ToxSci Advance Access originally published online on September 4, 2007
Toxicological Sciences 2007 100(2):333-344; doi:10.1093/toxsci/kfm230
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In Vitro Metabolism of 8-2 Fluorotelomer Alcohol: Interspecies Comparisons and Metabolic Pathway Refinement
* DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware 19714
The Sapphire Group, Inc., Dayton, Ohio 45431
1 To whom correspondence should be addressed at DuPont Haskell Laboratory for Health and Environmental Sciences H-1/1708, 1090 Elkton Road, Newark, DE 19714. Fax: (302) 366-5003. E-mail: diane.l.nabb-1{at}usa.dupont.com.
Received May 18, 2007; accepted August 25, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
The detection of perfluorinated organic compounds in the environment has generated interest in their biological fate. 8-2 Fluorotelomer alcohol (8-2 FTOH, C7F15CF2CH2CH2OH), a raw material used in the manufacture of fluorotelomer-based products, has been identified in the environment and has been implicated as a potential source for perfluorooctanoic acid (PFOA) in the environment. In this study, the in vitro metabolism of [3-14C] 8-2 FTOH and selected acid metabolites by rat, mouse, trout, and human hepatocytes and by rat, mouse, and human liver microsomes and cytosol were investigated. Clearance rates of 8-2 FTOH in hepatocytes indicated rat > mouse > human
trout. A number of metabolites not previously reported were identified, adding further understanding to the pathway for 8-2 FTOH metabolism. Neither perfluorooctanoate nor perfluorononanoate was detected from incubations with human microsomes. To further elucidate the steps in the metabolic pathway, hepatocytes were incubated with 8-2 fluorotelomer acid, 8-2 fluorotelomer unsaturated acid, 7-3 acid, 7-3 unsaturated acid, and 7-2 secondary fluorotelomer alcohol. Shorter chain perfluorinated acids were only observed in hepatocyte and microsome incubations of the 8-2 acids but not from the 7-3 acids. Overall, the results indicate that 8-2 FTOH is extensively metabolized in rats and mice and to a lesser extent in humans and trout. Metabolism of 8-2 FTOH to perfluorinated acids was extremely small and likely mediated by enzymes in the microsomal fraction. These results suggest that human exposure to 8-2 FTOH is not expected to be a significant source of PFOA or any other perfluorocarboxylic acids. Key Words: 8-2 fluorotelomer alcohol; glutathione conjugates; taurine conjugates; perfluorinated carboxylic acids; perfluorooctanoate; perfluorononanoic acid; fluorotelomer aldehydes; hepatocytes; hepatic clearance; microsomes.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Fluorotelomer alcohols (F(CF2)xC2H4OH, x = 6, 8, or 10, FTOHs) are a raw material used in the manufacture of fluorotelomer-based products which are used in a variety of applications including paints and coatings, paper, and textiles because of their unique surface properties (Kissa, 2001
). 8-2 fluorotelomer alcohol (8-2 FTOH, x = 8, 1H,1H,2H,2H-perfluorodecanol, CASRN 678-39-7) is the FTOH manufactured in the largest volume globally (Telomer Research Program, 2002). FTOHs have been shown to undergo abiotic and biotic degradation to form small amounts of perfluorocarboxylates (PFCAs) including perfluorooctanoic acid (PFOA) under environmental degradation conditions (Ellis et al., 2004; Wang et al., 2005a,b). PFCAs are persistent and have been detected broadly in the environment and humans (Calafat et al., 2006; Houde et al., 2006). The global historic contribution of fluorotelomers to PFCAs emissions in the environment has been estimated to be small, approximately 1% (Prevedouros et al., 2006). However, whether FTOH exposure and metabolism may contribute to quantifiable PFCAs in living systems has not been determined.
PFOA has been identified as a minor metabolite following oral administration of 8-2 FTOH to rats and mice (Fasano et al., 2006; Kudo et al., 2005; Martin et al., 2005). Martin et al. (2005) also reported that quantifiable PFCAs from rat hepatocyte incubations comprised only a small amount of the total oxidation products from 8-2 FTOH, with PFOA representing about 1.4% of the total dose. However, Kudo et al. (2005) proposed that the metabolic generation of PFOA and perfluorononanoic acid (PFNA) from dietary administration of 8-2 FTOH was associated with peroxisome proliferation observed in the livers of exposed mice, indicating the possibility that an amount of perfluorinated acids sufficient to produce biological consequences may be generated from 8-2 FTOH, at least in mice.
Initial 8-2 FTOH metabolic pathways in rats, including those leading to the production of PFOA, have been described (Fasano et al., 2006; Martin et al., 2005). The pathways were identified based on in vitro studies using rat hepatocytes (Fasano et al., 2006; Martin et al., 2005) and from in vivo rat studies (Fasano et al., 2006). A microbial metabolic pathway for 8-2 FTOH has also been proposed (Wang et al., 2005b). Currently, no metabolic pathways from in vitro or in vivo studies are available for 8-2 FTOH metabolism in mice, humans, or trout.
The 90-day subchronic, reproductive, and developmental toxicity of a commercial FTOH mixture dosed orally in rats has been reported (G. Ladics et al., 2005; E. Mylchreest et al., 2005). No functional reproductive or developmental effects were observed that did not adversely affect adult animals. The no-observed adverse effect level (NOAEL) was 25 mg/kg/day for subchronic toxicity and reproductive parameters and 200 mg/kg/day for developmental parameters. The NOAEL for developmental toxicity of 8-2 FTOH in rats was 200 mg/kg/day (Mylchreest et al., 2005). In the 90-day study of 8-2 FTOH in rats, liver and kidney effects were identified at 25 mg/kg/day, with liver lesions more pronounced in male rats while the observed kidney toxicity was more marked in female rats (DuPont-9478, Gregory S. Ladics and Eve Mylcreest).
A comprehensive description of the metabolic transformations and products in multiple species may advance the understanding of these results, help qualify the metabolite toxicities, serve as a basis for defining an animal model suitable for comparison to humans, and aid in assessing the potential for FTOH exposure to contribute to perfluorinated acids in living systems by metabolism. The objective of the current investigation was to study the in vitro pharmacokinetics of 8-2 FTOH in multiple species to determine the similarities and differences in biological disposition and metabolism rates and to more fully develop and refine 8-2 FTOH metabolic pathways. To accomplish these objectives, rat, mouse, human, and trout hepatocytes, and rat, mouse, and human microsomes and cytosols were incubated with [3-14C] 8-2 FTOH, and individual perfluorinated acids known to be present in the 8-2 FTOH metabolic pathway: 8-2 fluorotelomer acid (8-2 FTA), 8-2 fluorotelomer unsaturated acid (8-2 FTUA), 7-3 acid, 7-3 
-ß unsaturated acid (7-3 UA), as well as 7-2 secondary fluorotelomer alcohol (7-2 sFTOH). In this study, metabolites were identified using liquid chromatography/mass spectrometry (LC/MS), gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/accurate radioisotope counting (LC/ARC) methodologies. The availability of 14C radiolabeled 8-2 FTOH allowed the quantification of some of the metabolites that lacked standards and aided in the calculation of the material balance from the test incubations. For convenience, a formal listing of the chemical substances and conjugates identified by this in vitro study and their abbreviations is provided in the supplementary data section (Supplementary Table 7).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Chemicals and biological materials.
The test material, radiolabeled [3-14C] 8-2 FTOH, was synthesized by PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA). The radiolabeled test material had a specific activity of 54.3 mCi/mmol and a radiochemical purity of > 98%. The material was stored at < –70°C. 1H,1H,2H,2H-perfluorodecanol (CF3(CF2)7CH2CH2OH, 8-2 FTOH, CASN 678-39-7) was supplied by E. I. du Pont de Nemours and Company (Wilmington, DE). The sample had a purity of > 99%. Perfluoropentanoic acid (F(CF2)4COOH, PFPeA, purity = 97%, molecular weight (MW) = 264), perfluorohexanoic acid (F(CF2)5COOH, PFHxA, purity = 98%, MW = 314), PFOA (F(CF2)7COOH, purity = 98.7%, MW = 414), PFNA (F(CF2)8COOH, purity = 99%, MW = 464), and 8-2 FTOH (F(CF2)8CH2CH2OH, purity = 97.6%, MW = 464) were purchased from Oakwood Products, Inc. (West Columbia, SC). The perfluoroheptanoic acid (F(CF2)6COOH, PFHpA, purity = 99%, MW = 364) was purchased from Sigma-Aldrich (Milwaukee, WI), and 7-2 sFTOH (F(CF2)7CH(OH)CH3, purity = 98%, MW = 414), 8-2 FTA (F(CF2)8CH2COOH, purity = 97%, MW = 478), 7-3 acid (F(CF2)7CH2CH2COOH, purity = 99%, MW = 442), 8-2 FTUA (F(CF2)7CF = CHCOOH, purity = 98%, MW = 458), 7-3 UA (F(CF2)7CH = CHCOOH, purity = 98.4%, MW = 440), dual-labeled PFOA internal standard (F(CF2)613CF213COOH, 13C-PFOA, purity = 96.6%, MW = 416), and deuterated 8-2 FTOH internal standard (F(CF2)7 13CF2CD2CD2OH, D8-2 FTOH, purity = 96%, MW = 469) were custom synthesized by E. I. du Pont de Nemours and Company. The aldehyde standards 2H, 2H-perfluorooctanal (F(CF2)6CH2C(O)H, 6-2 fluorotelomer aldehyde [6-2 FTAL], MW = 362) and 2H, 2H-perfluorodecanal (F(CF2)8CH2C(O)H, 8-2 fluorotelomer aldehyde [8-2 FTAL], MW = 462) were custom synthesized by E. I. du Pont de Nemours and Company. The 2,4-dinitrophenylhydrazine (DNPH, purity 97%), bovine serum albumin (BSA), and Percoll were purchased from Sigma-Aldrich, and concentrated hydrochloric acid (36.5–38.5%) was purchased from J.T. Baker (Phillipsburg, NJ). Liver perfusion medium, liver digest medium, hepatocyte wash medium (HWM), fetal bovine serum (FBS), Liebovitz-15 (L-15) medium, and Dulbecco's modified eagle medium (DMEM) were obtained from Invitrogen (San Diego, CA). Male rat, mouse, and human liver microsomes were obtained from In Vitro Technologies (Baltimore, MD). Male rat, mouse, and human liver cytosol were obtained from XenoTech (Lenexa, KS). Human hepatocytes were obtained from CellzDirect (Research Triangle Park, NC). The first hepatocyte donor was a 68-year-old Caucasian male with no history of smoking or alcohol or drug abuse. The second donor was a 54-year-old Caucasian male with no history of smoking, alcohol, or drug abuse. The final donor was an 80-year-old Caucasian male with no history of alcohol or drug abuse, smoking history unknown.
Animals.
Male Sprague-Dawley rats (Crl:CD(SD)) and CD mice (Crl:CD1(ICR)) were obtained from Charles River Laboratories (Raleigh, NC). Upon arrival, all animals were housed in quarantine for at least 4 days. Animals were provided tap water ad libitum and fed PMI Nutrition International (Richmond, IN), LLC Certified Rodent LabDiet 5002 ad libitum. Animal rooms were maintained at a temperature of 18°C–26°C and a relative humidity of 30–70%. Animal rooms were artificially illuminated (fluorescent light) on a 12-h light/dark cycle. Rats and mice were 8–12 weeks of age at the time of hepatocyte isolation.
Male juvenile rainbow trout (Oncorhynchus mykiss), approximately 18 months old and approximately 10–12 in. in length, were purchased from Limestone Springs Fishing Preserve (Richland, PA). The fish were held for at least 1 week in continuous-flowing well water at a water temperature of approximately 10°C under a 16:8 light/dark cycle. The fish were fed with AquaMax Starter Fingerling 300 5D03 (PMI Nutrition International) once daily. The fish were fasted for 24 h prior to surgical manipulation of the liver.
Hepatocyte isolation.
Rat and mouse hepatocytes were prepared by two-stage collagenase perfusion using the method of Seglen (1976), and trout hepatocytes were isolated using a 2-step collagenase perfusion method (Klaunig et al., 1985), each with some modifications. Briefly, rats and mice were anesthetized with 80 mg/kg sodium pentobarbital, and trout were anesthetized with 0.15 g/l tricaine methane sulphonate (MS222). Livers were perfused with buffer containing ethylenediaminetetraacetic acid (EDTA) for approximately 10 min at flow rates of 5 ml/min for mice or 10 ml/min for rats and trout. This was followed by an approximate 10-min perfusion with buffer containing collagenase. Perfusion media were maintained at 37°C for mice and rats and at 10°C for trout. At the conclusion of the perfusion, livers were transferred to weigh boats containing either L-15 with 5% FBS for mice and rats or DMEM with 5% BSA for trout and the liver capsule was opened and shaken gently to release the cells. Cells were filtered through 100-µm nylon mesh, washed twice with HWM (mice and rats) or DMEM with 5% BSA (trout), and then purified by centrifugation through 40% Percoll at 4°C. The resulting pellet was washed twice and resuspended in L-15 medium and maintained on ice. Human hepatocytes were purified by centrifugation through Percoll prior to shipment by the vendor. Upon receipt, human hepatocytes were washed twice with L-15 medium, diluted to a concentration of 5 x 106 cells/ml, and maintained on ice until used. Cell viability and yield were determined by trypan blue exclusion. In all cases, hepatocyte viability was > 85%.
Microsome, cytosol, and hepatocyte incubations.
Microsome and cytosol incubations with a NADPH-regenerating system consisting of 100mM sodium phosphate buffer, 10mM glucose-6-phosphate, 0.1mM EDTA, 15mM magnesium chloride, 0.53mM NADP, and 2 units of glucose-6-phosphate dehydrogenase were carried out in 40 ml glass vials containing 5 ml of suspension. Initial experiments were performed using 0.5, 1.0, and 2.0 mg of protein with varying 8-2 FTOH concentrations of 5, 20, or 100µM. These test conditions were used to determine optimal protein concentration and substrate concentrations. Based on these experiments, final microsome test incubations contained 1 mg/ml microsomal protein while cytosol incubations contained 2 mg/ml cytosolic protein. Hepatocyte incubations were carried out in 40 ml glass vials containing 6 ml (trout), 5 ml (rat and mouse), or 3 ml (human) of hepatocyte suspension. Initial experiments were performed at cell concentrations of 1 x 106 and 5 x 106 cells/ml with varying 8-2 FTOH concentrations of 5, 20, or 100µM. Final test reactions were performed using 5 x 106 cells/ml. Microsome, cytosol, and hepatocyte suspensions were preincubated for 5 min at 37°C (rat, mice, human) or 10°C (trout) with gentle orbital shaking. All reactions were initiated by the addition of [14C] 8-2 FTOH or the acid substrates (8-2 FTA, 8-2 FTUA, 7-3 acid, and 7-3 UA) using a pipette. The substrate concentrations were 20µM for [14C] 8-2 FTOH and the acid metabolites (determined to be below metabolic saturation). All dose solutions were prepared in acetonitrile. The amount of organic solvent introduced into the incubation solution did not exceed 0.5% of the total reaction volume. Acetonitrile was chosen as the organic solvent based on data from Easterbrook et al. (2001) which reported using acetonitrile as a substrate at concentrations of 0.1–2% with no apparent effects on multiple activities studied in human hepatocytes. After the addition of test compound, the incubation vials were immediately sealed with threaded poly(tetrafluoroethylene)-lined septum caps and incubated for 2 (rat, mice, and human) or 3 hours (trout). While incubating, the headspace of each reaction vial was purged at 10-min intervals by inserting a 60-ml syringe that was fixed to a small cartridge containing C18 packing material through the septum cap, a separate needle was inserted through the septum cap for an air inlet, and 50 ml of air was pulled through the cartridge column. One cartridge column was used for each reaction. Optimization of the test system was challenging; see supplementary data for a comprehensive description of the process to determine the rationale for using a purged system. Additional hepatocyte reactions were tested using 220µM 8-2 FTOH for structure elucidation. Heat-inactivated hepatocytes or microsome and cytosol incubations with no NADP were used as negative controls. Microsome, cytosol, and hepatocyte incubations using 8-2 FTOH as the substrate were run in triplicate, except for human hepatocyte incubations that were run in duplicate from 3 separate donors. To further elucidate the steps in the metabolic pathway, microsome, cytosol, and hepatocyte incubations (except human) using 8-2 FTA, 8-2 FTUA, 7-3 acid, and 7-3 UA as the substrate were run in duplicate, while only a single human hepatocyte incubation was tested.
Sample preparation.
Aliquots taken from the incubation samples were quenched by pipetting 0.25 ml of sample and 0.50 ml of acetonitrile-containing internal standard into 1.7-ml disposable polypropylene centrifuge tubes at 0, 10, 20, 30, 60, and 120 min (an additional 180-min time point was included for trout). The internal standard was prepared in acetonitrile with 0.225% acetic acid and contained 13C-PFOA (3.75 ppb) and D8-2 FTOH (450 ppb). The tubes were capped and vortexed for 10 min. After vortexing, the samples were centrifuged at 15,000 x g for 10 min at 10°C. The supernatant was then transferred into a high-performance liquid chromatography (HPLC) vial and frozen at – 20°C until analyzed. The remaining pellet was reconstituted in 150 µl of distilled, deionized water, and the entire sample was counted by liquid scintillation counting (LSC). At the end of each experiment, the C18 cartridges were eluted with 3 ml of acetonitrile and a portion (1.5 ml) of the eluate was counted by LSC. Prior to LC/MS/MS or LC/ARC analysis, the samples were thawed, vortexed, and analyzed directly or diluted using acetonitrile-containing internal standard. Prior to LC/MS analysis for aldehydes, an aliquot of the extract was derivatized with DNPH. The calibration standards, fortification samples, and samples were derivatized with DNPH by mixing 1 part sample, 1 part DNPH solution, and 1 part acetonitrile. The DNPH derivatization solution consisted of 0.3% (w/v) of DNPH, 51% (vol/vol) nanopure water, 29% (vol/vol) concentrated hydrochloric acid, and 20% (vol/vol) acetonitrile. After DNPH derivatization, the samples were vortexed before LC/MS analysis.
LC/MS/MS quantitative analysis.
LC/MS/MS was used to quantify the metabolites for which standards were available. Sample extracts were analyzed by LC/MS/MS using an Applied Biosystems SCIEX API-4000 (Foster City, CA) interfaced to an Agilent 1100 HPLC (Agilent Technologies, Palo Alto, CA). The HPLC column used was an Agilent Zorbax 2.1 x 150 mm RX-C8 (5 µm). An in-line Agilent Zorbax SB-C18 2.1 x 150 (5 µm) was placed between the pump and the autosampler to minimize background levels of perfluorocarboxylic acids present in the instrument components. The data were processed using the Applied Biosystems Analyst software version 1.4.1. The gradient HPLC method used HPLC grade water (A) and acetonitrile (B) each containing 0.15% acetic acid as mobile phases with a gradient of 10% B for 1.0 min then to 50% B at 1.1 min held until 2 min then to 95% B at 7.5 min, held until 10.0 min, then to 10% B at 10.1 min, and held until 13.5 min with flow rate of 0.4 ml/min. The MS system was operated in the negative ionization mode with a Turbo Spray source and capillary voltage of – 4500 V and a source temperature of 100°C.
The molecular reaction monitoring (MRM) transitions (m/z) for each analyte were the anions of PFPeA (263 > 219), PFHxA (313 > 269), PFHpA (363 > 319), perfluorooctanoate (PFOA; 413 > 369 and 415 > 369), perfluorononanoate (PFNA; 463 > 419 and 465 > 421), 8-2 FTOH (523 > 59.1 and 525 > 59.1), 7-2 sFTOH (473 > 59.1 and 475 > 59.1), 8-2 FTA (477 > 393 and 479 > 395), 7-3 acid (441 > 337 and 443 > 339), 8-2 FTUA (457 > 393 and 459 > 395), 7-3 UA (439 > 369 and 441 > 371), 13C-PFOA (415 > 370), and D8-2 FTOH (528 > 59.1). The MRM transitions for the perfluorinated carboxylates (and 13C-PFOA internal standard) correspond to the [M-H] anion with the neutral loss of 12CO2 or 14CO2. The MRM transitions for the FTOHs (and D8-2 FTOH internal standard) correspond to the [M+ Acetate] adduct anion with the loss of the neutral M leaving the acetate (m/z = 59.1) anion. The transitions for the fluorotelomer acids correspond to the [M-H] anion with the neutral loss of [12CO2 or 14CO2 + 2HF]. The 13C-PFOA was used as an internal standard for both the perfluorocarboxylic acids and fluorotelomer acids classes of compounds. The D8-2 FTOH was used as an internal standard for the FTOHs. The limit of quantitation is generally limited to the low calibration standard concentration multiplied by the initial sample dilution factor of 3x. However, some sample results were quantified just below the low-level calibration standard whenever the analyte peak area was above the low calibration standard peak area, and the internal standard ratio was within 80% of the low-level calibration standard peak ratio (Table 1).
|
LC/ARC detection of [14C] 8-2 FTOH and 14C-labeled metabolites.
The acetonitrile extracts from the in vitro experiments with [14C] 8-2 FTOH were analyzed using an LC/ARC. Due to the radiolabel on carbon 3, only those metabolites with 8 carbons or more are shown in the LC/ARC chromatograms. The LC/ARC system was composed of an 1100 HPLC system (Agilent Technologies), a 500 TR Series Flow Scintillation Analyzer (Packard Instrument Company, Meriden, CT), and a stop flow system and software from AIM Research Company (Newark, DE). The chromatographic method to resolve and detect 14C-labeled metabolites utilized a Zorbax SB-C18, 4.6 x 150 mm, 5 µm particle size column (Agilent Technologies). The mobile phase was 10mM ammonium acetate in water (A) and methanol (B) with a gradient starting at 5% B for 2 min, then to 100% B at 115 min, and held at 100% B until 135 min at a flow rate of 1.0 ml/min. The injection volume was 100 µl, the fraction size for stop flow counting was 10 s, and the fraction counting time was 60 s. Chromatograms were integrated using LC/ARC software, and the metabolite structures were assigned to the integrated peaks based on the analysis of the collected fractions and confirmation using mass spectrometric methods. From the total radioactivity injected, and the integrated peak areas, expressed in disintegrations per minute (DPM), the percentage of each peak represented of the on-column injection was determined.
Metabolites resolved by LC/ARC were fraction collected and analyzed by HPLC coupled to a linear ion trap mass spectrometer. The metabolites present in the collected fractions were identified by confirming the presence of 14C-labeled molecular ions and corresponding precursor ion spectra compared with those obtained for previously identified metabolites (Fasano et al., 2006). In addition, the acetonitrile extracts were injected directly onto the LC/MS/MS system and screened for any potential new metabolites. The LC/MS/MS system was composed of a Q-Trap 4000 mass spectrometer (Applied Biosystems), an 1100 HPLC system (Agilent Technologies), and a CTC PAL autosampler (Leap Technologies, Carrboro, NC). Chromatographic separation was achieved using a Zorbax SB-C18, 2.1 x 30 mm, 3.5 µm particle size (Agilent Technologies) column, and a mobile phase composed of 2mM ammonium acetate in water (A) and methanol (B) with a gradient starting at 40% B for 1 min, then to 100% B at 6 min, and held at 100% B until 10 min at a flow rate of 0.3 ml/min. The MS system was operated in the negative ionization mode with a Turbo Spray source; capillary voltage was – 4500 V and the source temperature 450°C.
Structural confirmation of aldehyde and ketone metabolites.
Although 6-2 FTAL and 8-2 FTAL standards were available, their purity was unknown and thus used for metabolite identification only. To qualitatively determine if an aldehyde or ketone was present in each of the incubation types/species, area counts from live incubations were compared to negative controls. DNPH-derivatized samples were analyzed by LC/MS using an Agilent 1100 HPLC interfaced to a Waters Micromass Quattro Micro (Waters Corporation, Milford, MA). The mass spectrometer was operated in electrospray negative ionization selected ion recording mode. The capillary voltage was 3.0 kV, source temperature 120°C, and cone voltage 15 V. The HPLC was equipped with an Agilent Zorbax 2.1 x 150 mm RX-C8 with 5-µm-particle size analytical column. The areas were processed using MassLynx version 4.0. The gradient HPLC method used nanopure water with 0.15% acetic acid and acetonitrile mobile phases with a gradient starting at 70% B for 1.5 min then to 100% B at 8.0 min, held until 13 min, then to 70% B at 13.1 min, and held until 19.0 min with a flow rate of 0.4 ml/min. Structure elucidation was performed by the use of several additional instruments including GC/MSD (6890 Plus GC and 5973 Mass Selective Detector, Agilent Technologies), GC/TOF (Model 6890N GC, Agilent Technologies and Time-of-Flight: GCT, Waters Corporation), LC/TOF (Model 2795 Alliance HT/Q-TOF, Waters Corporation), and LC/MS/MS with SCIEX API-4000 (Applied Biosystems).
Kinetic data analysis.
Noncompartmental kinetic analysis was carried out using WinNonlin version 4.0 (Pharsight, Mountain View, CA) based on the following calculations. The concentration data were plotted versus time and a line of the form
 | (1) |
is the elimination rate constant (/min). The half-life (t1/2) was determined by dividing the natural logarithm of 2 by (Obach, 1997). The concentration curve was then integrated from zero to infinity by using the equation
 | (2) |
 | (3) |
 | (4) |
). The corresponding values for humans were a liver weight equivalent to 2.5% of body weight (25 g/kg) and a hepatocellularity of 1.37 x 108 cells/g liver (Arias et al., 1982). Corresponding values for trout were a liver weight equivalent to 1.27% of body weight (12.7 g/kg) (Nichols et al., 1990) and a hepatocellularity of 2.0 x 108 cells/g liver (Law et al., 1991). Equation 2 illustrates the calculation for rodents.
 | (5) |
Similar equations were used to calculate microsomal intrinsic clearance using the amount of protein (milligrams) in the incubation system, giving CLI in units of ml/min/mg protein. The values for liver were 35, 49, and 58 mg microsomal protein/gram of tissue for mouse (Csanády et al., 1992; Medinsky et al., 1994), rats (Baarnhielm et al., 1984; Boogaard et al., 2000; Chiba et al., 1990; Csanády et al., 1992; Joly et al., 1975), and humans (Baarnhielm et al., 1986; Boogaard et al., 2000; Csanády et al., 1992; Lipscomb et al., 2003a,b), respectively. Multiplying this value by the grams of liver weight per kilograms of body weight and milligrams of protein per gram of liver converted the CLI in units to ml/min/kg. The rate of PFOA production was calculated using the amount of PFOA (ng/ml) detected in each reaction over time (0–2 h for rats, mice, and humans, 0–3 h for trout). No metabolism was observed from incubations with cytosol from any species tested. See supplemental information for concentration data of 8-2 FTOH used in the clearance calculations listed above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
[3-14C] 8-2 FTOH: Material Balance
Aliquots of the sample extract, pellet, and cartridge elution (reaction headspace) were subjected to LSC to determine recovery of radioactivity (material balance) for each incubation. In incubations where greater amounts of metabolites were detected, the pellet sample had higher concentrations of radioactivity suggesting that the radioactivity bound to the pellet was most likely from a metabolite and not the parent when compared to negative controls (Table 2). In all the negative control incubations, the amount of radioactivity in the headspace was greater than the amount generated from the live incubations. The presence of [14C] 8-2 FTOH in the headspace of the control incubations confirmed that it was volatile under the test conditions. The headspace cartridge elutions were analyzed by LC/MS and found to contain primarily [14C] 8-2 FTOH. The percentage of radioactivity from the headspace air of hepatocyte incubations was greater in the heat-inactivated samples (22–63%) than in the live reactions (4–28%), confirming uptake and metabolism in the liquid phase of the parent telomer alcohol in the live reactions. In rat and mouse microsomal incubations, headspace air radioactivity was also found to be greater in the control incubations (39–80%) than in the live incubations (11–39%), but headspace air from the human live and control incubations had similar amounts of radioactivity (75 and 77%, respectively), confirming that there was very little metabolism of [14C] 8-2 FTOH. The material balance from the 3 fractions in each of the incubation systems ranged from 73.8 to 103% (Table 2). Cytosol incubations did not show metabolism of 8-2 FTOH from any of the species tested as evidenced by similar amounts of radioactivity in both live and negative control samples (20–27%; data not shown).
|
Metabolism of [14C] 8-2 FTOH
The 8-2 FTOH terminal half-life (T1/2), as determined from the hepatocyte and microsome incubations, was measurable in all species tested (Table 3). Rodents eliminated 8-2 FTOH (T1/2 = 9.9–12.9 min) about 3 times faster than humans (T1/2 = 35.9 min) and about nine times faster than trout (T1/2 = 103 min), which exhibited the slowest elimination half-life of the species studied.
|
Intrinsic clearance of 8-2 FTOH was more rapid in rodent hepatocytes (55.1–82.7 ml/min/kg) than in human (1.50 ml/min/kg) or trout hepatocytes (1.73 ml/min/kg). For rats, comparable clearance rates of 8-2 FTOH were calculated using microsomes and hepatocytes (82.7 vs. 91.6 ml/min/kg, respectively); mouse hepatocytes cleared 8-2 FTOH at only one-third the rate of mouse microsomes (55.1 vs. 147 ml/min/kg, respectively) (Table 3). Hepatocyte intrinsic clearance was determined by scaling the in vitro values up to the organ level using species-specific relative liver weight and hepatocellularity (Houston, 1994
). Clearance rates were adjusted to account for loss of parent compound due to volatility using the rate of loss in the negative control incubations. This adjustment was important to avoid overestimating the clearance rates particularly in human and trout incubation samples where the rate of loss of 8-2 FTOH was only slightly faster in live versus negative controls and only limited metabolism was observed.
Rate of Formation of PFOA
The relative amount of PFOA produced in hepatocyte incubations with [14C] 8-2 FTOH indicated mouse > rat > human trout. Rat and mouse hepatocytes produced 5-fold and 11.9-fold more PFOA than human hepatocytes, respectively (Table 4). The rate of formation of PFOA in hepatocytes was 0.143, 0.061, 0.012, and 0.007 pmol/min/106 cells in mouse, rat, human, and trout, respectively. The amount of PFOA produced from microsomal incubations indicated mouse > rat. Neither PFOA nor PFNA production was observed in human microsomal reactions. The rate of formation of PFOA in microsomes was 5.07 and 4.10 pmol/min/mg of protein in mouse and rat, respectively. PFOA production in mouse microsomal reactions were likely underestimated due to the unexpected presence of endogenous and cytosolic glutathione-S-transferase (GST) leading to a significant amount of the 8-2 uFTOH glutathione (8-2 uFTOH-GS) conjugate. To confirm the presence of GST and its effect on metabolism, an additional experiment was performed using mouse microsomes with the addition of 50µM glutathione (GSH), which significantly increased the amount of 8-2 uFTOH-GS conjugate. Due to the fact that conjugation is a preferred pathway for this compound, this would suggest that the amount of PFOA observed in the mouse incubations would be less than in incubations with no GST.
|
The percent of 8-2 FTOH metabolized to PFOA, based on the molar concentration detected at the end of the incubation periods, was extremely small, 0.47, 0.24, 0.02, and 0.02% for mouse, rat, human, and trout hepatocytes, respectively, and 0.59 and 1.94% for mouse and rat microsomes, respectively (Table 5). Neither PFOA nor PFNA was detected in human microsomes by the end of the 120-min incubation period (limit of detection (LOD) for PFOA and PFNA was 0.2 and 0.1 ng/mL respectively). Mouse hepatocytes produced about 24-fold more PFOA compared to humans and trout hepatocytes, rat hepatocytes produced about 12-fold more PFOA than humans and trout hepatocytes, and mouse hepatocytes produced about 2-fold more PFOA than rats. Comparing the amount of metabolites produced in mammalian hepatocyte incubations at 30 min to those at 120 min demonstrates how some metabolites (8-2 FTA and 8-2 FTUA) were produced but were further metabolized, while several others are terminal metabolites under these test conditions (PFOA, PFNA, and PFHpA). This was not evident in trout incubations due to the slow rate of metabolism of 8-2 FTOH and the duration of the trout incubations (3 h). A higher abundance of metabolites may have been observed if the duration of the trout incubations was increased. Table 5 illustrates the formation of acid metabolites that could be quantified using standards. This study provides quantitative evidence that the production of acid metabolites from 8-2 FTOH in rat, mouse, trout, and human microsomes and hepatocytes is minimal. Table 5 does not include other metabolites present in the incubation samples for which standards were not available. These unquantifiable metabolites include the conjugates and aldehydes identified in this study and by others (Fasano et al., 2006
; Martin et al., 2005).
|
Distribution of Terminal Conjugates by LC/ARC Chromatography
LC/ARC analysis was used to quantitate metabolites for which no standards were available and to aid in the identification of unknown metabolites. A representative LC/ARC radiochromatogram from rat hepatocytes after 2 h of incubation is presented in Figure 1. A representative LC/ARC radiochromatogram from heat-inactivated rat hepatocytes is presented in Figure 2. Several conjugated metabolites were identified and quantified using LC/ARC analysis including 8-2 FTOH glucuronide, 8-2 uFTOH-GS, and 8-2 FTUA-GS. Recovery of injected radioactivity from all samples ranged from 75 to 100%. When compared to the expected DPM based on dosed 14C, the actual amount of radioactivity in the sample extract varied depending on the type of sample (live vs. negative controls) due to the volatile nature of the parent compound. Because of the lower than expected DPM in the extract samples from volatility loss, the relatively low substrate concentration (20µM), and broad indistinguishable peaks in the chromatogram, identifying unknown metabolites was difficult. Unfortunately, fraction collection of these unidentified radioactive peaks did not produce enough signal for identification when analyzed by LC/MS. This was also true for rat hepatocyte incubations at the higher substrate concentration (220µM). In human, trout, and rat hepatocyte incubations, the 8-2 FTOH glucuronide represented approximately 9.8, 7.1, and 1.3% of the total radioactivity (dose), respectively; 8-2 FTOH glucuronide was not detected in mouse hepatocytes. All species produced measurable amounts of 8-2 uFTOH-GS, with the highest levels in mouse hepatocytes (11.8%), comparable levels in rat (4.8%) and human hepatocytes (3.5%), and the lowest levels in trout hepatocytes (3.0%). Rats were the only species tested that yielded detectable levels of the 8-2 FTUA-GS (4.3%).
|
|
Detection of Novel Metabolites
Several metabolites not previously reported were identified in this study. These metabolites are 7-2 ketone, 7-3 ß-keto acid, 7-3 aldehyde (7-3 AL), 7-3
-ß unsaturated aldehyde (7-3 UAL), and 7-3 acid taurine conjugate (7-3 TA) (Supplementary Fig. 6). 7-3 UAL was tentatively identified by Fasano et al. (2006) following oral dosing in rats in vivo. The aldehydes and ketones were detected as DNPH derivatives. Due to the fact that there were no authentic standards available for these novel metabolites, structure elucidation was performed using samples from this study and information gained from preliminary in vitro studies with rat hepatocytes and microsomes using nonlabeled 8-2 FTOH. Structure elucidation was performed using various mass spectrometric techniques. Details describing how these structures were determined are provided in the supplementary data. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Elaboration of 8-2 FTOH Metabolic Pathway
An 8-2 FTOH metabolic pathway is presented in Figure 3 which integrates this study with previous work (Fasano et al., 2006
; Martin et al., 2005). The in vitro experiments performed here, using [14C] 8-2 FTOH and the acid metabolites as substrates in the various species and systems (Table 6), support this more detailed understanding of the metabolic pathway.
|
|
Consistent with previous studies (Fasano et al., 2006
; Martin et al., 2005), 8-2 FTOH was metabolized to the glucuronide and sulfate conjugates and also oxidized to 8-2 FTAL, which, by hydrogen fluoride (HF) elimination, yielded unsaturated 8-2 fluorotelomer aldehyde (8-2 FTUAL). 8-2 FTUAL formed the GSH conjugate 8-2 FTUAL-GS, which was reduced to the unsaturated alcohol conjugate (8-2 uFTOH-GS). Martin et al. (2005) reported findings 8-2 FTAL and 8-2 FTUAL when rat hepatocytes were incubated with 8-2 FTOH and that they were unstable in aqueous solution. Their instability in aqueous solution was confirmed in this investigation. Extract samples stored for several days at < –20°C prior to DNPH derivatization showed minimal amounts of 8-2 FTAL and 8-2 FTUAL. These analytes were subsequently confirmed in rat hepatocyte incubation samples derivatized immediately after collection. Subsequently, 8-2 FTAL is oxidized to 8-2 FTA, with elimination of HF to form 8-2 FTUA and conjugation with GSH (8-2 FTUA-GS). This has been previously reported (Fasano et al., 2006; Martin et al., 2005) and has been confirmed by the current in vitro experiments. The vast majority of 14C 8-2 FTOH metabolism in all species studied leads to 8-2 FTOH, FTAL, FTUAL, FTA, and FTUA conjugates with no further chemical transformation.
Martin et al. (2005) hypothesized the ß-oxidation of 8-2 FTUA to PFOA or PFHxA. The in vitro experiments conducted for this current work were designed to further clarify this pathway. Although Wang et al. (2005a) hypothesized that ß-oxidation of 8-2 FTA and 8-2 FTUA to form PFOA could not occur based on current knowledge of the enzymology of this pathway, we found that PFOA and PFHpA were produced in hepatocyte incubations using 8-2 FTA and 8-2 FTUA as substrates. While not conclusive, a proposed ß-oxidation-like process in the pathway is supported by results of the current investigations. 8-2 FTUAL and 8-2 FTUA are the principal substrates that proceed down 2 distinct yet chemically similar pathways, Pathway A and Pathway B (Fig. 3). The in vitro experiments performed here add support to the proposed pathway of 8-2 FTA -oxidation to PFNA, PFHpA, or PFPeA. When 8-2 FTA was used as the substrate in hepatocyte reactions, measurable concentrations of PFPeA were detected in rat hepatocytes, while measurable concentrations of PFHpA were detected in both the rat and the mouse hepatocyte reactions.
A new pathway is proposed based on the detection of the 7-2 ketone and 7-2 sFTOH from reactions with microsomes and hepatocytes following administration of 8-2 FTOH, 8-2 FTA, and 8-2 FTUA as substrates. This pathway would require the formation of 2 intermediates (identified in the pathway by brackets in Fig. 3) that were not detected in these experiments, but their characteristics can be proposed based on typical fatty acid oxidation to form 7-2 sFTOH which then forms the conjugate 7-2 sFTOH-GLU. The amount of 7-2 sFTOH produced in rodent hepatocyte incubations using 8-2 FTOH as the substrate was less than 1%. In rat hepatocyte incubations using 7-2 sFTOH as the substrate, PFOA was only 0.4% of dose while 7-2 sFTOH-GLU was the major metabolite. This would indicate that in incubations using 8-2 FTOH as the substrate, the 0.2–0.5% of PFOA formed in hepatocyte incubations (rat and mouse, respectively) was not produced from the 7-2 sFTOH but some other pathway.
Fasano et al. (2006) proposed metabolic conversion of 7-3 UA to both PFOA and 7-3 acid. However, when incubated in hepatocytes, 7-3 UA proceeded exclusively to 7-3 acid and ultimately to conjugation with taurine. PFOA was not detected when 7-3 acid or 7-3 UA was used as substrates in hepatocyte preparations (Table 6). Extensive metabolism of 7-3 acid and 7-3 UA was observed in hepatocytes by a rapid decrease in the substrate concentration over time when compared to heat-inactivated hepatocytes in which substrate concentrations remained constant over the incubation period.
The conjugate, 7-3 acid taurine, is also proposed to be produced via a series of reactions from 8-2 FTUAL. 8-2 FTUAL conjugation with GSH forms 8-2 FTUAL-GS, which then proceeds by defluorination to 7-3 UAL and reduction to 7-3 AL. Our results suggest that 7-3 AL was oxidized to 7-3 acid, likely mediated by aldehyde dehydrogenase, with final conjugation to taurine (7-3 TA). The metabolites 7-3 UAL and 7-3 AL have not been reported previously and were detected in either microsome or hepatocyte preparations when 8-2 FTOH was used as the substrate.
Finally, the formation of PFOA seems most likely by fluoride ion release from 8-2 FTUAL by hydrolysis to form the intermediate 7-3 ß-hydroxy FTUAL. Fluoride ion release has been reported for a sulfuryl fluoride fumigant (Mendrala et al., 2005), although how easily 8-2 FTUAL is hydrolyzed depends on the stability of the fluorine bond on the ß-carbon. Further metabolism of 7-3 ß-hydroxy aldehyde to 7-3 ß-keto aldehyde and subsequent decarboxylation would lead to PFOA.
In Vitro Kinetics and Metabolism of 8-2 FTOH and Acid Metabolites
8-2 FTOH was found to be rapidly metabolized principally to conjugates by mice and rats and to a lesser extent humans and trout (Table 2). The rainbow trout (Oncorhynchus mykiss) was selected for these investigations because it has been used as a representative fish species to predict bioconcentration/bioaccumulation potential of the perfluorinated carboxylic acids, including PFOA (Martin et al., 2004). The predicted in vivo intrinsic clearance values were equal to or below normal hepatic blood flows (approximately 90, 80, 17, and 9 ml/min/kg body weight for mice, rats, humans, and trout, respectively) (Brown et al., 1997; Nichols et al., 1990), indicating that blood flow–limited clearance would not be expected for 8-2 FTOH metabolism in vivo. Hepatocytes from all 4 species and microsomes from rats, mice, and humans were found to be capable of metabolizing 8-2 FTOH, while cytosols from rats, mice, and humans were not. These results indicate the importance of membrane-bound enzymes (microsomes) in the metabolism of 8-2 FTOH and certain downstream metabolites. Metabolism occurred in hepatocyte incubations when 8-2 FTA, 8-2 FTUA, 7-3 acid, and 7-3 UA were used as substrates. Metabolites identified for 8-2 FTA and 8-2 FTUA incubations included PFOA, PFHpA, 7-3 UA, 7-3 acid, 7-2 ketone, 7-2 sFTOH, and 7-3 TA. The 7-3 TA was identified in hepatocyte incubations where 7-3 acid and 7-3 UA were used as substrates. No PFCAs were formed from 7-3 acid or 7-3 UA, demonstrating that beta oxidation is not an active metabolic pathway.
In Vitro Metabolic Production of PFOA and PFNA
PFOA and PFNA were produced during incubations with mouse, rat, human, and trout hepatocytes and with mouse and rat liver microsomes at starting concentrations of 20µM 8-2 FTOH (a concentration below metabolic saturation) incubated for 2 h. The percent of dose of PFOA in rat hepatocytes was very small (0.24%), using a starting concentration of 20µM 8-2 FTOH. This percentage was approximately sixfold lower than the 1.4% reported by Martin et al. (2005) using rat hepatocytes at a concentration of 18µM and 4 h of incubation. Our value for PFNA, 0.06% in rat hepatocytes was also lower than that reported by Martin et al. (2005) in which PFNA was < 0.2%. Our results and those reported by Martin et al. (2005) were qualitatively similar in that both experiments indicate that PFOA and PFNA were only minor metabolites in the total oxidation of 8-2 FTOH. Our attempts to duplicate previous experiments (Martin et al., 2005) conducted in an open flask incubated for 4 h at 37°C resulted in a decrease in the volume of hepatocyte suspension due to evaporation. The evaporation of the buffer from the hepatocyte suspension resulted in concentration of the metabolic products. The reduction or evaporative loss and the shorter incubation duration likely explain the difference between our results and those reported previously. The optimization of the test system described in the supplementary data also contains an explanation of the experimental differences between the current study and the study of Martin et al. (2005) (Supplementary Table 8).
The rates of PFOA produced in vitro using hepatocytes from the four species were determined (Table 4), and this facilitated comparisons (of relative rates) between the species on a "per million cells" basis. The relative rates for whole-body formation of PFOA from 8-2 FTOH can also be determined from these rates (using the cellularity data, total liver weights for a given body weight, etc.), and this provides an additional relevant species comparison. The in vitro rates provided in Table 2 were recalculated to 54.9, 23.4, and 2.47 nmol/h/kg body weight for mice (0.03 kg), rats (0.250 kg), and humans (70 kg), respectively, and represent a measure of whole-body capacity to produce PFOA from exposures to 8-2 FTOH (see supplementary data for calculations). These comparisons indicate that mice would produce about 22-fold more PFOA than humans and rats would produce about 9.5-fold more PFOA than humans. This suggests that a rodent model may be overly sensitive for predicting the toxicity of PFOA in humans. Similar results could be obtained for PFNA; however, due to the fact that only the final time point yielded enough quantifiable PFNA, kinetic rates could not be calculated for from these experiments. These whole-body capacities should prove useful in comparisons with future in vivo results.
Although the results of the current work have improved the understanding of 8-2 FTOH metabolism, additional work will be needed to resolve the pathway for formation of the shorter chain perfluorinated acids. Current ongoing in vivo investigations in our laboratory with 8-2 FTOH in rats should provide an important link to the in vitro results presented here and provide additional insights into the potential mechanisms of toxicity reported in the rat following subchronic oral dosing. When more quantitative in vivo metabolic results are available, the in vitro kinetics of 8-2 FTOH metabolism will prove valuable when extrapolation of kinetic rates between species are desired and routes of exposure are considered.
|
SUPPLEMENTARY DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Supplementary data, Tables 7–9, and Figures 4–9 are available online at http://toxsci.oxfordjournals.org/.
|
ACKNOWLEDGMENTS |
|---|
The authors thank Shawn Gannon, Dr William Zimmerman, and Leigh Carson (The Sapphire Group, Inc.) for their helpful thoughts and insights regarding the proposed 8-2 FTOH metabolic pathway. We also thank Dr Raymond Kemper for his initial involvement in the in vitro testing of 8-2 FTOH in 2005, which led to the work reported in this manuscript. Finally, we thank Robert Mingoia, Ching Hui Yang, Keith Prickett, and Richard Rossi for their expert technical assistance in the conduct of these experiments.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Arias IM, Popper H, Schacher D, Shafritz DA. The Liver: Biology and Pathobiology (1982) New York: Raven Press.
Baarnhielm C, Dahlback H, Skanberg I. In vivo pharmacokinetics of felodipine predicted from in vitro studies in rat, dog and man. Acta Pharmacol. Toxicol. (1986) 59:113–122.[Medline]
Baarnhielm C, Skanberg I, Borg KO. Cytochrome P-450-dependent oxidation of felodipine—a 1,4-dihydropyridine—to the corresponding pyridine. Xenobiotica (1984) 14:719–726.[Web of Science][Medline]
Boogaard PJ, De Kloe KP, Bierau J, Kuiken G, Borkulo PED, Van Sittert NJ. Metabolic inactivation of five glycidyl ethers in lung and liver of humans, rats and mice in vitro. Xenobiotica (2000) 30:485–502.[CrossRef][Web of Science][Medline]
Brown RP, Delp MD, Lindstedt SL, Rhomberg LR, Beliles RP. Physiological parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health (1997) 13:407–484.[Web of Science][Medline]
Cadenas E, Muller A, Brigelius R, Esterbauer H, Sies H. Effects of 4-hydroxynonenal on isolated hepatocytes. Biochem. J. (1983) 214:479–487.[Web of Science][Medline]
Calafat AM, Kuklenyik Z, Caudill SP, Reidy JA, Needham LL. Perfluorochemicals in pooled serum samples from United States Residents in 2001 and 2002. Environ. Sci. Technol. (2006) 40:2128–2134.[Medline]
Chiba M, Fujita S, Suzuki T. Pharmacokinetic correlation between in vitro hepatic microsomal enzyme kinetics and in vivo metabolism of imipramine and desipramine in rats. J. Pharm. Sci. (1990) 79:281–287.[CrossRef][Web of Science][Medline]
Csanády GA, Guengerich FP, Bond JA. Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats and mice. (Erratum in Carcinogenesis 14, 784). Carcinogenesis (1992) 13:1143–1153.
Easterbrook J, Lu C, Sakai Y, Li A. Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes. Drug Metab. Dispos. (2001) 29:141–144.
Ellis DA, Martin JW, De Silva AO, Mabury SA, Hurley MD, Sulbaek Andersen MP, Wallington TJ. Degradation of fluorotelomer alcohols: A likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. (2004) 38:3316–3321.[Medline]
Fasano WJ, Carpenter SC, Gannon SA, Snow TA, Stadler JC, Kennedy GL, Buck RC, Korzeniowski SH, Hinderliter PM, Kemper RA. Absorption, distribution, metabolism, and elimination of 8-2 fluorotelomer alcohol in the rat. Toxicol. Sci. (2006) 91:341–355.
Haynes RL, Szweda L, Pickin K, Welker ME, Townsend AJ. Structure-activity relationships for growth inhibition and induction of apoptosis by 4-hydroxy-2-nonenal in raw 264.7 cells. Mol. Pharmacol. (2000) 58:788–794.
Houde M, Martin JW, Letcher RJ, Solomon KR, Muir DCG. Biological monitoring of polyfluoroalkyl substances: A review. Environ. Sci. Technol. (2006) 40:3463–3473.[Medline]
Houston JB. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. (1994) 47:1469–1479.[CrossRef][Web of Science][Medline]
Joly JG, Doyon C, Peasant Y. Cytochrome P-450 measurement in rat liver homogenate and microsomes. Its use for correction of microsomal losses incurred by differential centrifugation. Drug Metab. Dispos. (1975) 3:577–586.[Abstract]
Kissa E. Fluorinated Surfactants and Repellents. Second Edition Revised and Expanded. In: Surfactant Science Series, CRC (2001) Vol. 97, 2nd ed. 1–615.
Klaunig JE, Ruch RJ, Goldblatt PJ. Trout hepatocyte culture: Isolation and primary culture. In Vitro Cell Dev. Biol. (1985) 21:221–228.[CrossRef][Web of Science][Medline]
Kudo N, Iwase Y, Okayachi H, Yamakawa Y, Kawashima Y. Induction of hepatic peroxisome proliferation by 8-2 telomer alcohol feeding in mice: Formation of perfluorooctanoic acid in the liver. Toxicol. Sci. (2005) 86:231–238.
Ladics G, Stadler JC, Makovec GT, Everds NE, Buck RC. Subchronic toxicity of a fluoroalkylethanol mixture in rats. Drug Chem. Toxicol. (2005) 28:135–158.[CrossRef][Web of Science][Medline]
Law FCP, Abedini S, Kennedy CJ. A biologically based toxicokinetic model for pyrene in rainbow trout. Toxicol. Appl. Pharmacol. (1991) 110:390–402.[CrossRef][Web of Science][Medline]
Lipscomb JC, Teuschler LK, Swartout JC, Popken D, Cox T, Kedderis GL. The impact of cytochrome P450 2E1-dependent metabolic variance on a risk-relevant pharmacokinetic outcome in humans. Risk Anal. (2003a) 23:1221–1238.[CrossRef][Web of Science][Medline]
Lipscomb JC, Teuschler LK, Swartout JC, Striley CAF, Snawder JE. Variance of microsomal protein and cytochrome P450 2E1 and 3A forms in adult human liver. Toxicol. Mech. Methods (2003b) 13:45–51.[CrossRef]
Martin JW, Mabury SA, O'Brien PJ. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interact. (2005) 155:165–180.[CrossRef][Web of Science][Medline]
Martin JW, Whittle DM, Muir DCG, Mabury SA. Perfluoroalkyl contaminants in a food web from Lake Ontario. Environ. Sci. Technol. (2004) 38:5379–5385.[Medline]
Medinsky MA, Leavens TL, Csanády GA, Gargas ML, Bond JA. In vivo metabolism of butadiene by mice and rats: A comparison of physiological model predictions and experimental data. Carcinogenesis (1994) 15:1329–1340.
Mendrala AL, Markham DA, Eisenbrandt DL. Rapid uptake, metabolism, and elimination of inhaled sulfuryl fluoride fumigant by rats. Toxicol. Sci. (2005) 86:239–247.
Mylchreest E, Munley SM, Kennedy GL Jr. Evaluation of the developmental toxicity of 8-2 telomer B alcohol. Drug Chem. Toxicol. (2005) 28:315–328.[CrossRef][Web of Science][Medline]
Nichols JW, McKim JM, Andersen ME, Gargas ML, Clewell HJ III, Erickson RJ. A physiologically based toxicokinetic model for the uptake and disposition of waterborne organic chemicals in fish. Toxicol. Appl. Pharmacol. (1990) 106:433–447.[CrossRef][Web of Science][Medline]
Obach RS. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab. Dispos. (1997) 27:1350–1359.
Prevedouros K, Cousins IT, Buck RC, Korzeniowski SH. Sources, fate and transport of perfluorocarboxylates. Environ. Sci. Technol. (2006) 40:32–44.[Medline]
Seglen PO. Preparation of isolated liver cells. Methods Cell Biol. (1976) 13:29–83.[Medline]
Telomer Research Program. Telomer Research Program Update—Presented to USEPA OPPT. (2002) U.S. Environmental Protection Agency. Public Docket AR226-1141, Washington, DC.
Wang N, Szostek B, Folsom PW, Sulecki LM, Capka V, Buck RC, Berti WR, Gannon JT. Aerobic biotransformation of 14C-labeled 8-2 telomer B alcohol by activated sludge from a domestic sewage treatment plant. Environ. Sci. Technol. (2005a) 39:531–538.[Medline]
Wang N, Szostek B, Folsom PW, Sulecki LM, Capka V, Buck RC, Berti WR, Gannon JT. Fluorotelomer alcohol biodegradation—direct evidence that perfluorinated carbon chains breakdown. Environ. Sci. Technol. (2005b) 39:7516–7528.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||