Biotransformation of N-Ethyl-N - American Chemical Society

Rochester, New York 14624, and 3M Medical Department, Corporate Toxicology,. 3M Center 220-2E-02, St. Paul, Minnesota 55133. Received October 30, 2003...
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Chem. Res. Toxicol. 2004, 17, 767-775

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Biotransformation of N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide by Rat Liver Microsomes, Cytosol, and Slices and by Expressed Rat and Human Cytochromes P450 Lin Xu,† Daria M. Krenitsky,† Andrew M. Seacat,‡ John L. Butenhoff,‡ and M. W. Anders*,† Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14624, and 3M Medical Department, Corporate Toxicology, 3M Center 220-2E-02, St. Paul, Minnesota 55133 Received October 30, 2003

Perfluorooctanesulfonic acid (PFOS) and its derivatives have been used in a range of industrial and commercial applications, including the manufacture of surfactants, adhesives, anticorrosion agents, and insecticides. PFOS is found at detectable concentrations in human and wildlife tissues and in the global environment. N-Substituted perfluorooctanesulfonamides are believed to be degraded to PFOS and, therefore, contribute to the accumulation of PFOS in the environment. N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide (N-EtFOSE) is converted to PFOS in experimental animals. The objective of this study was to elucidate the pathways for the biotransformation of N-EtFOSE, which is a major precursor and component of PFOS-based compounds. N-EtFOSE and several putative metabolites were incubated with liver microsomes and cytosol and with liver slices from male Sprague-Dawley rats. Microsomal fractions fortified with NADPH catalyzed the N-deethylation of N-EtFOSE to give N-(2hydroxyethyl)perfluorooctanesulfonamide (FOSE alcohol) and of FOSE alcohol to give perfluorooctanesulfonamide (FOSA). These N-dealkylation reactions were catalyzed mainly by male rat P450 2C11 and P450 3A2 and by human P450 2C19 and 3A4/5. Rat liver microsomal fractions incubated with UDP-glucuronic acid catalyzed the O-glucuronidation of N-EtFOSE and FOSE alcohol and the N-glucuronidation of FOSA. Cytosolic fractions incubated with NAD+ catalyzed the oxidation of FOSE alcohol to perfluooctanesulfonamidoacetate (FOSAA). The oxidation of N-EtFOSE to N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) was observed in liver slices but not in cytosolic fractions. FOSA was biotransformed in liver slices to PFOS, albeit at a low rate. These results show that the major pathway for the biotransformation of N-EtFOSE is N-dealkylation to give FOSA. The biotransformation of FOSA to PFOS explains the observation that PFOS is found in animals given N-EtFOSE.

Introduction PFOS1,2 and related N-alkylperfluorooctanesulfonamides have been in commercial production for consumer applications since the 1950s (1). Because of their chemical stability as compared with other chlorinated and brominated organic chemicals, they have been used in numerous industrial and commercial applications, such as the manufacture of surfactants, lubricants, waxes, gloss finish enhancers, adhesives, anticorrosion agents, stain repellents, fire-fighting foams, food wrapper coatings, and * To whom correspondence should be addressed. Tel: 585-275-1678. Fax: 585-273-2652. E-mail: [email protected]. † University of Rochester Medical Center. ‡ 3M Medical Department. 1 Abbreviations: P450, cytochrome P450; N-EtFOSE, N-ethyl-N-(2hydroxyethyl)perfluorooctanesulfonamide (5); PFOS, perfluorooctanesulfonic acid (10); FOSE alcohol, (2-hydroxyethyl)perfluorooctanesulfonamide (4); FOSA, perfluorooctanesulfonamide (9); FOSAA, perfluooctanesulfonamidoacetate (8); N-EtFOSA, N-ethylperfluorooctanesulfonamide (6); N-EtFOSAA, N-ethylperfluorooctanesulfonamidoacetate (7); POSF, perfluorooctanesulfonyl fluoride; UDPGA, UDPglucuronic acid; THPFOS, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulfonic acid; TFA, trifluoroacetic acid. 2 The structures of the compounds studied and the identification numbers are shown in Figure 8.

insecticides (2, 3). PFOS is a persistent compound and has been identified in serum and tissue samples from the general human population (4) and from occupationally exposed workers (5). Moreover, PFOS has been found in various wildlife species (6, 7), in surface waters, and in other environmental areas (8-10). PFOS and other fluorochemicals have been found in the serum of American Red Cross blood donors (11). The presence of PFOS in human and wildlife species has raised concerns about its potential long-term health effects. Although no PFOS-associated acute or chronic toxicity has been associated with the concentrations of PFOS found in humans, the cumulative toxicity of PFOS is observed in rats and nonhuman primates (12, 13). The toxic effects observed in rats and nonhuman primates include reduced body weights, decreased serum cholesterol concentrations, and increased liver weights. Although PFOS and N-EtFOSE are not selective developmental toxicants in either rats or rabbits (14), in a twogeneration rat and mouse reproduction study, both maternal and developmental toxicity are observed, including reduction in maternal weight gains, neonatal

10.1021/tx034222x CCC: $27.50 © 2004 American Chemical Society Published on Web 05/01/2004

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Chem. Res. Toxicol., Vol. 17, No. 6, 2004 Scheme 1. Synthesis of FOSE Alcohol 4

mortality, and delays in growth and development (15, 16). Also, PFOS has been reported to cause hypolipidemia (17), peroxisomal proliferation (18), inhibition of gap junctional intercellular communication, and increased permeability of cell membranes (19, 20). N-EtFOSE, an N-alkylperfluorooctanesulfonamide, has been reported to cause an increase in hepatocellular adenomas in rats (21). Toxicity to freshwater organisms at current environmental concentrations of PFOS has not been reported (22). It is believed that PFOS is derived from the metabolic or environmental degradation of N-alkylperfluorooctanesulfonamide derivatives. The metabolism of N-EtFOSE to PFOS is observed in Sprague-Dawley rats (23), but the enzymology of the metabolism of N-alkylperfluorooctanesulfonamide derivatives to PFOS has not been elucidated. The objective of the present studies was to elucidate pathways for the biotransformation of NEtFOSE 5 and its putative metabolites in rat liver microsomes, cytosol, and slices and to investigate the enzymology of the biotransformation of N-EtFOSE.

Experimental Procedures Materials. N-EtFOSE 5, N-EtFOSA 6, N-EtFOSAA 7, FOSAA 8, FOSA 9, PFOS 10, and THPFOS were supplied by the 3M Co. (St. Paul, MN). POSF was obtained from Oakwood Products, Inc. (West Columbia, SC). FOSE alcohol 4, N-EtFOSE O-glucuronide 13, and FOSA N-glucuronide 11 were obtained by synthesis. All other chemicals were of HPLC grade and were obtained from Sigma-Aldrich, Inc. (St. Louis, Mo.), unless specified otherwise. Synthesis of FOSE Alcohol. FOSE alcohol 4 was synthesized in three steps (Scheme 1). In a round-bottomed flask purged with dry nitrogen gas, POSF (1, 1.0 g, 2.0 mmol) was dissolved in 20 mL of anhydrous pyridine. The flask was cooled to 0 °C, and aminoacetaldehyde dimethylacetal (0.6 mL, 5.5 mmol) was added dropwise over 10 min. The flask was allowed to warm to room temperature overnight. The reaction mixture was extracted with ethyl acetate (3 × 10 mL), and the organic layer was chromatographed on a silica gel column (3 × 12 cm, ethyl acetate/hexane, 30:70) to give N-(2,2-dimethoxyethyl)perfluorooctanesulfonamide (2, 940 mg, 80%). Amide 2 was dissolved in 40 mL of chloroform containing 50% TFA. The solution was stirred for 3 h at room temperature, the solvent was evaporated in vacuo, and the product was chromatographed on a silica gel column (3 × 12 cm, ethyl acetate/hexane, 45:55) to yield N-(2-oxoethyl)perfluorooctanesulfonamide (3, 621 mg, 90%). Aldehyde 3 was dissolved in anhydrous ethanol at room temperature. Sodium borohydride (100 mg, 2.63 mmol) was added, and the reaction mixture was allowed to stand for 30 min. The reaction mixture was extracted with ethyl acetate (3 × 10 mL) and chromatographed on a silica gel column (3 × 12 cm, ethyl acetate/hexane, 80:20) to yield FOSE alcohol 4 as a white solid (525 mg, 84%). ESI-MS [M - H]-: m/z 542. 1H NMR

Xu et al. (CDCl3, δ, 400 MHz): 5.56 (s, 1H), 3.81 (t, J ) 4.94 Hz, 2H), 3.47 (t, J ) 5.27, 2H). Synthesis of FOSA N-Glucuronide 11. FOSA N-glucuronide 11 was synthesized by a modification of the method of Ahmad and Powell (24). FOSA (9, 300 mg, 0.6 mmol) and benzyl triethylammonium chloride (200 mg, 1 mmol) were dissolved in 10 mL of 1.25 M aqueous NaOH solution. A solution of acetobromo-R-D-glucuronic acid methyl ester (300 mg, 0.75 mmol) in 10 mL of chloroform was added, and the mixture was stirred vigorously and heated at reflux for 3 h. After it was cooled, the solution was brought to pH 5 by addition of 1 M acetic acid. The chloroform phase was separated and extracted successively with water and brine; the solution was dried with anhydrous magnesium sulfate, filtered, and evaporated under vacuum to give the product (400 mg). The product (400 mg) in 21 mL of methanol was added to 10 mL of 1 M NaOH solution cooled to 0 °C. The mixture was stirred at 4 °C for 30 min. The reaction mixture was concentrated to approximately 10 mL under vacuum and then extracted with methylene chloride (2 × 10 mL). The aqueous phase was separated, brought to pH 7.0 with 1 M HCl, and then lyophilized. The crude product was purified by chromatography on a reverse-phase C18 column (MeOH/H2O/NH4OAc, 60:40:0.1) to give FOSA N-glucuronide 11 as a white solid (135 mg, yield 32%). ESI-MS [M + Na H]-: m/z: 696.2. 1H NMR (CD3OD, δ, 400 MHz): 4.40 (d, J ) 8.36 Hz, 1H), 3.69 (t, J ) 6.76 Hz, 1H), 3.53 (d, J ) 6.61 Hz, 1H), 3.50 (t, J ) 8.45 Hz, 1H), 3.22 (t, J ) 8.56 Hz, 1H). Synthesis of N-EtFOSE O-Glucuronide 13. N-EtFOSE (5, 200 mg, 0.35 mmol) and acetobromo-R-D-glucuronic acid methyl ester (300 mg, 0.75 mmol) were dissolved in 10 mL of anhydrous chloroform. The mixture was stirred for 10 min at room temperature and then cooled to -15 °C. Silver triflate (128.3 mg, 0.50 mmol) was added, and the mixture was allowed to warm to room temperature over 2 h (25); 3 mL of triethylamine and 10 mL of water were added. The reaction mixture was shaken, and the chloroform phase was separated and extracted successively with water and brine. The chloroform extract was dried with anhydrous magnesium sulfate, filtered, and evaporated under vacuum to give the product (200 mg). The remaining steps were identical to those in the synthesis of FOSA Nglucuronide conjugate 11. The product (200 mg) dissolved in 21 mL of methanol was added to 10 mL of 1 M NaOH at 0 °C. The product was purified by reverse-phase HPLC (3.9 mm × 300 mm, µBondapak C18, Waters, Corp. Milford, MA.) MeOH/H2O/ NH4OAc: 60:40:0.1 gave N-EtFOSE O-glucuronide 13 (112 mg, yield 43%). ESI-MS [M - H]-: m/z 746.1. 1H NMR (CD3OD, δ, 400 MHz): 4.31 (d, J ) 7.5 Hz, 1H), 4.19 (t, 5.19 Hz, 1H), 3.78 (m, 2H), 3.65 (m, 2H), 3.57 (d, 9.14 Hz, 1H), 3.39 (t, 8.9 Hz, 2H), 3.36 (t, 9.30 Hz, 1H), 3.22 (t, 8.59 Hz, 1H), 1.29 (t, 7.01 Hz, 3H). Preparation of Subcellular Fractions. The animal use protocol was reviewed and approved by the University of Rochester University Committee on Animal Resources. Male Sprague-Dawley rats (200-230 g, Taconic, Inc., Germantown, NY) were euthanized by decapitation after anesthetization with CO2. The livers were removed immediately, weighed, minced, and homogenized with a Potter-Elvehjem glass-Teflon tissue homogenizer in sufficient cold 0.25 M sucrose to obtain a ratio of 3 g of liver tissue per mL of 0.25 M sucrose. The homogenate was centrifuged at 10 000g for 30 min at 4 °C. The 10 000g supernatant was centrifuged at 100 000g for 60 min at 4 °C to give the microsomal and cytosolic fractions. The microsomal and cytosolic fractions were dialyzed overnight with two buffer changes against 30 volumes of 0.1 M potassium phosphate (pH 7.4). Both dialyzed microsomes and cytosol were diluted with 0.1 M potassium phosphate buffer (pH 7.4) before storage at -80 °C. Protein concentrations were determined by the method of Lowry with bovine serum albumin as the standard (26). Microsomal and Cytosolic Incubations. The incubation mixtures contained 0.5 mg microsomal or cytosolic protein/mL and 0.5 mM NADPH or NAD+ in a final volume of 500 µL of 100 mM phosphate buffer (pH 7.4). Reactions were initiated by

Biotransformation of N-EtFOSE addition of 200 µM substrate and were incubated for 30 min at 37 °C (26). The reactions were quenched by addition of 50 µL of neat TFA (15% final concentration). Tetrabutylammonium hydrogen sulfate (50 µL of a 1.0 M solution; final concentration, 0.1 M) was added, and the reaction mixtures were extracted with ethyl acetate (200 µL × 3). The organic phase was dried under a stream of dry nitrogen gas, and the residue was dissolved in 0.2 mL of methanol. A sample of the methanol solution was analyzed by LC-MS. For quantitative analysis, THPFOS was used as the internal standard (4). Incubations with Expressed Rat and Human P450s. Microsomes from baculovirus-infected Sf9 insect cells expressing rabbit P450 reductase and each individual human P450 1A2, 2C9, 2C19, 2D6, 3A4, and 3A5 were purchased from Panvera Corp. (Madison, WI). Microsomes from baculovirus-infected Sf9 insect cells expressing human NADPH-P450 reductase, cytochrome b5, and each individual human P450 2B6 and 2E1 were purchased from Gentest Corp. (Woburn, MA). Microsomes from baculovirus-infected Sf9 insect cells expressing rat P450 reductase and each individual rat P450 1A2, 2B1, 2C11, 2E1, and 3A2 were also purchased from Gentest Corp. N-EtFOSE 5 was incubated with 30 pmol of P450, 200 µM substrate, and 0.5 mM NADPH in a final volume of 500 µL of 100 mM phosphate buffer (pH 7.4); the reaction mixtures were incubated for 12 min. FOSE alcohol 4 was incubated with the same additions except that 20 pmol of P450 was added; the reaction mixtures were incubated for 20 min. Other additions and conditions were the same as those described in microsomal and cytosolic incubation section. Kinetic parameters (Km and Vmax) were determined with Prism 4.0 (GraphPad Software, San Diego, CA) for nonlinear regression analysis. Glucuronidation Experiments. The complete incubation mixtures contained microsomal protein (1.0 mg/mL), 1 mM substrate, 1 mM MgCl2, 5 mM saccharolactone, 50 µg of alamethicin, and 5 mM UDPGA in a final volume of 200 µL of 100 mM phosphate buffer (pH 7.4). Reactions were initiated by addition of the substrate. Control incubation mixtures lacked UDPGA. The reaction mixtures were incubated for 30 min at 37 °C. The reactions were quenched by addition of 20 µL of neat TFA (15% final concentration), and the mixtures were centrifuged for 3 min at 16 000g. The supernatants were filtered through 0.2 µm filter (NALGENE, VWR, Rochester, NY) and injected into LC-MS for analysis. Liver Slice Experiments. Freshly excised rat livers were placed in 40 mL of ice-cold 100 mM phosphate buffer (pH 7.4). Liver cores were obtained manually with an 8-mm stainlesssteel coring tube. The core was immediately placed in ice-cold 100 mM phosphate buffer (pH 7.4) until used. Liver slices (200250 µm in thickness) were prepared with a tissue slicer (Vitron, Inc., Tucson, AZ) in ice-cold RPMI media 1640 (pH 7.4, GIBCO, Invitrogen Corp., Carlsbad, CA). Slices were transferred to vials that contained 1.7 mL of oxygenated (95% O2, 5% CO2) RPMI media 1640 (pH 7.4) with 10% fetal bovine serum (GIBCO, Invitrogen Corp.) in the incubation chamber. The liver slices were incubated for 1 h in an incubator purged with 95% O2/5% CO2 after which time the slices were transferred to fresh vials containing the substrate (42 µM) and 1.7 mL of the same media. The liver slices were incubated for 6 h, and reactions were terminated by addition of 1.7 mL of ice-cold acetonitrile (27). The acetonitrile extracts were centrifuged for 3 min at 16 000g, and the supernatants were filtered and analyzed by LC-MS. Inhibition Studies. The nonselective P450 inhibitors 1-octylamine and 1-benzylimidazole (28, 29) and the NADPH-P450 reductase inhibitor diphenyleneiodonium chloride (30, 31) were used to investigate the role of the P450 in the biotransformation of N-EtFOSE and its metabolites. To determine the major rat liver P450s involved in the biotransformation of N-EtFOSE 5 and its metabolites, these isoform selective rat P450 inhibitors were used as follows: furafylline (1A2), diphenhydramine (2B1/ 2), orphenadrine (2B1/2), cimetidine (2C11), ajmalicine (2D1), disulfiram (2E1), diallyl sulfide (2E1), clotrimazole (3A), and troleandomycin (3A) (32-37). Furafylline was added at 20 or

Chem. Res. Toxicol., Vol. 17, No. 6, 2004 769 100 µM, and clotrimazole was added at 0.5, 5, or 50 µM. Other inhibitors were added at concentrations of 10, 100, or 1000 µM. In experiments with 1-octylamine, 1-benzylimidazole, diphenhydramine, ajmalicine, and diallyl sulfide, the inhibitors were added simultaneously with the substrates. The reaction mixtures were incubated for 12 min. Other additions and conditions were same as those described in the microsomal and cytosolic incubation section. Incubation mixtures that contained cimetidine, clotrimazole, diphenyleneiodonium chloride, disulfiram, furafylline, orphenadrine, and troleandomycin were incubated at 37 °C for 5 min before the substrates were added; other additions and conditions were the same as described above. LC-MS Analyses. The LC-MS conditions reported by Hansen et al. (4) were typically used as follows: the mobile phase was 2 mM ammonium acetate in methanol (solvent A) and 2 mM ammonium acetate in water (solvent B). An Agilent HP1100 HPLC system (Agilent Technologies, Wilmington, DE) fitted with an analytical C18 column (2 mm × 150 mm, 2 µm particle size, Waters) was used. The column was held at ambient temperature and was eluted at a flow rate of 0.3 mL min-1. The samples were eluted from the HPLC column with a stepped gradient: 0 min, 65% A; 10 min, 85% A; 20 min, 95% A; 22 min, 95% A; 23 min, 65% A. The eluate was analyzed with a UV diode array detector and by electrospray mass spectroscopy. LC-MS analyses were performed with an Agilent LC/MSD ion trap mass spectrometer (Agilent Technologies) with an electrospray interface operated in the negative ion mode: dry temp, 350 °C; nebulizer, 40 psi; drying gas, 9 L min-1; skim 1, -44.6 V; capillary exit, -77.9 V; trap drive, 43.1. Each chemical was detected in selected daughter ion mode from MS/MS at unit resolution (PFOS 10, m/z 499 f 280; FOSA 9, m/z 498 f 478; FOSAA 8, m/z 556 f 498; N-EtFOSAA 7, m/z 584 f 526; N-EtFOSA 6, m/z 526 f 362; FOSE alcohol 4, m/z 542 f 388; THPFOS, m/z 427 f 407).

Results Biotransformation of N-EtFOSE 5 in Rat Liver Microsomes and Cytosol. N-EtFOSE 5 was incubated with rat liver microsomal fractions and NADPH for 30 min. Analysis of extracts of the reaction mixtures by LCMS in the negative ion mode showed the formation of FOSE alcohol 4 (m/z 542 f 388) and FOSA 9 (m/z 498 f 478). The retention times of the two metabolites were identical with those of synthetic FOSE alcohol 4 and FOSA 9. The biotransformation of N-EtFOSE 5 to FOSE alcohol 4 was linear with time for 12 min and then decreased at longer incubation times. To determine the kinetics of the biotransformation of N-EtFOSE 5 to FOSE alcohol 4, N-EtFOSE 5 (10-80 µM) was used as the substrate. (FOSE alcohol 4 was not detectable when the substrate concentration was below 10 µM, and substrate inhibition occurred when the N-EtFOSE 5 concentration was greater than 100 µM.) The apparent Km and Vmax values were 4.06 ( 0.83 µM and 1.67 ( 0.05 pmol min-1 mg protein-1, respectively (mean ( SD, n ) 3) (Figure 1). FOSE alcohol 4 was biotransformed to FOSA 9 under the same incubation conditions. The biotransformation of FOSE alcohol 4 to FOSA 9 was linear with time for at least 30 min. FOSE alcohol 4 showed substrate inhibition at substrate concentrations greater than 80 µM. At substrate concentrations between 5 and 50 µM, Michaelis-Menten kinetics were observed, and the apparent Km and Vmax values were 15.6 ( 2.2 µM and 19.4 ( 1.1 pmol min-1 mg protein-1, respectively (mean ( SD, n ) 3) (Figure 1). Although N-EtFOSA 6 was not detected when N-EtFOSE 5 was incubated with NADPH and microsomes, N-EtFOSA 6 was biotransformed to FOSA 9 at a relatively high rate in the presence of rat liver microsomal fractions (data not shown).

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Figure 2. MS/MS spectrum of N-EtFOSE O-glucuronide 13 [M - H]- ion (m/z 746). The fragment ion at m/z 526 is derived from cleavage of the C-N bond. The fragment ion at m/z 419 is formed by cleavage of the C-S bond.

Figure 1. Lineweaver-Burk plots of FOSE alcohol 4 formation from N-EtFOSE 5 (A, 2) and FOSA 9 formation from FOSE alcohol 4 (B, 9) catalyzed by rat microsomes. Rat liver microsomes were incubated with 10-80 µM N-EtFOSE 5, and FOSE alcohol 4 formation was measured as described in the Experimental Procedures. The results of triplicate reactions are shown. The Vmax and Km were derived from nonlinear regression analysis of the experimental data.

When N-EtFOSE 5 was incubated with rat liver cytosol in the presence of NAD+, no metabolites were detected by LC-MS analysis. Incubation of FOSE alcohol 4 with rat liver cytosol in the presence of NAD+ resulted in the formation of FOSAA 8. The biotransformation of FOSE alcohol 4 to FOSAA 8 was linear with time for at least 40 min. When other metabolites, such as FOSA 9, FOSAA 8, and N-EtFOSAA 7, were incubated with rat liver microsomal fractions in the presence of NADPH or incubated with rat liver cytosolic fractions in the presence of NAD+, no metabolites were detected. Glucuronidation of N-EtFOSE 5, FOSE Alcohol 4, and FOSA 9 in Rat Liver Microsomes. O-Glucuronidation of N-EtFOSE 5 was observed when N-EtFOSE 5 was incubated with rat liver microsomes in the presence of UDPGA. Structural identification of N-EtFOSE O-glucuronide 13 was first performed by LC-MS analysis, which showed a [M - H]- ion at m/z 746. Fragmentation of the parent [M - H]- ion m/z 746 gave a daughter ion at m/z 526, which resulted from the loss of N-ethoxy glucuronide by cleavage of the C-N bond (Figure 2). The structure was further confirmed by comparison with a synthetic standard: the biosynthesized glucuronide conjugate showed the same retention time on LC and fragmentation patterns on MS/MS as the synthetic standard. O-Glucuronidation of FOSE alcohol 4 to give FOSE alcohol O-glucuronide 12 was observed under the same incubation conditions but at an apparent lower rate than for N-EtFOSE 5. Because FOSE alcohol 4 glucuronide contains two acidic protons (sulfonyl amide and carboxylic acid), the sodium salt of glucuronide was observed as the molecular ion [M + Na - H]- at m/z 740.1 in the

Figure 3. MS/MS spectrum of FOSE alcohol O-glucuronide 12 [M + Na - H]- ion (m/z 740). The fragment ion at m/z 542 is formed by cleavage of the acetal bond in the glucuronic acid moiety.

Figure 4. MS/MS spectrum of FOSA N-glucuronide 11 [M + Na - H]- ion (m/z 696). The fragment ion at m/z 540 is formed by cleavage of the acetal and C2-C3 bonds in the glucuronic acid moiety. The fragment ion at m/z 498 is derived from loss of the glucuronic acid moiety.

negative ion mode. The major fragment ion shown by MS/ MS analysis was m/z 542, which was attributed to cleavage of the acetal bond (Figure 3). FOSA 9 underwent N-glucuronidation under the same incubation conditions to give FOSA N-glucuronide 11 but at an apparent lower rate as compared with the Oglucuronidation of N-EtFOSE 5 and FOSE alcohol 4. N-Glucuronidation of FOSA was also identified as the sodium salt by LC-MS analysis. The [M + Na - H]- ion at m/z 696 gave two major daughter ions of m/z 540 and m/z 498 by MS/MS analysis, which confirmed the identification of the metabolite as FOSA N-glucuronide 11 (Figure 4). Biotransformation of N-EtFOSE 5 in Rat Liver Slices. The biotransformation of N-EtFOSE 5 and its metabolites in rat liver slices was similar to that found in rat liver microsomal and cytosolic fractions. The products formed from N-EtFOSE 5 and its metabolites in rat liver slices are shown in Table 1. These data showed that N-EtFOSE 5 underwent N-dealkylation to give FOSE alcohol 4 and FOSA 9. N-EtFOSE 5 was also oxidized to give N-EtFOSAA 7. No evidence for the N-deethylation of N-EtFOSE 5 to NEtFOSA 6 was observed. The N-EtFOSE 5 metabolite

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Table 1. Metabolites Formed from Each Substrate in Rat Microsomes, Cytosol, and Liver Slicesa substrate

a

microsomes

cytosol

N-EtFOSE 5

FOSE alcohol 4, FOSA 9, and O-glucuronide 13

ND

FOSE alcohol 4

FOSA 9 and O-glucuronide 12

FOSAA 8

N-EtFOSA 6 FOSA 9 FOSAA 8 or N-EtFOSAA 7

FOSA 9 N-glucuronide 11 ND

ND ND ND

liver slices FOSE alcohol 4, FOSA 9, N-EtFOSAA 7, and O-glucuronide 13 FOSA 9, FOSAA 8, and O-glucuronide 12 FOSA 9 PFOS 10 and N-glucuronide 11 ND

ND, no metabolite detected.

Figure 5. Effects of rat P450 isoform selective inhibitors on FOSE alcohol 4 formation from N-EtFOSE 5. Incubation mixtures contained 0.5 mg/mL rat microsomal proteins, 100 µM N-EtFOSE 5, and 0.5 mM NADPH and were incubated for 12 min at 37 °C. The inhibitors were added at three different concentrations (10, 100, or 1000 µM) except furafylline and clotrimazole. Rates of FOSE alcohol 4 formation are shown as the percentage of control incubation without the inhibitor. Data are shown as means ( SD, n ) 3. *Furafylline was added at 20 or 100 µM. **Clotrimazole was added at 0.5, 5, or 50 µM.

FOSE alcohol 4 underwent N-dealkylation to give FOSA 9 and oxidation to give FOSAA 8. N-EtFOSA 6 was N-deethylated to form FOSA 9. FOSA 9 was biotransformed in liver slices to PFOS 10, but the biotransformation of FOSA 9 was not detected in either microsomes or cytosol. N-EtFOSE 5 and FOSE alcohol 4 were also converted to O-glucuronide conjugates 12 and 13, and FOSA 9 was biotransformed to FOSA N-glucuronide 11. These data are consistent with the results obtained in microsomal incubations. Acyl glucuronide formation of FOSAA 8 and N-EtFOSAA 7 was not detected. N-EtFOSE 5 Dealkylation by Rat Liver P450s. To confirm a role for the P450s in the N-deethylation of N-EtFOSE 5, the effects of the nonselective P450 inhibitors 1-octylamine and 1-benzylimidazole on N-dealkylation of N-EtFOSE 5 were measured. The inhibitory effect was measured at three different concentrations of inhibitors (Figure 5). 1-Octylamine and 1-benzylimidazole (100 µM) decreased the rate of N-deethylation by 71 and 79%, respectively. The selective NADPH-P450 reductase inhibitor diphenyleneiodonium chloride, at a concentration of 10 µM, decreased the rate of N-deethylation by 94%. These data indicated that P450s catalyzed the N-dealkylation of N-EtFOSE 5. The effect of selective inhibitors of P450 subfamilies on the N-deethylation of N-EtFOSE 5 was examined to

identify the major rat P450 isoforms involved in its biotransformation (Figure 5). The P450 1A2 inhibitor furafylline (20 and 100 µM) decreased activity by 62 and 67%, respectively. Diphenhydramine hydrochloride and orphenadrine (100 µM), which are inhibitors of P450 2B1/2, decreased activity by 68 and 75%, respectively. Cimetidine (P450 2C11 inhibitor) decreased activity by 30% at 10 µM and 70% at 100 µM. Disulfiram and diallyl sulfide (100 µM), which are inhibitors of P450 2E1, decreased activity by 89 and 40%, respectively. Clotrimazole, which is a more selective inhibitor of rat P450 3A than troleandomycin, decreased activity by 25% at 5 µM and by 55% at 50 µM. In contrast, the P450 2D1 and P450 3A inhibitors ajmalicine and troleandomycin had no effect on N-dealkylation activity at 10 and 100 µM. To confirm the identity of the P450s that catalyze the major N-dealkylation of N-EtFOSE 5, N-EtFOSE 5 was incubated with these expressed rat P450s: 1A2, 2B1, 2C11, 2E1, and 3A2, whose involvement was implicated from inhibition studies. Results showed that P450 2B1, 2C11, and 3A2 catalyzed the N-deethylation of 100 µM N-EtFOSE 5 at rates of 6.6, 14.7, and 15.6 pmol min-1 nmol P450-1, respectively (Figure 6A). N-EtFOSE 5 was also incubated with several expressed human P450s (1A2, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5). Human P450 2C19, 3A4, and 3A5 catalyzed the

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Figure 6. Rate of FOSE alcohol 4 formation from N-EtFOSE 5 (100 µM) catalyzed by expressed rat (A) and human (B) liver P450s (30 pmol). Reaction rates were linear with time for 12 min. ND, not detected.

N-deethylation of 100 µM N-EtFOSE 5 at rates of 267, 23.6, and 204 pmol min-1 nmol P450-1, respectively (Figure 6B). Rat P450s catalyzed the N-deethylation of N-EtFOSE 5 at relatively low rates. The rate for the deethylation of 100 µM N-EtFOSE 5 by human P450 2C19 and 3A5 was 10-times faster than those of rat P450s (Figure 6). The kinetics (Km and Vmax) of the human P450 2C19- and 3A5catalyzed N-dealkylation of N-EtFOSE 5 was measured at substrate concentrations of 5-200 µM. The apparent Km, Vmax, and Vmax/Km values for the N-deethylation of N-EtFOSE 5 were 11.6 ( 0.8 µM, 304 ( 5 pmol min-1 nmol P450-1, and 26.2 ( 1.5 pmol min-1 nmol P450-1 µM-1 for human P450 2C19 and 46.8 ( 5.6 µM, 288 ( 13 pmol min-1 nmol P450-1, and 6.15 ( 0.52 pmol min-1 nmol P450-1 µM-1 for human P450 3A5 (mean ( SD, n ) 3). Subsequently, the metabolite FOSE alcohol 4 (50 µM) from N-deethylation of N-EtFOSE 5 was incubated with the same series of rat and human P450s to determine the major P450s involved in the N-dealkylation of FOSE alcohol 4. The results showed that rat P450 2C11 and human P450 2C19 catalyzed the N-dealkylation of FOSE alcohol 4 at rates of 0.69 and 1.55 nmol min-1 nmol P450-1, respectively (Figure 7). The apparent Km, Vmax, and Vmax/Km values for the N-dealkylation of FOSE alcohol 4 were 11.4 ( 0.7 µM, 0.87 ( 0.02 nmol min-1 nmol P450-1, and 0.076 ( 0.003 nmol min-1 nmol P450-1 µM-1 for rat P450 2C11 and 21.9 ( 2.0 µM, 2.23 ( 0.09 nmol min-1 nmol P450-1, and 0.102 ( 0.005 nmol min-1 nmol P450-1 µM-1 for human P450 2C19 (mean ( SD, n ) 3). The rat P450s 3A2, 1A2, and 2B1 catalyzed the N-dealkylation of 50 µM FOSE alcohol 4 at rates of 98, 39, and 30 pmol min-1 nmol P450-1, respectively.

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Figure 7. Rate of FOSA 9 formation from FOSE alcohol 4 (50 µM) catalyzed by expressed rat (A) and human (B) liver P450s (20 pmol). Reaction rates were linear with time for 20 min. ND, not detected.

Discussion The objective of the present research was to investigate the biotransformation of N-EtFOSE 5 and its putative metabolites in rat liver microsomes, cytosol, and slices. The data show that N-EtFOSE 5 and some of its metabolites are substrates for microsomal and cytosolic enzymes. The proposed pathways for the biotransformation of N-EtFOSE 5 are shown in Figure 8. The phase I metabolic pathway involved two N-dealkylation and two oxidation steps. N-EtFOSE 5 underwent N-deethylation rat liver microsomes to give FOSE alcohol 4, which also underwent dealkylation to give FOSA 9. The finding that the efficiency of the N-deethylation of N-EtFOSE 5 (Vmax/ Km ) 0.41 ( 0.09 pmol min-1 mg protein-1 µM-1) to give FOSE alcohol 4 (Vmax/Km ) 1.24 ( 0.12 pmol min-1 mg protein-1 µM-1) was low as compared with the biotransformation of FOSE alcohol 4 to FOSA 9 indicates that the initial N-deethylation of N-EtFOSE 5 may be a ratelimiting step in its biotransformation. The N-dealkylation of FOSE alcohol 4 would presumably yield glycoaldehyde, which, by analogy with ethylene glycol, would be expected to be metabolized to oxalic acid (38). Although no evidence was found for the biotransformation of N-EtFOSE 5 to N-EtFOSA 6, the N-deethylation of N-EtFOSA 6 to give FOSA 9 presumably proceeded at a relatively high rate. Hence, if N-EtFOSA 6 was formed from N-EtFOSE 5 at a relatively slow rate, its facile N-deethylation to FOSA 9 may have prevented detection of N-EtFOSA 6 as an intermediate. Hence, the biotransformation of N-EtFOSE 5 to FOSE alcohol 4 and to N-EtFOSA 6 may constitute the rate-limiting steps in the metabolism of N-EtFOSE 5. N-EtFOSE 5 and FOSE alcohol 4 also were oxidized to give N-EtFOSAA 7 and FOSAA 8, respectively, which were apparently not biotransformed to other products. The NAD+- and cytosol-dependent oxidation of FOSE

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Figure 8. Proposed biotransformation pathways of N-EtFOSE 5.

alcohol 4 to FOSAA 8 indicated the involvement of NAD+dependent oxidoreductases and, perhaps, aldehyde dehydrogenase in the biotransformation of FOSE alcohol 4. In addition, N-EtFOSE 5 was biotransformed to N-EtFOSAA 7 in rat liver slices. N- and O-Glucuronidation were the only phase II metabolic pathways detected. N-EtFOSE 5 was biotransformed to its O-glucuronide 13 at a relatively high rate, which may be attributed to its high hydrophobicity. FOSE alcohol 4, and FOSA 9 formed O- and N-glucuronides 12 and 11, respectively, at low rates. These glucuronides may, however, undergo enterohepatic circulation, which may increase their half-life for elimination and, hence, their tendency to accumulate in the body (39). Although sulfonamides are generally considered to be metabolically stable (40, 41), FOSA 9 was biotransformed to PFOS 10 in rat liver slices at a low rate, but no biotransformation was observed in microsomal, cytosolic, and 9000g supernatant fractions. This is apparently the first observation of the metabolic hydrolysis of a sulfonamide. These data explain the observation that PFOS 10 is formed in rodents and nonhuman primates given N-EtFOSE 5. Other N-alkylperfluorooctanesulfonamide derivatives could be converted to PFOS in a similar manner. The biotransformation of FOSA 9 to PFOS 10 could not, however, be detected in microsomal and cytosolic fractions; hence, the enzymes involved in the biotransformation FOSA 9 to PFOS 10 could not be identified. The mechanism of the enzymatic hydrolysis of FOSA 9 to PFOS 10 is not known. Because the proton in the sulfonamide group of FOSA 9 is labile, it is possible to speculate that the mechanism of PFOS 10 formation from FOSA 9 proceeds through a N-glucuronide intermediate. Conversion of the amine group of FOSA Nglucuronide 11 to an imine by opening of the pyran ring, protonation of the imine to give an iminium ion, and SN2 attack by hydroxide would afford the sulfonate. Precedent for an analogous hydrolysis of a sulfonamide has been

reported (42). Other mechanisms for the hydrolysis of sulfonamides have been reported, but these involve intramolecular nucleophilic catalysis or carbanion formation in β-sultams (43-45). The finding that rat hepatic microsomal fractions catalyze two NADPH-dependent dealkylation steps in the biotransformation of N-EtFOSE 5 indicated a role for the P450s. A role for the P450s was confirmed by the observed inhibition of the biotransformation of N-EtFOSE 5 by the nonselective P450 inhibitors 1-octylamine and 1-benzylimidazole. Further evidence for a role of the P450s was obtained by the complete inhibition of the N-deethylation of N-EtFOSE 5 by the NADPH-P450 reductase inhibitor diphenyleneiodonium chloride. To identify the P450s responsible for the N-dealkylation reactions, inhibition of N-dealkylase activity by several P450 isoform selective inhibitors was examined. The N-deethylation of N-EtFOSE 5 was inhibited 90% by P450 2E1 inhibitor disulfiram, indicating that P450 2E1 catalyzed the reaction. The N-deethylation of NEtFOSE 5 was also inhibited by about 70% by the P450 2C11 inhibitor cimetidine, the P450 2B1 inhibitors orphenadrine and diphenhydramine hydrochloride, and the P450 1A2 inhibitor furafylline, indicating roles for P450s 2C11, 2B1, and 1A2. Clotrimazole decreased the activity by 25% at a concentration of 5 µM, which is the clotrimazole IC50 value on P450 3A2-dependent reactions in rat microsomes (36). Clotrimazole (50 µM) concentration inhibited N-dealkylase activity by 55%. These results indicated that P450 3A2 was also involved in N-deethylation. The P450 3A inhibitor troleandomycin did not decrease the activity at 100 µM, perhaps because troleandomycin is not a selective inhibitor of P450 3A isoforms in male rats (46). In summary, the N-deethylation of N-EtFOSE 5 was catalyzed by several rat P450s: 1A2, 2B1, 2C11, 2E1, and 3A2. Studies with expressed rat P450s showed that P450 3A2, 2C11, and 2B1 were major P450 isoforms that catalyzed the N-deethylation of N-EtFOSE 5. P450 1A2

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and 2E1 did not catalyze the N-dealkylation of NEtFOSE 5. These results indicated that disulfiram and furafylline do not exhibit the same selectivity for rat P450s as for human hepatic P450s. Eagling et al. (46) demonstrated similar results. P450s 2C11 and 3A2 are the major isoforms present in hepatic microsomes of mature, male rats (200 g or 7 weeks old) (47) and catalyze the metabolism of many xenobiotics (48). Rat P450 2C11 also catalyzed the N-dealkylation of FOSE alcohol 4 to FOSA 9 but apparently at a faster rate than the Ndeethylation of N-EtFOSE 5. This observation was consistent with the results observed with the N-dealkylation of N-EtFOSE 5 in male rat liver microsomes and indicated that the initial N-deethylation of N-EtFOSE 5 may be a rate-limiting step in its biotransformation in rats. Human P450 2C19 and 3A4/5 both biotransformed N-EtFOSE 5. Human P450 2C19 had the lowest Km and the highest specific activity among the isoforms studied for the N-dealkylation of both N-EtFOSE 5 and FOSE alcohol 4. The Vmax/Km of the second step in the Ndealkylation of N-EtFOSE 5 was greater than the Vmax/ Km of the first N-dealkylation, which was catalyzed by human P450 2C19. Human P450 3A5 had a lower efficiency to catalyze the N-dealkylation of N-EtFOSE 5 than did human P450 2C19. Human P450 3A5 also catalyzed the N-dealkylation FOSE alcohol 4. Because of the relative abundance of P450 3A4 and 3A5 (47) in human liver as compared with P450 2C19 (49), human P450 3A4/5 would be expected to contribute significantly to the disposition of N-EtFOSE 5 in humans. In summary, these studies identified pathways for the biotransformation of N-EtFOSE 5 and showed that the terminal metabolite is the metabolically inert PFOS 10. Moreover, the results demonstrated the metabolic degradation of N-alkyl perfluorooctanesulfonamide derivatives to PFOS 10. In addition, oxidation of N-EtFOSE 5 to the hydrophilic metabolite N-EtFOSAA 7 and oxidation of its metabolite, FOSE alcohol 4, to the more hydrophilic metabolite FOSAA 8 would also promote elimination of N-EtFOSE 5 from the body. FOSAA 8 and N-EtFOSAA 7 have been identified in the bile and liver of rats and nonhuman primates given N-EtFOSE 5.3 Furthermore, the formation of N-EtFOSE O-glucuronide 13 and FOSE alcohol O-glucuronide 12 would foster their elimination from the body. Hence, the biotransformation of N-EtFOSE 5 and its metabolites would primarily constitute a detoxication pathway. However, the formation of FOSA 9 and PFOS 10 represents bioactivation to potentially toxic metabolites, although PFOS 10 is formed from FOSA 9 at a comparatively low rate. After subchronic dietary dosing of rats with N-EtFOSE 5, FOSA 9 did not accumulate; rather, PFOS 10 was the metabolite found in the greatest concentration in the liver (23). This observation likely represented the combination of the metabolism of FOSA 9 to PFOS 10 and the detoxication of FOSA 9 through N-glucuronidation and elimination. The N-EtFOSE concentrations used for this study are physiologically relevant, as lowered cholesterol and increased liver to body weight ratios were observed in rats fed N-EtFOSE at liver PFOS concentrations of approximately 46 and 160 µM, respectively (23). Therefore, as suggested by Butenhoff and Seacat (23), the 3

Seacat, A. M., and Butenhoff, J. L. Unpublished observations.

Xu et al.

effects from N-EtFOSE 5 treatment in subchronic toxicity studies may largely be due to formation of PFOS 10.

Acknowledgment. These studies were supported by the 3M Co. We also thank Wayne B. Anderson, Hoffman B. M. Lantum, and Neil Price for helpful suggestions and advice.

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