Biotransformation of the 8:2 fluorotelomer acrylate in rainbow trout. 2. In vitro incubations with liver and stomach S9 fractions

Abstract

The biotransformation of the 8:2 fluorotelomer acrylate (C(8) F(17) CH(2) CH(2) OC(O)CH = CH(2) , 8:2 fluorotelomer-based acrylate [FTAc]) was quantitatively investigated in cytosolic (S9) fractions isolated from rainbow trout stomach and liver. The in vitro studies presented in this manuscript compliment the whole body 8:2 FTAc dietary exposure study, presented as a companion paper. The S9 fractions were prepared in our laboratory, using fish that had previously been used as control animals in our in vivo study. Before 8:2 FTAc incubations, general carboxylesterase activity was determined using paranitrophenyl acetate (PNPA) as the substrate with formation of paranitrophenol monitored using an ultraviolet-vis spectrometer. In the 8:2 FTAc incubations, the degradation of the parent compound and 8:2 fluorotelomer alcohol (FTOH) formation was monitored by gas chromatography-mass spectrometry. Incubations were performed in triplicate, over a range of concentrations encompassing two orders of magnitude, and the initial rate of 8:2 FTOH or paranitrophenol formation was determined. Enzyme kinetic parameters were determined by plotting the initial rate versus concentration, using nonlinear regression analysis. The maximum initial velocities of the enzyme-catalyzed reaction (V(max)) in the PNPA incubations were 614 ± 18 nmol/min/mg and 147 ± 16 nmol/min/mg for the liver and stomach fractions, respectively. These values are much faster than other phase I and II metabolism reactions. The calculated intrinsic clearance rates (CL(int)) for the 8:2 FTAc incubations were 1.7 and 0.40 ml/min/mg protein, respectively. These results show that the esterase activity toward the 8:2 FTAc is only fourfold greater in the liver as compared with the stomach. These trends demonstrate the potential for considerable extrahepatic metabolism of the 8:2 FTAc before uptake into the internal tissues, ultimately limiting the overall bioaccumulation.