Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes
Introduction
The discovery that blood of the general human population contained organic fluorine compounds was first established in 1968 [1]. At that time, it was hypothesized that the unknown contaminants were similar in structure to perfluorooctanoic acid (PFOA) [2], however, it was not until 2001 that unambiguous identification and quantification of PFOA was reported in human blood by high pressure liquid chromatography tandem mass spectrometry (HPLC/MS/MS) [3]. Prompted by the detection of longer chained perfluoroalkyl carboxylates (PFCAs) in wildlife samples [4], [5], Kuklenyik et al. [6] recently demonstrated that the blood of American adults is also contaminated with a homologous series of PFCAs (CF3(CF2)yCOO−, where y = 7–10), including perfluorononanoate (PFNA, y = 7), perfluorodecanoate (y = 8), perfluoroundecanoate (y = 9), and perfluorododecanoate (y = 10). Surprisingly, the exposure sources for all of these substances are not understood.
The US Environmental Protection Agency has appealed for data regarding the sources of PFOA due to the risk of adverse developmental effects in human offspring [7]. Chronic human exposure to PFCAs is also of concern given the non-genotoxic tumorogenicity of PFOA in rats [8] and the inhibitory effect of PFOA and perfluorodecanoate on gap-junction intercellular communication [9]. The toxicological information pertaining to PFCAs is limited largely to PFOA and perfluorodecanoate, however, PFNA and perfluoroundecanoate produce effects that are similar to those elicited by PFOA and perfluorodecanoate [10], [11]. The half-life of PFOA in human blood is estimated to exceed 4 years [12], and although the pharmacokinetics of longer PFCAs has not been examined in humans, longer perfluoroalkyl chains equate to longer elimination half-lives in experimental animals [13], [14], [15]. It can be generalized that all PFCAs resist catabolism and phase II conjugation, and are poorly excreted in humans.
To mitigate any future identifiable risks associated with PFCAs, it is necessary to understand their source(s) of exposure. Human PFCA exposure may result from two broad hypothetical scenarios: (i) direct exposure to PFCAs in commercial products, household dust, or ingestion of food and water containing PFCAs, or alternatively, (ii) via similar exposure routes to precursor molecule(s) that can be metabolized to PFCAs. The only documented direct use of long-chain PFCAs, other than PFOA, is as polymerization aids in fluoropolymer processing [16], but they are also fluoropolymer thermolysis products [17]. These sources may result in some human exposure to PFCAs but these are not examined here. Rather, based on the widespread detection of a series of fluorotelomer alcohols (FTOHs; CF3(CF2)xC2H4OH; where x = 3, 5, 7, 9) in ambient air [18], [19], we hypothesize that the later route of exposure is responsible, at least in part, for current human PFCA concentrations. For example, it is established that 8:2 FTOH (e.g. where x = 7) is metabolized to PFOA in rats [20], however, it is unknown to what extent other PFCAs are also formed from FTOHs. It is also not known if reactive intermediates are formed, and hence if there are any additional adverse health consequences to be expected upon FTOH exposure.
FTOHs belong to a class of telomerized fluorochemicals, having an estimated global production of 5 × 106 kg/year [21], that find use in a diverse range of commercial and industrial applications including paints, coatings, polymers, adhesives, waxes, polishes, electronics, and caulks [22]. Presumably as a result of their widespread use, 6:2, 8:2, and 10:2 FTOH (e.g. x = 5, 7, and 9) are now widespread in the North American atmosphere and human exposure can be expected. Although the magnitude of human exposure to FTOHs has not been assessed, their widespread distribution in ambient air warranted a comprehensive examination of their metabolic fate. Herein, we report results from in vivo and in vitro metabolism studies of the metabolic products and pathways for a series of FTOHs. Results from tandem mass spectrometry experiments are reported here for an 8:2 FTOH exposed rat to validate the early metabolite identification work of Hagen et al. [20], but we focus on results from various isolated rat hepatocyte incubations dosed with 8:2 FTOH, or its synthesized intermediates, in an effort to elucidate the metabolic pathways leading to several novel and reactive metabolites. The respective metabolite profiles for 4:2, 6:2, 8:2, and 10:2 FTOH are also compared in isolated rat hepatocytes.
Section snippets
Chemicals
HPLC grade methanol and acetonitrile, ammonium acetate (>97%), 2,4-dinitrophenylhydrazine (97%; DNPH), PFOA (98%), PFNA (97%), pyrazole (98%) and aminobenzotriazole (98%) were purchased from Aldrich Chemical Co. Hydrochloric Acid was obtained from EM Science (Gibbstown, NJ, USA). 4:2 (97%), 6:2 (97%), 8:2 (97%), and 10:2 (97%) FTOH were purchased from Oakwood Products, Inc. (West Columbia, SC). The major impurity of 8:2 FTOH was the allylic alcohol (CF3(CF2)6CF = CHCH2OH) [23], and all FTOHs were
Identification of acid metabolites
Mass spectrometric analysis of hepatocyte or rat tissue extracts confirmed the three acid metabolites previously reported by Hagen et al. for 8:2 FTOH [20]: PFOA, 8:2 FTCA, and 8:2 FTUCA (Fig. 1). Confirmation was based on retention time and product ion spectra that matched those of authentic standards. These acids were formed quickly from 8:2 FTOH and were confirmed in rat tissues (blood, liver, kidney) 6-h post dose, and also in isolated hepatocytes incubated for 1–3 h with 8:2 FTOH. A novel
Acid metabolites and unaccounted molar balance
The in vivo detection of PFOA, 8:2 FTCA, and 8:2 FTUCA validates, with mass spectral evidence, the early 8:2 FTOH metabolite identification work of Hagen et al. performed by gas chromatography of methyl ester derivatives and using a helium microwave plasma detector [20]. Some fraction of the mass balance attributed to FTUCA in this study may result from metabolism of the allylic alcohol impurity (maximum 3%, based on purity), but certainly FTUCA is also a metabolite of 8:2 FTOH, as evidenced by
Acknowledgements
The Natural Science and Engineering Research Council (NSERC) of Canada is thanked for funding this research through a postdoctoral fellowship grant (Martin). Funding for materials, supplies, and instrument time was provided through NSERC Discovery Grants (O’Brien and Mabury), and an NSERC Strategic Grant (Mabury). Dr. David Ellis, Joyce Dinglasan, and Yun Ye (Department of Chemistry, University of Toronto) are thanked for the synthesis of several polyfluorinated metabolite standards.
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