title: Experimental characterization of the mechanism of perfluorocarboxylic acids' liver protein bioaccumulation: the key role of the neutral species.
abstract: Perfluorocarboxylic acids (PFCAs) of chain length greater than seven carbon atoms bioconcentrate in the livers of fish. However, a mechanistic cause for the empirically observed increase in the bioconcentration potential of PFCAs as a function of chain length has yet to be determined. To this end, recombinant rat liver fatty acid-binding protein (L-FABP) was purified, and its interaction with PFCAs was characterized in an aqueous system at pH 7.4. Relative binding affinities of L-FABP with PFCAs of carbon chain lengths of five to nine were established fluorimetrically. The energetics, mechanism, and stoichiometry of the interaction of perfluorooctanoic acid (PFOA) with L-FABP were examined further by isothermal titration calorimetry (ITC) and electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS). Perfluorooctanoic acid was shown to bind to L-FABP with an affinity approximately an order of magnitude less than the natural ligand, oleic acid, and to have at least 3:1 PFOA:L-FABP stoichiometry. Two distinct modes of PFOA binding to L-FABP were observed by ESI-MS/MS analysis; in both cases, PFOA binds solely as the neutral species under typical physiological pH and aqueous concentrations of the anion. A comparison of their chemical and physical properties with other well-studied biologically relevant chemicals showed that accumulation of PFCAs in proteins as the neutral species is predictable. For example, the interaction of PFOA with L-FABP is almost identical to that of the acidic ionizing drugs ketolac, ibuprofen, and warfarin that show specificity to protein partitioning with a magnitude that is proportional to the K(OW) (octanol-water partitioning) of the neutral species. The experimental results suggest that routine pharmacochemical models may be applicable to predicting the protein-based bioaccumulation of long-chain PFCAs.
authors.affiliation: Department of Chemistry, Trent University, 1600 West Bank Drive, Peterborough, Ontario K9J 7B8, Canada.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Woodcroft
authors.forename: David A
authors.initials: DA
authors.lastname: Ellis
authors.forename: Steven P
authors.initials: SP
authors.lastname: Rafferty
authors.forename: Darcy C
authors.initials: DC
authors.lastname: Burns
authors.forename: Raymond E
authors.initials: RE
authors.lastname: March
authors.forename: Naomi L
authors.initials: NL
authors.lastname: Stock
authors.forename: Kyle S
authors.initials: KS
authors.lastname: Trumpour
authors.forename: Janet
authors.initials: J
authors.lastname: Yee
authors.forename: Kim
authors.initials: K
authors.lastname: Munro
mesh_terms: D000818:Animals; D002210:Caprylates / Q000737:chemistry / Q000378:metabolism; D002264:Carboxylic Acids; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005399:Fishes / Q000378:metabolism; D005466:Fluorocarbons / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000737:chemistry / Q000378:metabolism
chemical_list: D002210:Caprylates; D002264:Carboxylic Acids; D050556:Fatty Acid-Binding Proteins; D005466:Fluorocarbons; D014874:Water Pollutants, Chemical; C023036:perfluorooctanoic acid