Article pubs.acs.org/est
Covalent Binding of Fluorotelomer Unsaturated Aldehydes (FTUALs) and Carboxylic Acids (FTUCAs) to Proteins Amy A. Rand† and Scott A. Mabury*,† †
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *
ABSTRACT: Fluorotelomer unsaturated carboxylic acids and aldehydes (FTUCAs and FTUALs) are intermediate compounds that form from the biotransformation of fluorotelomer-based compounds. Previous evidence that FTUCAs and FTUALs bind to biological nucleophiles has indicated that protein binding might give rise to toxicity resulting from protein function disruption. The current study assesses the reactivity of FTUALs and FTUCAs by probing the covalent interactions of FTUALs and FTUCAs with proteins present in rat liver microsomes and bovine blood plasma. The FTUALs exhibited significant levels of protein covalent binding, with binding levels ranging from 20.1 (±2.8)% to 71.3 (±19.5)% in microsomes and 24.0 (±1.5)% to 82.5 (±14.0)% in blood plasma. By contrast, the FTUCAs did not exhibit any apparent covalent binding. Bovine serum albumin (BSA) was extracted and isolated from the plasma after incubation of 8:2 FTUAL (5−100 μM). Electrospray ionization mass spectrometry (ESI-MS) was used to investigate the stoichiometry of 8:2 FTUAL covalently bound to BSA; three measurable FTUAL adducts were formed with BSA. To compare the percent binding results from the FTUAL microsome incubation experiments, 8:2 FTOH was incubated in microsomes to determine the protein binding associated with the biotransformation of 8:2 FTOH. Results from this study showed that the biotransformation of 8:2 FTOH yielded 26.1 (±3.0)% binding, and was statistically similar to the percent binding associated with 8:2 FTUAL exposure (p > 0.05), indicating that the binding of 8:2 FTUAL to proteins might be a primary fate in the biotransformation of 8:2 FTOH.
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INTRODUCTION Polyfluoroalkyl phosphate diesters (diPAPs) are a class of fluorotelomer-based compounds that have been observed at μg/L concentrations in human blood.1,2 DiPAPs are used to greaseproof food contact paper products and have been observed to migrate from the paper into food and food simulants.3−5 It is suggested that humans are exposed to diPAPs through ingestion of foods in contact with diPAPtreated paper.3,4 Human exposure to diPAPs is significant because PAPs (mono- and di-) have been shown to biotransform,6,7 hydrolyzing to produce the corresponding fluorotelomer alcohol (FTOH)8 and subsequently producing perfluorinated carboxylates (PFCAs) with carbon chain lengths that range from C4 to C11.1,2 Much focus has been directed toward PFCAs with carbon chain lengths ≥C8 due to their biological persistence and observation in human sera at μg/L concentrations.9−16 However, studies have shown that the PFCAs that form from biotransformation of fluorotelomerbased species such as the diPAPs or 8:2 FTOH (i.e., perfluorohexanoate (PFHxA); perfluorooctanoate (PFOA); perfluorononanoate (PFNA)) make up 601.1 transition was used for quantification. Table S10 shows the derivatized8:2 FTUAL sample concentrations corresponding to the 8:2 FTOH and 8:2 FTUAL biotransformations. The supernatants had low concentrations of 8:2 FTUAL, and derivatized concentrations above the LOQ (LOQ = 50 ppt) ranged from 0.8 ± 0.1 ppb to 2.1 ± 1.2 ppb (n = 3). In the microsomes incubated directly with 8:2 FTUAL, the concentrations of 8:2 FTUAL-DNPH was high in the volatile extracts, with concentrations of 1150.3 ± 110.3 ppb and 1123.0 ± 5.6 ppb for the experiments with and without NADPH, respectively. These large concentrations reflect the volatility of 8:2 FTUAL, suggesting that partitioning into the headspace might be a possible fate for 8:2 FTUAL. However, when 8:2 FTUAL was formed from the transformation of 8:2 FTOH, the concentration trapped on the DNPH cartridge was small (1.5 ± 0.4 ppb) indicating that when formed as an intermediate, the fate of 8:2 FTUAL is through either oxidative transformation or protein covalent binding rather than partitioning to the headspace.
Implications. Human exposure to PAPs occurs through their use as grease-proofing agents in food packaging materials52 and potentially from their incorporation into personal care and cosmetic products.5,53 Due to their commercial applicability, they have been found at low ppb concentrations in human sera,1,2 and are considered a significant contributor to the PFCA load currently observed in humans.54 Despite the importance of PFCAs, this study shows that we must also determine the significance of PFCA precursors in order to fully elucidate the effect of human exposure to fluorotelomer-based compounds. This study demonstrates the ability of FTUALs to bind with plasma proteins, including BSA. Although it is unknown whether FTUALs are transported into plasma, detection of serum albumin adducts in vivo might be an important biomarker for the indirect exposure to PFCAs from PAPs. This is important since the sources and relative significance of direct versus indirect exposure to PFCAs currently remains unclear. Since FTUALs are a metabolite of the biotransformation of fluorotelomer-based compounds,17,19,20 FTUAL-protein adducts might be used to measure the relative importance of indirect versus direct exposure to PFCAs. Future research in our lab seeks to elucidate the potential for protein binding in vivo using a rat model in order to determine whether adduct formation is specific to liver proteins, or whether FTUALs can be transported to plasma. Furthermore, this study demonstrates that the primary fate of FTUALs is not solely oxidation to PFCAs, but also formation of adducts to the protein fraction of cellular and intracellular systems; this binding accounts for a significant portion of the unaccounted mass-balance of fluorotelomer-based transformations. We currently do not know whether the in vivo conjugation pathway to GSH is the primary fate of FTUALs; however, the results presented here demonstrate that other surrounding proteins might compete with GSH to form adducts. This is concerning, since the formation of covalent protein adducts is often associated with toxicity.26,55−57 Although the extent to which humans may be exposed to FTUALs may be small given the low ppb concentrations of the diPAPs in sera, elucidating the potential toxicity resulting from protein binding is warranted. To determine mechanisms of toxicity associated with this pathway, specific proteins must be targeted in order to determine whether adduct formation poses risk from exposure to fluorotelomer-based compounds.
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ASSOCIATED CONTENT
S Supporting Information *
List of chemicals, synthetic procedures, instrumental details, TOF-IC mass balance percentages, LC-MS/MS supernatant concentrations, GC/MC FTOH concentrations, and timefinding studies. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; phone: (416)-978-1780. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Deborah Zamble for use of a cold room and to Mark Nitz for use of a lyophilizer. We also give thanks to Leo Yeung 1661
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for assistance with the TOF-IC and with LC-MS/MS method development for the 8:2 FTUAL-DNPH samples (University of Toronto, Toronto, ON, Canada). Scott A. Mabury acknowledges receipt of an NSERC Discovery award.
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