Protein Binding Associated with Exposure to Fluorotelomer Alcohols

Article pubs.acs.org/est

Protein Binding Associated with Exposure to Fluorotelomer Alcohols (FTOHs) and Polyfluoroalkyl Phosphate Esters (PAPs) in Rats Amy A. Rand† and Scott A. Mabury†,* †

University of Toronto, Department of Chemistry, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *

ABSTRACT: The biotransformation of fluorotelomer-based compounds such as fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are sources of exposure to perfluorinated carboxylates (PFCAs), leading in part to the observation of significant concentrations of PFCAs in human blood. The biotransformation of FTOHs and PAPs yield intermediate metabolites that have been observed to covalently modify proteins. In the current investigation, the extent of covalent protein binding in Sprague−Dawley rats upon exposure to 8:2 FTOH and the 6:2 polyfluoroalkyl phosphate diester (6:2 diPAP) was quantified. The animals were administered a single dose of 8:2 FTOH or 6:2 diPAP at 100 mg/kg by oral gavage to monitor biotransformation and extent of protein binding within the liver, kidney, and plasma. In the 8:2 FTOH-dosed animals, perfluorooctanoate (PFOA) was produced as the primary PFCA, at 623.13 ± 59.3, 459.5 ± 171.8, and 397.3 ± 133.0 ng/g in the plasma, liver, and kidney, respectively. For the animals exposed to 6:2 diPAPs, perfluorohexanoate (PFHxA) was the primary PFCA produced, with maximum concentrations of 57.4 ± 6.5, 9.0 ± 1.2, and 25.3 ± 1.2 ng/g in the plasma, liver, and kidney, respectively. Protein binding was observed in the plasma, liver, and kidney after 8:2 FTOH and 6:2 diPAP exposure, with the most significant binding occurring in the liver (>100 nmol/g protein). This is the first study to link the exposure and in vivo biotransformation of fluorotelomer-based compounds to covalent protein binding.

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PFCAs.11−18 Due to the production of PFCAs from FTOH transformation, current efforts are in place to remove 100% of the FTOH residuals on commercial products by 2015.19,20 PFCAs that have carbon chain lengths ≥8 are bioaccumulative and have been observed in human sera at ng/mL concentrations.21−27 Additionally, animal studies have demonstrated that perfluorooctanoate (PFOA), the C8 PFCA congener, is associated with peroxisome proliferation, and developmental and immunological effects.28,29 Although the biotransformation of 8:2 FTOH produces PFCAs, specifically PFOA, perfluorononanoate (PFNA), and perfluorohexanoate (PFHxA), the yields are low, at 2− 3%.12,14,18 While not entirely elucidated, the remaining mass balance of the 8:2 FTOH biotransformation has been attributed to the formation of intermediate metabolites and phase II metabolic conjugates, including 8:2 FTOH-glucuronide and 8:2 FTOH-sulfate.12,14,18 Two intermediate metabolites, the 8:2 fluorotelomer unsaturated carboxylic acid (FTUCA) and 8:2 the fluorotelomer unsaturated aldehyde (FTUAL), undergo conjugation with glutathione (GSH).12,14,15,18 FTUALs (6:2, 8:2, 10:2) have not only been observed to form conjugates with GSH, but also with amino acids (cysteine, lysine, arginine, and

INTRODUCTION Fluorotelomer-based surfactants are high-production chemicals utilized for water- and grease-proofing applications. Among these surfactants are the polyfluoroalkyl phosphates (PAPs), which are primarily applied to food contact papers. PAPs are commercially produced as a mixture of several polyfluorinated chain lengths (i.e., 4:2, 6:2, 8:2, and 10:2) and can have one (monoPAP) or two (diPAP) polyfluorinated tails. These chemicals have been observed to migrate from food contact paper into food, especially water−oil emulsions such as butter.1,2 As such, diPAPs are considered a potential source of human exposure to fluorotelomer-based compounds.3 DiPAPs have been detected at low ng/mL concentrations in the sera of humans, and the distribution of diPAP congeners observed in these studies showed that the 6:2, 6:2/8:2, and 8:2 diPAPs were most prevalent.4,5 The observation of diPAPs in human blood is an indicator that humans are exposed to fluorotelomer-based commercial materials. This warrants attention because diPAPs have been shown to be enzymatically hydrolyzed to produce the corresponding fluorotelomer alcohol (FTOH),6 which subsequently yields perfluorinated carboxylates (PFCAs) (Figure 1).7−9 FTOHs are building blocks in many fluorotelomer-based commercial applications, and as a result have been found as residuals in fluorotelomer-based manufactured products.10 The biotransformation of FTOHs, primarily 8:2 FTOH, has been extensively studied, as FTOHs can oxidize to produce © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2421

October 2, 2013 January 20, 2014 January 25, 2014 January 26, 2014 dx.doi.org/10.1021/es404390x | Environ. Sci. Technol. 2014, 48, 2421−2429

Environmental Science & Technology

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Figure 1. General biotransformation pathway for x:2 diPAP and x:2 FTOH (x = 6 or 8).

histidine), and two model plasma proteins.30 In comparison, FTUCAs conjugate only to small, thiol-containing moieties (i.e., GSH and cysteine).30 Recent work in our lab has also shown that FTUALs bind covalently to proteins in both plasma and liver microsomes.31 This work provided evidence that a major portion of the unaccounted mass balance from 8:2 FTOH biotransformation might be due to covalent binding of FTUALs to proteins, as it was shown that 58% of the mass balance was bound to the microsomal protein fraction. This could have toxic consequences, as protein inactivation and disruption of function are sometimes associated with protein binding.32 The current study was based on work that was previously done using plasma and rat liver microsomes. The biotransformation of 8:2 FTOH and 6:2 diPAP was carried out using rats to assess the extent of protein binding in tissues that accumulate the fluorotelomer biotransformation products: the liver, kidney, and blood plasma. Although there have been many studies on the biotransformation of 8:2 FTOH,11−15,18,33 and a few elucidating the biotransformation pathway of diPAPs,7−9 in this study we determine the extent of covalent protein binding associated with the biotransformation of fluorotelomer-based compounds, thus providing insight into a novel pathway with potential for toxicity.

12 h light/dark cycle with food and water available ad libitum. Two groups of 12, 6−8 week old male rats were administered either 8:2 FTOH or 6:2 diPAP, each at 100 mg/kg, at 4 mL/kg by oral gavage. Using a mortar and pestle, 8:2 FTOH was ground to a fine power and mixed with 0.5% methylcellulose, resulting in a cloudy but homogeneous solution. Adapted from a previous study, the 6:2 diPAP dosing solution was made using 50:50 propylene glycol:water, with 0.1% soy lecithin.9 For both the 8:2 FTOH and 6:2 diPAP experiment, four control rats were administered a respective clean vehicle at 4 mL/kg. Blood, liver, and kidney samples were collected at 0.5, 2, 6, and 24 h subsequent to dosing, with three dosed and one control rat for each time point. At each time point, the animals were anaesthetized using an aqueous solution of approximately 2000 mg/kg urethane (≥99.8%, Sigma-Aldrich, St. Louis, MO). Approximately 10 mL of blood was then collected by cardiac puncture and stored in a 10 mL BD Vacutainer embedded with lithium heparin at 4 °C. Animals were sacrificed by cervical dislocation after which the liver and kidney were collected and stored at −20 °C until analysis. Toxic End Points. The dosing concentrations chosen for 8:2 FTOH and 6:2 diPAP were either equal to or lower than those reported in previous studies where no clinical sign of toxicity was observed.7,34 No fatalities were observed during the course of the experiment, and there were no physical signs of stress at any point during either study. A Mann−Whitney U test was used to determine that the liver somatic index was not statistically different among the exposure groups (8:2 FTOH and 6:2 diPAP) compared to the control animals, indicating that the animals were not under any metabolic stress after chemical exposure (p = 0.05). Extraction Procedure. Plasma was isolated from whole blood by centrifuging (1300g) at 4 °C for 10 min. The plasma fraction was then transferred to a lithium heparin-coated Vacutainer using a lithium heparin-coated syringe to prevent

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MATERIALS AND METHODS Materials. A list of all standards, reagents, and purification methods used in this study is provided in the Supporting Information (SI). Animal Care and Sampling. This research was conducted under an animal use protocol approved by the University of Toronto Animal Care Committee. All animals were treated humanely with regards to alleviation of suffering. Sprague− Dawley rats were obtained from Charles River Laboratories Inc. (Sherbrooke, QC, Canada). The animals were exposed to a 12/ 2422

dx.doi.org/10.1021/es404390x | Environ. Sci. Technol. 2014, 48, 2421−2429


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coagulation, and stored at 4 °C until analysis. A fraction of whole liver (∼1.5 g) and kidney (∼0.5 g) was obtained by wrapping them each in aluminum foil and submerging in liquid nitrogen until frozen. The frozen tissues were then broken into pieces, and several pieces were placed in 50 mL polypropylene centrifuge tubes. A small volume of 1% potassium chloride was added to the liver (0.2 mL) and kidney (0.1 mL) samples. The tissues were homogenized for 1 min with a mechanical mixer before adding ice-cold acetonitrile to the liver (2.5 mL) and kidney (1.5 mL) samples. The mixtures were further homogenized for 2 min and transferred to clean 15 mL polypropylene centrifuge tubes. The plasma samples (0.5 mL) were extracted by adding 0.5 mL ice-cold acetonitrile. The liver, kidney, and plasma samples were each centrifuged (6000g for the liver and kidney, and 14 000g for the plasma) for 10 min at 4 °C, and the supernatant was transferred into a clean tube. The extraction was repeated (n = 3), and the solvent fractions were combined for LC-MS/MS analysis. To remove noncovalently bound protein adducts from the precipitated protein fraction, the fraction was washed with the following solvents:35,36 a mixture of methanol:water (80:20 v/ v) containing 10% (w/v) trichloroacetic acid; methyl tert-butyl ether (MTBE):methanol (50:50, v/v); and methanol:water (80:20, v/v). The resulting precipitated protein pellet was suspended in 0.5, 1, or 2 mL of 1 M NaOH for the plasma, kidney, or liver samples, respectively. The samples were analyzed for total organic fluoride using total organic fluoride combustion ion chromatography (TOF-CIC), and for total protein using the bicinchoninic acid (BCA) assay. The amount of covalent binding to proteins was determined by quantifying the mass of fluoride in the protein pellet relative to the mass of protein present in the extracted sample, and comparing the fluoride concentration values to the procedural blanks. For all experiments, background fluoride concentration was assessed using procedural blanks and determined to be approximate to the background level of fluoride from the whole instrumental analysis, at ∼8.3 ng F- (or 1.7 ng F/mL). For each set of tissue extractions, four blank samples were spiked with 50 ng of each internal standard, extracted, and analyzed using LC-MS/MS to assess recovery during each extraction procedure. Results from these quality control experiments are found in the SI (Tables S1 and S3). To determine the effectiveness of the method used to remove noncovalently bound metabolites, each of one blank liver, plasma, and kidney sample was spiked with 50 ng each of PFCAs, FTCAs, and FTUCAs, extracted, washed, and analyzed for fluoride in the protein fraction using TOF-CIC. Results from these experiments were all found to be below the instrument detection limit. BCA Assay for Protein Determination. Protein concentrations and recovery in whole and extracted plasma, liver, and kidney fractions were determined using the BCA assay, the details of which are found in the SI. Briefly, extracted and whole tissues were mixed with BCA, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 N NaOH. The mixture was left to sit for 30 min at 37 °C, after which the absorbance was read at 562 nm. Protein concentration was determined by comparing of the absorbance of the unknown samples to a standard bovine serum albumin (BSA) curve. LC-MS/MS Analysis. The supernatant from the liver, kidney, and plasma extracts were analyzed by LC-MS/MS using a Waters Acquity LC system (Mississauga, ON, Canada) coupled to an API 4000 mass spectrometer (Applied Biosystems/MDS Sciex, Mississauga, ON, Canada). Analytes

of interest included the 6:2 diPAP parent compound; the saturated and unsaturated fluorotelomer carboxylic acid (FTCA and FTUCA) intermediate products; the FTOH-sulfate, FTOH-glucuronide, and FTUCA- and FTUAL-GSH phase II metabolites; and the PFCAs (SI Table S2). In the 8:2 FTOHdosed rats, concentrations of monitored compounds were below the limits of detection in control rat tissues. For the 6:2 diPAP experiment, there were low levels ( 0.05). All statistical tests were done using nonparametric methods. The Kruskall-Wallis test was used to determine the differences between the plasma, liver, and kidney PFCA concentrations after 6:2 diPAP and 8:2 FTOH exposure (p = 0.05). The Mann−Whitney U test was used to determine differences between two tissue groups (i.e., kidney vs plasma, liver vs plasma, and kidney vs liver, p = 0.05). TOF-CIC Analysis. Concentrations of total organic fluoride in the whole tissues and tissue protein extracts were determined using ion chromatography (IC).37 Briefly, samples were combusted in a furnace (Automatic Quick Furnance (AQF100), Mitsubishi Chemical Analytech, Japan) at 900−1000 °C. Sample combustion converted organofluorines into hydrogen fluoride (HF), which was transferred into an absorption unit where HF separated into H+ and F−. The concentration of F− in the solution was analyzed using IC (ICS-2100, Dionex Co. Ltd., Sunnyvale, CA). Methanesulfonic acid was used as an internal standard to correct for volume differences in the absorption unit. A representative chromatograph for the TOFCIC can be found in the SI (Figure S7). Procedural blanks of whole and extracted tissues and sample replicates (n = 3) were employed.

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RESULTS AND DISCUSSION 8:2 FTOH Exposure. As this was the first study to examine the extent of protein binding as a result of exposure to fluorotelomer-based compounds, the 8:2 FTOH was chosen

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Figure 2. Liver, plasma, and liver concentrations (ng/g ww) of 7:3 FTCA, 8:2 FTCA, 8:2 FTUCA, 7:3 FTUCA, PFOA, PFNA, and PFHpA produced from exposure to 8:2 FTOH. Error bars represent standard error for n = 3 sample replicates.

because of the extensive amount of research elucidating its transformation mechanism. By contrast, the 6:2 FTOH, although expected to proceed similarly, has never been studied in a mammalian system. Past studies have shown that 8:2 FTOH transforms biologically to primarily produce PFOA, where the calculated half-life of 8:2 FTOH was between 1 and 5 h.14 Here, although the 8:2 FTOH half-life was not calculated, the production of PFOA was observed within 0.5 h of transformation, at concentrations (± standard error) of 115.0 ± 35.3, 175.0 ± 95.4, and 66.8 ± 24.6 ng/g in the plasma, liver, and kidney, respectively, as presented in Figure 2. Over the course of the 24 h experiment, the PFOA concentration increased to maximum concentrations of 623.13 ± 59.3, 459.5 ± 171.8, and 397.3 ± 133.0 ng/g in the

plasma, liver, and kidney, respectively. This increase was expected, as a previous study showed that the half-life for PFOA in male rats was between 112 and 217 h.14 The other PFCAs observed in the biotransformation of 8:2 FTOH were PFNA and PFHpA; PFNA formed at higher concentrations than PFHpA in the liver (Figure 2). The relative concentration differences, where [PFOA] > [PFNA] > [PFHpA], have been observed in previous transformation studies of 8:2 FTOH, where PFOA was the primary product formed as a result of the “β-like oxidation” process, followed by PFNA through αoxidation, and PFHpA through oxidation of the 7:3 FTUCA intermediate.14,18 For a complete description of PFCA concentrations in the liver, kidney, and plasma at all time 2424



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presented in SI Figure S2. Maximum plasma concentrations can be found in SI Table S7, where 5:3 FTCA was the highest (1265.3 ± 314.8 ng/g), followed by 6:2 FTCA (471.4 ± 132.8 ng/g), 5:3 FTUCA (183.7 ± 57.3 ng/g) and 6:2 FTUCA (16.4 ± 3.9 ng/g). Calculated plasma half-lives were 11.4 ± 3.0, 18.0 ± 7.2, and 30.2 ± 4.5 h for 6:2 FTUCA, 6:2 FTCA, and 5:3 FTUCA, respectively. These half-lives were about 10-times longer than those seen for 8:2 FTOH likely due to the extra biotransformation step from 6:2 diPAP to 6:2 FTOH. The relative half-life duration was similar to 8:2 FTOH, where 6:2 FTUCA and 6:2 FTCA were significantly shorter than 5:3 FTUCA. Similar to the plasma, concentrations in the liver decreased in the following order: 5:3 FTCA (423.2 ± 105.6 ng/g) > 6:2 FTCA (23.8 ± 9.4 ng/g) > 5:3 FTUCA (10.7 ± 3.1 ng/g) > 6:2 FTUCA (nd). The concentration levels in the kidney exhibited a similar trend, where 5:3 FTCA (367.6 ± 40.4 ng/g) > 6:2 FTCA (148.4 ± 44.4 ng/g) > 5:3 FTUCA (59.2 ± 18.5 ng/g) > 6:2 FTUCA (0.13 ± 0.13 ng/g). Detection of intermediates in all tissues was surprising, as D’eon and Mabury only detected intermediates in a few plasma samples and suggested that the biotransformation of diPAP might proceed differently than that of 8:2 FTOH.9 Here, the production of intermediate acids in all tissues, including blood plasma, was observed. This difference might arise from the larger dose (100 mg/kg versus 50 mg/kg) and plasma volumes extracted in this study (∼0.5 mL versus 0.3 mL).9 At 24 h, the maximum PFCA concentration compared to the initial dose was 0.007 ± 0.001%, and the total amount of acid metabolites (intermediates and PFCAs) was 0.095 ± 0.014% of the initial dose. In the 8:2 FTOH transformation experiment, the PFCA concentrations in tissues represented approximately 94% of the total acid metabolites produced (0.051% out of a total 0.055%). In the 6:2 diPAP transformation experiment, PFCAs represented approximately 7.4% of the total acids produced after 24 h, indicating that the biotransformation of 6:2 diPAP and production of PFCAs occurred more slowly than the biotransformation of 8:2 FTOH. This was also indicated by the relatively longer intermediate half-lives calculated from the 6:2 diPAP transformation. Detection of Phase II Metabolites. Although standards did not exist for any of the phase II metabolites, they were monitored based on mass transitions obtained from previous reports.9,12,14,18 In both the 8:2 FTOH and 6:2 diPAP exposure experiments, phase II metabolites were detected in the plasma, liver, and kidney, as presented in SI Table S9. 6:2 FTOHglucuronide and 6:2 FTOH-sulfate were detected after 0.5 h of 6:2 diPAP exposure. The 6:2 FTUCA-GSH conjugate was not detected. The 8:2 FTOH-glucuronide and 8:2 FTOH-sulfate were detected in liver, kidney, and plasma. The 8:2 FTUCAGSH was primarily detected in liver between 0.5 and 6 h, and in the plasma at the 2 h time point only. Although the 8:2 FTUAL-GSH transition was monitored, there was no evidence of its formation. Covalent Protein Binding. In order to assess the extent of covalent binding to proteins in the liver, kidney, and plasma, an extraction was performed on each tissue to remove any noncovalently bound analytes. This method was based on a standardized method by Evans et al.35 There were a couple of differences between the method used by Evans et al. and the one presented herein. First, the method by Evans et al. involved collecting the protein precipitate using a cell/membrane harvester prior to washing the protein. In our study, the

points, see SI Table S5. After 24 h, the concentration of PFCAs relative to the original dose of 8:2 FTOH was 0.051 ± 0.006%. Intermediate metabolites which formed from the 8:2 FTOH biotransformation were 8:2 FTCA, 8:2 FTUCA, 7:3 FTCA, and 7:3 FTUCA. The maximum concentrations of all intermediates formed at less than 24 h in plasma, with concentrations of 617.4 ± 154.9 ng/g for 8:2 FTCA, 571.7 ± 107.9 ng/g for 8:2 FTUCA, 236.2 ± 25.5 ng/g for 7:3 FTCA, and 5.2 ± 1.0 ng/g for 7:3 FTUCA, shown in Figure 2. Calculated half-lives were 3.5 ± 0.1, 3.6 ± 0.2, 7.1 ± 0.7, and 9.7 ± 0.7 h for the 8:2 FTCA, 8:2 FTUCA, 7:3 FTCA, and 7:3 FTUCA, respectively. The longer presence of 7:3 FTCA and 7:3 FTUCA might indicate that these metabolites were forming from precursors (i.e., 8:2 FTCA and 8:2 FTUCA) still present in the body, which was also noted to be the case by Butt et al. in rainbow trout.38 In the liver, maximum concentrations were 35.8 ± 17.0 ng/g for 8:2 FTUCA, 184.5 ± 65.7 ng/g for 8:2 FTCA, and 170.0 ± 62.8 ng/g for 7:3 FTCA; 7:3 FTUCA was not detected. All metabolites were detected in the kidney, with 8:2 FTUCA having the highest maximum concentration of 571.7 ± 107.9 ng/g, followed by 7:3 FTCA at 236.2 ± 25.5 ng/ g and 7:3 FTUCA at 3.6 ± 0.2 ng/g. Comparison of intermediate concentrations between tissues showed no significant difference (Kruskall-Wallace test, p = 0.05) in concentrations after 24 h. 6:2 diPAP Exposure. The 6:2 diPAP was chosen for the exposure study because of its bioavailability compared to the 8:2 and 10:2 diPAPs, at 74% compared to 5% and < LOD, respectively.9 The biotransformation of 6:2 diPAP proceeds through an enzyme-mediated hydrolysis of the phosphate ester linkage to produce the β-oxidation PFCA product (PFHxA), the α-oxidation PFCA product (PFHpA), and shorter chain length PFCAs (PFPeA). Although the transformation of 6:2 FTOH in mammalian systems has never been studied, the biotransformation of 6:2 diPAP in this study corroborated with the relative biotransformation pathway based on 8:2 FTOH transformation, where PFHxA was produced at the highest concentration, followed by PFHpA and PFPeA, as shown in SI Figure S2. All PFCAs were detected after 0.5 h, and continued to increase and level out over time. Generally, the maximum concentrations occurred at 24 h, although not in all cases (for a complete description of analyte concentrations, see SI Table S7). In the plasma, maximum PFCA concentrations were 57.4 ± 6.5 ng/g (2 h), 72.3 ± 15.5 ng/g (24 h), and 7.4 ± 0.1 ng/g (6 h) for the PFHxA, PFHpA, and PFPeA, respectively. In the kidney, maximum concentrations for PFHxA, PFHpA, and PFPeA were 25.3 ± 1.2 ng/g (2 h), 15.4 ± 3.2 ng/g (24 h), and 8.9 ± 0.4 ng/g (24 h), respectively. The liver had the lowest PFCA concentrations at 9.0 ± 1.2, 5.6 ± 1.3, and 3.4 ± 0.3 ng/ g for the PFHxA, PFHpA, and PFPeA, respectively, which contrasts with what previous studies have shown.7,14 D’eon observed highest PFCA concentrations in the liver, followed by the plasma and kidney. This, as well as the high 6:2 diPAP concentrations observed in liver at 24 h, suggests that the true concentrations in liver were likely not captured within this sampling period. After 24 h, the % biotransformation of 6:2 diPAP in liver was approximately 0.1% as calculated by determining the area under the concentration−time curve (AUC24) (SI Figure S3). D’eon and Mabury calculated the 6:2 diPAP half-life to be 3.9 ± 0.7 days, which also corroborates with an incomplete transformation after 24 h.9 The intermediates produced from exposure to 6:2 diPAP were 5:3 FTCA, 6:2 FTCA, 5:3 FTUCA and 6:2 FTUCA, as 2425



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Table 1. Maximum Protein Binding Concentrations (nmol/g) in Plasma, Kidney, And Liver Resulting from Exposure to 8:2 FTOH and 6:2 diPAP covalent binding (mean ± S.E.)

8:2 FTOH 6:2 diPAP

liver

kidney nmol/g protein

plasma

2661.1 ± 249.4 (6 h) 103.8 ± 29.5 (24 h)

216.1 ± 60.4 (24 h) 15.4 ± 2.5 (24 h)

198.4 ± 26.3 (6 h) 22.1 ± 2.5 (24 h)

Figure 3. Normalized concentrations (%) of protein binding of corresponding to 6:2 diPAP and 8:2 FTOH exposure in liver, plasma, kidney. Concentration values (nmol/g) were normalized to the highest binding concentration observed in all tissues collected over all time points for each of the dosed-exposure experiments. Both graphs have been normalized to 100%. Error bars represent standard error for n = 3 sample replicates.

in the liver and other tissues after 8:2 FTOH and 6:2 diPAP exposure, respectively. In the kidney and plasma, binding was approximately 1 order of magnitude lower than that observed in the liver. This low binding value might not be surprising despite the similarities in acid metabolite levels observed in the kidney and liver; since metabolism primarily occurs in the liver, the production of the FTUAL would also occur in the liver. If binding is associated with production of the FTUAL, the high reactivity and corresponding brief lifetime of the FTUAL might permit it to react readily with liver proteins but limit its ability to migrate to the kidney or plasma. The lifetime of FTUALs has not been accurately quantified in any biological system, but the 8:2 FTUAL has been reported as undetected in both live and dead hepatocytes after two hours.12 However, since some binding was observed in both kidney and plasma, this suggests that the FTUAL may have a long enough lifetime to escape its site of formation and migrate into the kidney and plasma.41 Alternatively, FTUAL-GSH conjugates formed in the liver can facilitate the migration of the reactive intermediate to the kidney and plasma. Subsequently, the GSH-conjugate can dissociate to yield the free FTUAL.35 Martin et al. detected the 8:2 FTUAL-GSH conjugate in rat hepatocytes,12 and its corresponding reduced conjugate, the 8:2 unsaturated FTOH (8:2 uFTOH)-GSH, has been observed in rat and human hepatocytes, and rat bile and feces.12,14,18 Although the MS/MS transition for 8:2 FTUAL-GSH was monitored in this study, there was no evidence of conjugate formation in any tissue, although this could have been attributed to the unoptimized instrumental parameters rather than lack of formation in vivo. The feasibility for 8:2 FTUAL-GSH to migrate into other tissues and release free FTUAL is unknown, although evidence of migration of the 8:2 FTUAL-GSH from the liver is

centrifugation method was used after precipitation to collect the protein in each tissue. However, a comparison of covalent binding data from the two methods determined that the methods produced similar results.39 Second, the method by Evans et al. used 80% methanol to wash the protein. Here, three kinds of methanol solvent mixtures were used, based on other studies that effectively used these solvent mixtures to examine the in vitro and in vivo covalent binding of several drug metabolites.40,36 To determine the total amount of fluoride bound to the extracted protein fraction, the extracted fractions were analyzed using TOF-CIC. Extracted tissues were also analyzed for the total amount of protein using the BCA assay. The amount of covalent binding was expressed as the amount of fluoride (nmol) bound to the mass of protein (g) in the extract. Maximum concentrations of bound fluoride from exposure to 6:2 diPAP and 8:2 FTOH can be found in Table 1. Significant protein binding occurred in all tissues examined, where binding in liver ≫ kidney ≈ plasma. Since the liver is the primary site of biotransformation for FTOHs and diPAPs, it was appropriate that liver protein binding was readily observed, presuming that binding is correlated with biotransformation. Our previous study using rat liver microsomes indicated that, in the presence of NADPH to initiate biotransformation of 8:2 FTOH, protein binding comprised much of the mass balance compared to the negative controls (without NADPH) that inhibited biotransformation.31 This indicated that binding was due to the production of an 8:2 FTOH metabolite. In the same study, FTUALs were shown to readily bind with proteins, suggesting that it was the production of the FTUALs that contributed to the protein binding observed.31 Thus, the 8:2 FTUAL and 6:2 FTUAL might be contributing to most of the binding observed 2426



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contribution is unknown. Although the presence of these pathways might decrease the amount of binding that would otherwise occur, this study shows that binding is still significant. Uetrecht has reported that drugs given at a daily dose of 10 mg or less are rarely associated with a significant degree of drug reactions and corresponding toxicity.44 Nevertheless, protein binding data is part of the many factors that contribute to the fate of a drug candidate. The covalent protein binding observed in this study should be considered in the many contributing factors that govern the consequence of this exposure. The biotransformation of 8:2 FTOH and 6:2 diPAP after 24 h yielded 0.055 ± 0.008% and 0.095 ± 0.013%, respectively, of total acid metabolites produced compared to the initial dose. This demonstrates that the production of acid metabolites might not be the only fate of fluorotelomer biotransformation. As suggested by previous studies, additional pathways contributing to the mass balance of 6:2 diPAP and 8:2 FTOH biotransformation might involve the formation of phase II conjugates.12,14,18,45 Previous work in our lab has shown that a significant fraction of 8:2 FTOH biotransformation in rat liver microsomes corresponded to protein binding. Here, the extent of protein binding compared to the total fluoride in each tissue was calculated based on results from the TOF-CIC. Binding ranged from 4 to 40% for 6:2 diPAP and 12−43% for 8:2 FTOH in all tissues over the 24 h period, suggesting that an appreciable fraction of the mass balance of 8:2 FTOH and 6:2 diPAP biotransformation after 24 h of exposure contributes to protein binding. For this study, male rats were used to assess the transformation of fluorotelomer-based compounds and corresponding protein adduct levels. However, transformation rates, half-lives, and metabolite levels can vary significantly depending on the sex and species used. For example, the halflives of PFCAs in male and female rats were calculated to be 0.10 and 0.05 days, respectively for PFHxA, 5.63 and 0.08 days for PFOA, 29.5 and 2.44 days for PFNA, and 39.9 and 58.6 days for PFDA.46 The data from our investigation provided a picture regarding what transpires in one sex of one species. Results may differ depending on intersex and interspecies variations.

supported by the observation of the 8:2 uFTOH-GSH in bile and feces.14,15 Fasano et al. proposed that the 8:2 uFTOH-GSH may form from conjugation of 8:2 FTUAL in the liver and/or direct GSH conjugation of 8:2 FTUAL in the kidney;14,15 however, glutathione S-transferase activity in the kidney is much lower than in the liver, suggesting that conjugates formed in the liver may be responsible for extrahepatic effects.42 The protein binding resulting from exposure to 8:2 FTOH was approximately 10-times greater than the binding observed after exposure to 6:2 diPAP. This provides further evidence that the amount of protein binding is likely directly correlated to the biotransformation of that compound to produce reactive intermediates. More binding of 6:2 diPAP might have occurred past 24 h, as its half-life in plasma is approximately 3.9 ± 0.7 days,9 whereas 8:2 FTOH biotransforms comparatively more quickly, with a half-life between 1 and 5 h.14 Given that the transformation of 6:2 diPAP in this study had a calculated biotransformation of 0.1%, the levels of binding observed here may increase 1−3 orders of magnitude with increasing transformation. This suggests that, compared to the 8:2 FTOH transformation, there may be increased levels of binding with a similar extent of transformation. When the amount of binding was plotted over time (Figure 3), the maximum levels of binding for 8:2 FTOH transformation occurred prior to 24 h. For the 6:2 diPAP transformation, the levels of binding continued to increase, suggesting that the full extent of binding might occur past 24 h. However, the impact of protein binding depends on the halflives of protein adducts in vivo; based on the protein half-lives, protein adducts might be eliminated in the 6:2 diPAP-exposed rats prior to reaching the maximum levels of binding. For example, the half-life of serum albumin in rats is 2.5 days.43 This indicates that serum albumin adducts might be eliminated before 6:2 diPAP has transformed by 50%. Therefore, the maximum levels of protein adducts after 6:2 diPAP transformation may not be reached depending on the elimination half-lives of proteins, which may reduce the effect of these adducts in vivo. Covalent protein binding has important implications for drug design and development, since bioactivation of drugs to produce reactive intermediates is a common mechanism to form covalent protein adducts, with potential for toxicity.32 For example, acetominaphen (APAP), bromobenzene, 4-ipomeanol, and furosemide, are known hepatotoxins, where hepatic necrosis is associated with binding levels of approximately 1000 nmol/g protein.35 For drug development, the threshold level for in vivo covalent binding is 20-fold less than that which induces toxicity, at 50 nmol/g total liver protein.35 Both 6:2 diPAP and 8:2 FTOH had protein binding levels that exceeded 100 nmol/g in the liver, where the maximum binding level for 8:2 FTOH in liver was 2661.1 ± 249.4 nmol/g. Both compounds exceeded the pharmaceutical threshold level for covalent protein binding, and 8:2 FTOH exhibited binding on target with the listed hepatoxins, which had binding levels ranging from 1900 to 5600 nmol/g liver protein, after intraperitoneal (IP) doses ranging from 45 to 400 mg/kg.35 The threshold limit is often one of many factors that contribute to the ultimate fate of a drug candidate. Several drugs, including APAP, undergo bioactivation and are still commercially marketed. Many of these factors are influenced by the extent of dose or exposure, and the rate of clearance. We know that the elimination of fluorotelomer-based compounds is influenced by phase II conjugation pathways, but the extent of their

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IMPLICATIONS This study demonstrates that covalent binding to proteins occurs upon exposure to fluorotelomer-based compounds. Rats were exposed to a single dose of 8:2 FTOH or 6:2 diPAP and significant binding was observed. To further understand the risk of protein binding, it will be necessary to determine how the level of protein adducts change depending on the route of exposure and exposure level, and subsequently extrapolate this to the behavior in exposed humans. Additionally, to elucidate the consequence of this binding, we must understand the specific proteins which these reactive intermediates are targeting. Since plasma protein binding was also observed, serum albumin (SA), the most abundant protein in plasma, might be a target.47 Studies have shown that reactive electrophiles produced from metabolism of drugs might target SA; as such, SA or other protein adducts can be used as biomarkers to determine general human exposure to electrophiles produced as intermediates during the metabolism.41 Furthermore, when bovine plasma was incubated directly with FTUALs, adducts with BSA were observed.31 Although this adduct formation with BSA is preliminary evidence of targeted protein modification, it suggests that covalent binding to blood proteins such as SA might be a useful measure to elucidate the 2427



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extent of indirect versus direct exposure to fluorotelomer-based compounds. D’eon and Mabury have suggested diPAPs to be a major source of exposure to PFCAs.3 Industry is shifting toward C6and C4-based compounds which produce PFCAs that are less bioaccumulative and potentially less toxic than those with chain-lengths greater than C7. However, continuous exposure to diPAPs, as observed by the increased concentrations of diPAPs in blood, might result in continuous exposure to PFCAs, such as PFHxA.4,9 Although FTOHs are being removed as residuals from commercial products, diPAPs are directly used in commercial applications.48 From this study and others, it has been demonstrated that the biotransformation of FTOHs and diPAPs closely mimic each other in terms of the metabolites produced; removal of FTOHs does not mean that the indirect exposure to PFCAs will cease.7−9 Additionally, exposure to diPAPs, even those with shorter carbon chain lengths, may lead to the continuous production of reactive intermediates and formation of protein adducts, which have unknown toxicity. Further work must be done to understand the consequence of protein binding, in addition to our concerted efforts to determine the effects of exposure to PFCAs.

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(5) D’eon, J. C.; Crozier, P. W.; Furdui, V. I.; Reiner, E. J.; Libelo, E. L.; Mabury, S. A. Observation of a commercial fluorinated material, the polyfluoroalkyl phosphoric acid diesters in human sera, wastewater treatment plant sludge, and paper fibers. Environ. Sci. Technol. 2009, 43, 4589−4594. (6) Jackson, D. A.; Mabury, S. A. Enzymatic kinetic parameters for polyfluorinated alkyl phosphate hydrolysis by alkaline phosphatase. Environ. Toxicol. Chem. 2012, 39, 1966−1971. (7) D’eon, J. C.; Mabury, S. A. Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): Exploring routes of human contamination. Environ. Sci. Technol. 2007, 41, 4799−4805. (8) Lee, H.; D’eon, J.; Mabury, S. A. Biodegradation of polyfluoroalkyl phosphates as a source of perfluorinated acids to the environment. Environ. Sci. Technol. 2010, 44, 3305−3310. (9) D’eon, J. C.; Mabury, S. A. Exploring indirect sources of human exposure to perfluoroalkyl carboxylates (PFCAs): Evaluating uptake, elimination, and biotransformation of polyfluoroalkyl phosphate esters (PAPs) in the rat. Environ. Health Perspect. 2010, 119, 344−350. (10) Dinglasan-Panlilio, M. J. A.; Mabury, S. A. Significant residual fluorinated alcohols present in various fluorinated materials. Environ. Sci. Technol. 2006, 40, 1447−1453. (11) Hagen, D. F.; Belisle, J.; Johnson, J. D.; Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographichelium microwave plasma detector-the biotransformation of 1H, 1 H,2H,2H perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981, 118, 336−343. (12) Martin, J. W.; Mabury, S. A.; O’Brien, P. J. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interact. 2005, 155, 165−180. (13) Kudo, N. Induction of hepatic peroxisome proliferation by 8−2 telomer alcohol feeding in mice: Formation of perfluorooctanoic acid in the liver. Toxicol. Sci. 2005, 86, 231−238. (14) Fasano, W. J. Absorption, distribution, metabolism, and elimination of 8−2 fluorotelomer alcohol in the rat. Toxicol. Sci. 2006, 91, 341−355. (15) Fasano, W. J.; Sweeney, L. M.; Mawn, M. P.; Nabb, D. L.; Szostek, B.; Buck, R. C.; Gargas, M. L. Kinetics of 8−2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats. Chem. Biol. Interact. 2009, 180, 281−295. (16) Liu, J.; Wang, N.; Buck, R. C.; Wolstenholme, B. W.; Folsom, P. W.; Sulecki, L. M.; Bellin, C. A. Aerobic biodegradation of [14C] 6:2 fluorotelomer alcohol in a flow-through soil incubation system. Chemosphere 2010, 80, 716−723. (17) Wang, N.; Szostek, B.; Buck, R. C.; Folsom, P. W.; Sulecki, L. M.; Gannon, J. T. 8−2 Fluorotelomer alcohol aerobic soil biodegradation: Pathways, metabolites, and metabolite yields. Chemosphere 2009, 75, 1089−1096. (18) Nabb, D. L.; Szostek, B.; Himmelstein, M. W.; Mawn, M. P.; Gargas, M. L.; Sweeney, L. M.; Stadler, J. C.; Buck, R. C.; Fasano, W. J. In vitro metabolism of 8−2 fluorotelomer alcohol: Interspecies comparisons and metabolic pathway refinement. Toxicol. Sci. 2007, 100, 333−344. (19) Environmental Performance Agreement (“Agreement”) Respecting Perfluorinated Carboxylic Acids (PFCAs) and Their Precursors in Perfluorochemical Products Sold in Canada; Environment Canada, Health Canada: Gatineau, QC, 2010. (20) U.S. EPA. 2010/2015 PFOA Stewardship Program. (21) Hansen, K. J.; Clemen, L. A.; Ellefson, M. E.; Johnson, H. O. Compound-specific, quantitative characterization of organic fluorochemicals in biological matrices. Environ. Sci. Technol. 2001, 35, 766− 770. (22) Yeung, L. W. Y.; So, M. K.; Jiang, G.; Taniyasu, S.; Yamashita, N.; Song, M.; Wu, Y.; Li, J.; Giesy, J. P.; Guruge, K. S.; Lam, P. K. S. Perfluorooctanesulfonate and related fluorochemicals in human blood samples from China. Environ. Sci. Technol. 2006, 40, 715−720. (23) Calafat, A. M.; Kuklenyik, Z.; Caudill, S. P.; Reidy, J. A.; Needham, L. L. Perfluorochemicals in pooled serum samples from

ASSOCIATED CONTENT

S Supporting Information *

A list of chemicals, instrumental details, pharmacokinetic calculations, kinetic plots, and LC-MS/MS concentration results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (416)-978-1780; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jessica D’eon for assistance in the development of the animal experiment. We also thank Kate Banks and Jean Kontogiannis for their assistance with the development of animal protocols and handling. We give thanks to Leo Yeung for assistance with the experimental extraction design and with the TOF-CIC analysis (University of Toronto, Toronto, ON, Canada), and to Holly Lee for the synthesis of the 6:2 diPAP. Thanks to Deborah Zamble for use of a cold room and to Mark Nitz for use of a lyophilizer. Scott A. Mabury acknowledges receipt of an NSERC Discovery award.

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REFERENCES

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Protein Binding Associated with Exposure to Fluorotelomer Alcohols

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