overall trend with concentration, but the protein bound fraction of individual branched PFOA isomers was lower than that of linear PFOA at all concentrations (Figure 4B). ANOVA showed a statistically significant isomer effect (p < 0.001), and posthoc pairwise comparisons showed the proportion of linear PFOA in the ultrafiltrate was significantly lower than for all other branched isomers (p < 0.05). 5728
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elute earlier by HPLC, Figure 3) and most had weaker protein binding to HSA (Figures 1 and 2) and total serum proteins (Figure 4B, Figures S3 and S4). The same explanation is useful for explaining the lower binding affinity of linear PFOA, relative to linear PFOS; the latter is more hydrophobic than PFOA and elutes later on an HPLC column. Bischel et al.58 also reported that, in general, the affinity of perfluoroalkyl substances for bovine serum albumin increases with hydrophobicity. Third, Salvalaglio et al.65 reported on the presence of binding sites at the core of the HSA molecule and therefore discussed how an increase in “size” of the ligand would reduce the ease of access to these sites because of steric hindrance. Given the more bulky structure of branched PFOS and PFOA isomers, compared to their linear counterparts, this could create steric hindrance and may be part of the explanation for lower binding affinity and fewer binding sites for the branched isomers. Other studies of perfluoroalkyl acid or fatty acid binding to HSA also noted the importance of electrostatic70 and hydrogen bonding interactions,71 respectively, in the associated binding domains; thus these factors also likely affect the binding phenomena observed here. This is the first study looking at the isomer-specific binding of PFOA or PFOS, and the data prove useful for explaining previously observed physiological phenomena. Nevertheless, the current study has some limitations. First, the pure individual linear and branched isomers of PFOS and PFOA are not available for purchase at sufficiently high concentrations from any commercial supplier, and the amounts donated for the current study were completely consumed to produce the singlereplicate data presented in Figures 1 and 2. Although these data were sufficient for the variant of Scatchard analysis here, more data points would have been valuable. More pure isomer material would also have allowed examining any influence of the residual methanol (2−10% in final solution) on quantitative Kd estimates. The strength of the binding to HSA and serum proteins was so strong that working at lower concentrations of isomer (which are commercially available) produced no detectable response in the ultrafiltrates. These facts may explain why there have been no reports of isomer specific protein binding previously and also explain why there is so little known about the toxicity of individual PFOS and PFOA isomers. The second limitation, compounded by the same reason as above, is that we did not compare the results of the present experiment using the Centrifree ultrafiltration device with the equilibrium dialysis method, which is still considered as the gold standard method in studies of protein binding. A third limitation is that we did not buffer the pH of albumin solutions (pH = 6.4) to a physiological pH of 7.4. This choice was made to avoid interference in the analytical method, but Zhang et al.56 have shown that this lower pH causes somewhat stronger binding of PFOS to albumin; thus this could partially account for the low Kd estimate for linear PFOS compared to previous reports for total PFOS (Table S1).
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AUTHOR INFORMATION
Corresponding Author
*Phone: 1-780-492-1190. Fax: 1-780-492-7800. E-mail: jon.
[email protected]; mail: 10-102 Clinical Sciences Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2G3. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.B. acknowledges scholarship funding from Alberta Innovates Health Solutions. J.W.M. acknowledges grant support for this project from NSERC Discovery. Alberta Health is thanked for support of daily laboratory operations.
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ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http://pubs.acs.org/ 5729
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