role of cytochrome p4503a in cysteine s-conjugates sulfoxidatıon and the nephrotoxicity of the...

Role of Cytochrome P4503A in Cysteine S-ConjugatesSulfoxidatıon and the Nephrotoxicity of the Sevoflurane

Degradatıon ProductFluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl Ether

(Compound A) in Rats

Pam Sheffels,† Jesara L. Schroeder,† T. Gul Altuntas,†,‡ H. Denny Liggitt,§ andEvan D. Kharasch*,†,|

Departments of Anesthesiology, Comparative Medicine, and Medicinal Chemistry,University of Washington, Seattle, Washington 98195-6540, and Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey

Received April 8, 2004

The volatile anesthetic sevoflurane is degraded to fluoromethyl-2,2-difluoro-1-(trifluoro-methyl)vinyl ether (FDVE) in anesthesia machines. FDVE is nephrotoxic in rats. FDVEundergoes glutathione conjugation, subsequent conversion to cysteine and mercapturic acidconjugates, and cysteine conjugate metabolism by renal â-lyase, which is a bioactivationpathway mediating nephrotoxicity in rats. Recent in vitro studies revealed cytochrome P4503A-catalyzed formation of novel sulfoxide metabolites of FDVE cysteine-S and mercapturic acidconjugates in rat liver and kidney microsomes. FDVE-mercapturic acid sulfoxides were moretoxic than other FDVE conjugates to renal proximal tubular cells in culture. Nevertheless,the occurrence and toxicological significance of FDVE sulfoxides formation in vivo remainunknown. This investigation determined, in rats in vivo, the existence, role of P4503A, andnephrotoxic consequence of FDVE conjugates sulfoxidation. Rats were pretreated withdexamethasone, phenobarbital, troleandomycin, or nothing (controls) before FDVE, and then,nephrotoxicity, FDVE-mercapturate sulfoxide urinary excretion, and FDVE-mercapturatesulfoxidation by liver microsomes were assessed. The formation of FDVE-mercapturic acidsulfoxide metabolites in vivo and their urinary excretion were unambiguously established bymass spectrometry. Dexamethasone and phenobarbital increased, and troleandomycin de-creased (i) liver microsomal FDVE-mercapturic acid sulfoxidation in vitro, (ii) FDVE-mercapturic acid sulfoxide urinary excretion in vivo, and (iii) FDVE nephrotoxicity in vivoassessed by renal histology, blood urea nitrogen concentrations, and urine volume and proteinexcretion. Urine 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid, reflecting â-lyase-dependentFDVE-cysteine S-conjugates metabolism, was minimally affected by the pretreatments. Theseresults demonstrate that FDVE S-conjugates undergo P4503A-catalyzed sulfoxidation in ratsin vivo, and this sulfoxidation pathway contributes to nephrotoxicity. FDVE S-conjugatessulfoxidation constitutes a newly discovered mechanism of FDVE bioactivation and toxicificationin rats, in addition to â-lyase-catalyzed metabolism of FDVE-cysteine S-conjugates.

Introduction

The volatile anesthetic sevoflurane undergoes base-catalyzed dehydrofluorination by the carbon dioxideabsorbents in anesthesia machines, forming FDVE1

(referred to as “compound A” in the sevoflurane labeling)(Figure 1; 1) as the major degradation product (1, 2).FDVE is nephrotoxic when administered to rats byinhalation or intraperitoneal injection (3-8). Many halo-alkenes are nephrotoxic in rats, and their toxicity isassociated with a multistep pathway involving GSHS-conjugate formation, enzymatic hydrolysis of the GSHS-conjugates to cysteine S-conjugates, renal uptake of

cysteine S-conjugates, and bioactivation by renal cysteineS-conjugate â-lyase to intermediates whose reaction withcellular proteins is associated with cell damage and death(9-11).

Considerable evidence supports the hypothesis thatFDVE S-conjugates formation, their renal uptake, andFDVE-cysteine conjugates metabolism by renal â-lyase

* To whom correspondence should be addressed. Tel: 206-543-4070.Fax: 206-685-3079. E-mail: [email protected].

† Department of Anesthesiology, University of Washington.‡ Department of Pharmaceutical Chemistry, Ankara University.§ Department of Comparative Medicine, University of Washington.| Department of Medicinal Chemistry, University of Washington.

1 Abbreviations: FDVE, fluoromethyl-2,2-difluoro-1-(trifluorometh-yl)vinyl ether; GSH, glutathione; GST, glutathione S-transferase; GGT/DP, γ-glutamyltransferase/dipeptidase; BUN, blood urea nitrogen;TAO, troleandomycin; PB, phenobarbital; DEX, dexamethasone; N-Ac-DFEC, N-acetyl-S-[1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)-ethyl]-L-cysteine; (E,Z)-N-Ac-FFVC, (E,Z)-N-acetyl-S-[1-fluoro-2-fluo-romethoxy-2-(trifluoromethyl)vinyl]-L-cysteine; (E,Z)-N-Ac-FFVC-SO,(E,Z)-N-acetyl-S-[1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl]-L-cysteine sulfoxide); N-Ac-DFEC-SO, N-acetyl-S-[1,1-difluoro-2-fluo-romethoxy-2-(trifluoromethyl)ethyl]-L-cysteine sulfoxide; LC-MS, liquidchromatography/mass spectrometry; LC-MS/MS, liquid chromatogra-phy/tandem mass spectrometry; GC-MS, gas chromatography/massspectrometry; NMR, nuclear magnetic resonance.

1177Chem. Res. Toxicol. 2004, 17, 1177-1189

10.1021/tx049899e CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 08/18/2004

mediate, in part, FDVE nephrotoxicity in rats. FDVEundergoes enzymatic and nonenzymatic GSH conjugationto form several FDVE-GSH conjugates, subsequentconversion to the corresponding FDVE-cysteine andmercapturic acid conjugates, and bioactivation of thecysteine conjugates by renal â-lyase. In rats, in vivo,FDVE reacts with GSH to form (R)- and (S)-S-[1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl]glu-tathione (Figure 1; 2) and (E)- and (Z)-S-[1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl]glutathione con-jugates (Figure 1; 3), which undergo cleavage to thecorresponding cysteine S-conjugates (Figure 1; 4 and 5)(12-14). N-Acetylation forms the nontoxic mercaptur-ates, (R)- and (S)-N-Ac-DFEC (Figure 1; 6) and (E,Z)-N-Ac-FFVC (Figure 1; 7), which are excreted in urine (12,14, 15). The cysteine S-conjugates are also metabolizedby rat renal â-lyase in vitro and in vivo to reactiveintermediates, which may bind to cellular macromol-ecules or undergo hydrolysis to 3,3,3-trifluoro-2-(fluo-romethoxy)propanoic acid (Figure 1; 8) (14-19). In rats,aminooxyacetic acid, an inhibitor of renal cysteine con-jugate â-lyase, and probenecid, an inhibitor of renalorganic anion transport and S-conjugate uptake, signifi-cantly diminished histologic and biochemical evidence ofFDVE nephrotoxicity (7, 8). FDVE-GSH and -cysteineS-conjugates replicated the nephrotoxicity of FDVE itself,while a FDVE-cysteine S-conjugate analogue, which isnot a â-lyase substrate, did not (20).

A second pathway of haloalkene S-conjugates bioacti-vation and toxification, involving hepatic microsomalcytochrome P450- or flavin monooxygenase-catalyzedsulfoxidation of haloalkene cysteine and mercapturic acidconjugates, has been identified (21-26). Sulfoxidation ofhaloalkyl cysteine S-conjugates can constitute a toxifi-

cation pathway, which is independent of â-lyase-mediatedbioactivation (22-26).

Recent results demonstrate that FDVE S-conjugatescan also undergo sulfoxidation. Rat liver microsomesoxidized N-Ac-DFEC and (Z)-N-Ac-FFVC to the corre-sponding sulfoxides, N-Ac-DFEC-SO (Figure 1; 12) and(Z)-N-Ac-FFVC-SO (Figure 1; 13) (27). Much lower ratesof (Z)-N-Ac-FFVC, but not N-Ac-DFEC, sulfoxidation alsooccurred with rat kidney microsomes. Among severalexpressed P450s and flavin monooxygenases, P4503Aisoforms were the major enzymes responsible for (Z)-N-Ac-FFVC and N-Ac-DFEC sulfoxidation in vitro, and ratliver microsomal sulfoxidation was induced by PB. DFECsulfoxidation with liver microsomes was also observed,predominantly nonenzymatic, forming S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl)ethyl)-L-cysteine sulfox-ide (DFEC-SO; Figure 1; 10). In addition, in culturedhuman proximal tubular HK-2 cells, both (Z)-N-Ac-FFVC-SO and DFEC-SO were somewhat more toxic thantheir corresponding GSH, cysteine, or mercapturic acidconjugates (28).

It is unknown, however, whether FDVE S-conjugatesundergo sulfoxidation in rats in vivo. Furthermore, thetoxicological significance of potential FDVE sulfoxidationalso remains unknown. Therefore, the objective of thisinvestigation was to test the hypothesis that FDVES-conjugates undergo sulfoxidation in rats in vivo andthat FDVE S-conjugates sulfoxidation may constitute apathway of FDVE toxification in rats. Experiments wereconducted in FDVE-treated rats to determine whetherFDVE S-conjugate sulfoxides are formed and excreted inurine, the influence of P4503A induction and inhibitionon FDVE sulfoxidation in vivo and in liver microsomes

Figure 1. Biotransformation of FDVE.

1178 Chem. Res. Toxicol., Vol. 17, No. 9, 2004 Sheffels et al.

from induced and inhibited rats, and the effect of P4503Ainduction and inhibition on FDVE nephrotoxicity.

Material and Methods

Chemicals. FDVE (99.92% purity) was provided by AbbottLaboratories (Abbott Park, IL). Dichloroacetic acid (DCAA),Magtrieve (CrO2), and benzophenone hydrazone were purchasedfrom Aldrich (Milwaukee, WI); methylene chloride and hydro-chloric acid were purchased from Fisher Scientific (Pittsburgh,PA); ethyl acetate (GC grade) was purchased from Burdick andJackson (Muskegon, MI); and anhydrous ether was purchasedfrom J. T. Baker (Phillipsburg, NJ). N-Ac-S-benzyl-DL-cysteinewas purchased from TCI America (Portland, OR). NADPH, TAO,PB, and DEX were obtained from Sigma-Aldrich Co. (St. Louis,MO), as were all other reagents unless specified. All stock drugsolutions and buffers were prepared using Milli-Q grade water(Millipore, Bedford, MA).

N-Ac-DFEC, (Z)-N-Ac-FFVC, (Z)-N-Ac-FFVC-SO, N-Ac-DFEC-SO, and DFEC-SO were synthesized as described previously(27), as was 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid (14).Diphenyldiazomethane was prepared by adding benzophenonehydrazone (25.8 mg) to methylene chloride (1.9 mL), followedby Magtrieve (189 mg); the mixture was reacted for 30 min withperiodic vortexing, release of the gas formed, and removal ofMagtrieve by centrifugation and filtration through Whatmanno. 2 paper (29).

Animals and Treatments. All animal experiments wereapproved by the University of Washington Animal Use Com-mittee in accordance with the American Association for Ac-creditation of Laboratory Animal Care guidelines. Male Fisher344 rats (250-300 g, Harlan, San Diego) were housed inindividual metabolic cages, provided food and water ad libitum,and maintained on a 12 h light-dark cycle. Baseline saphenousvein blood samples were obtained, and FDVE (0.25 mmol/kg in0.125 mmol/mL corn oil) was then administered by intraperi-toneal injection (7). Urine was collected on ice in 24 h intervals,beginning the day before FDVE injection, and stored at -80 °Cuntil analyzed.

Each treatment group contained 5-9 animals. The rats wererandomized to pretreatment with either DEX (50 mg/kg ip incorn oil 25 mg/mL, daily for 3 days before FDVE administration)(30), PB (0.1% in drinking water for 10 days before FDVEadministration), TAO (500 mg/kg ip, 2 h before FDVE admin-istration) (31), or corn oil (2 mL/kg for 3 days before FDVEadministration) as a control. TAO in saline was prepared freshdaily as described previously (32). Briefly, TAO was suspendedin 0.9% saline and approximately an equimolar volume of 1 MHCl was added until the TAO completely dissolved; then, thepH was adjusted to 4.0 with 1 M NaOH. The sample was dilutedin 0.9% saline to a final concentration of approximately 150 mgTAO/0.9 mL, and the rats were dosed by weight. The animalswere not pretreated for the experiment evaluating the timecourse of FDVE metabolites excretion. The animals wereanesthetized with pentobarbital 24 h after FDVE injection(except where noted) and sacrificed by cardiac exsanguination.The plasma and urine were stored at -20 and -80 °C,respectively, for later analysis. The kidneys were immediatelyexcised, trimmed, and cut in a midtransverse plane through thecortex and medullary pyramid, and a section was fixed in 10%neutral buffered formalin. The remainder was stored at -80°C. The formalin-fixed kidney sections were later embedded inparaffin, sliced, and stained with hematoxylin and eosin forhistological analysis. The livers were flushed with PBS (pH 7.4)and immediately frozen in an ethanol dry ice bath and thenstored at -80 °C. The liver microsomes were prepared aspreviously described (27).

Urine protein concentrations were measured spectrophoto-metrically using a microprotein kit (Sigma 611-A) according tothe manufacturers directions. Urine osmolality was determinedusing an Advanced Instruments (Norwood, MA) model 3D3

freezing point osmometer. Urine fluoride concentrations weremeasured using an Orion fluoride specific electrode (ThermoElectron Corp., Waltham, MA). The thawed urine was mixed,80 µL was added to 20 µL of low-level total ionic strengthadjuster buffer (1 M NaCl in 1 M acetic acid, pH 5.0-5.5 with5 M NaOH), and 40 µL was placed on the tip of an invertedfluoride electrode and then covered with a microscope slidecoverslip. The millivolt reading of the sample was converted tofluoride concentration using a standard curve (0.1-5 mMsodium fluoride in water). The plasma urea nitrogen (BUN) wasmeasured spectrophotometrically using the ThermoDMA Infin-ity Urea Nitrogen reagent (Thermo DMA, Louisville, CO)according to the manufacturer’s directions.

The kidneys were fixed in 10% neutral buffered formalin,trimmed, processed, and embedded in paraffin. Sections werestained with hematoxylin and eosin and periodic acid Schiff andexamined histologically by a pathologist who was unaware ofthe treatment group. Histological analysis was performed by aveterinary pathologist blinded to animal treatments. Histo-pathological changes were recorded in regard to location,character, and severity. The semiquantitative severity scoreconsisted of a range from 0 to 4 (respectively, normal, minimal,slight, moderate, and marked), which reflects the degree anddistribution of the tubular necrosis.

Incubation Conditions. (Z)-N-Ac-FFVC and N-Ac-DFEC(2 mM) were incubated with liver microsomes from DEX, PB,TAO, or corn oil pretreated rats (1 mg/0.25 mL) in 0.1 Mphosphate buffer (pH 7.4) containing NADPH (2 mM). Thereaction mixture was preincubated for 5 min at 37 °C in thepresence of NADPH, started by the addition of substrate, andterminated after 30 min with 20% perchloric acid. The controlreactions lacked NADPH. After the mixture was centrifuged,the supernatant (20 µL) was analyzed by LC-MS. Sulfoxideformation was quantified by comparing the peak areas withcalibration curves (r2 > 0.99) for (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO standard. The limits of quantification were 50 ng/mL for both (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO.

Determination of FDVE-Mercapturic Acid Conjugatesand Their Corresponding Sulfoxides in Urine. An internalstandard (2 µg of N-Ac-S-benzyl-DL-cysteine in 100 µL of water)was added to urine (0.5 mL) and then acidified to pH 2.0 with100 µL of 20% perchloric acid and extracted with diethyl ether(2 × 2 mL). The combined organic layers were evaporated todryness under nitrogen at 30 °C. The residue was dissolved in100 µL of acetonitrile and transferred to autosampler vials withlimited volume inserts for LC-MS and LC-MS/MS analysis.

N-Ac-DFEC, (E,Z)-N-Ac-FFVC, N-Ac-DFEC-SO, and (Z)-N-Ac-FFVC-SO were quantified using standard curves of peakarea ratios vs analyte concentration (r2 > 0.99) of analytes addedto control rat urine, which ranged from 0.2 to 100 µg/mL forN-Ac-DFEC-SO and (Z)-N-Ac-FFVC-SO and from 0.5 to 200 µg/mL for N-Ac-DFEC and (E,Z)-N-Ac-FFVC. Because individualstandards were not available, the peak areas for (E)- and (Z)-N-Ac-FFVC were quantified together as were those for thediastereomers of N-Ac-DFEC-SO.

The presence and identities of (Z)-N-Ac-FFVC-SO and N-Ac-DFEC-SO in urine were confirmed by LC-MS/MS using aQuattro II Tandem Quadrupole Mass Spectrometer (MicromassLtd., Manchester, United Kingdom) equipped with an atmo-spheric pressure ion source and ion spray interface. Nitrogenserved as the drying and nebulizing gas. For collision-induceddissociation, argon was used as the target gas at a pressure of1.0 × 10-3 mbar. The optimal cone voltage and collision energywere 20 V and 15 eV, respectively. All data were processed withWindows NT based Micromass MassLynxNT 3.2, MaxEnt, andBioLynx. Chromatographic separations of the authentic stan-dards FDVE-mercapturic acid sulfoxide conjugates and samplesfrom rat urine were performed on a Shimadzu LC-10AD binarypump with SIL-10ADVP autoinjector (Shimadzu ScientificInstruments Inc., Columbia, MD) and Supelcosil LC-18-DB (15cm × 3 mm i.d., 3 µm) reverse phase analytical column (SupelcoCo., Bellefonte, PA). Peak confirmation was achieved by mul-

Haloalkene Conjugates Sulfoxidation and Toxicity Chem. Res. Toxicol., Vol. 17, No. 9, 2004 1179

tireaction monitoring of the transitions from m/z 359.8 to 130.1(N-Ac-DFEC-SO) and m/z 339.8 to 130.1 [(Z)-N-Ac-FFVC-SO].

Mercapturates and their sulfoxides were identified andquantified by LC-MS, using an Agilent (Palo Alto, CA) 1100Series instrument equipped with a vacuum degasser, binarysolvent delivery system, well plate autosampler, and a SupelcosilLC-18-DB (15 cm × 3 mm, 3 µm) (Supelco Co.) reverse phaseanalytical column. The mobile phase (0.5 mL/min) was agradient of solvent A (0.05% trifluoroacetic acid in water) andsolvent B [acetonitrile:methanol (1:1) with 0.05% trifluoroaceticacid], starting at 10% B for the first minute, linearly increasedto 60% B over 11 min, then increased to 90% B over 1 min, heldthere for 2 min, and then reequilibrated with 10% B for 4.5 min.The mass spectrometer was equipped with an electrosprayinterface and operated in the positive ionization mode. Theinterface was maintained at 350 °C with a nitrogen nebulizationpressure of 35 psi and a drying gas flow of 11.0 L/min. Analysis(5 µL injection) was performed in the selected ion monitoringmode with the fragmentor set at 90 V and a capillary voltage of3500 V. The [M + H]+ ion, m/z 254, 324, 340, 344, and 360, wasmonitored for N-Ac-S-benzyl-DL-cysteine, (E,Z)-N-Ac-FFVC, (Z)-N-Ac-FFVC-SO, N-Ac-DFEC, and N-Ac-DFEC-SO, respectively.

Determination of 3,3,3-Trifluoro-2-(fluoromethoxy)pro-panoic Acid in Urine. Rat urine or standards (50 µL), 0.2 µgof DCAA (20 µL aqueous), and 250 µL of 0.1 N HCl (final pH2-3) were added to glass tubes and then extracted twice byvortexing with 1 mL of ether. The samples were centrifuged,the organic layer was removed, and 75 µL of diphenyldiaz-omethane derivatizing agent was added to the combined organic

layers. The samples were vortexed, reacted at 21 °C for 45 min,evaporated at 50 °C under nitrogen, reconstituted in 50 µL ofethyl acetate, and placed in autosampler vials with limitedvolume inserts. GC-MS analysis was performed on an Agilent6890-5973 MSD, using a DB-5 capillary column (30 m × 0.32mm × 0.25 µm) (J&W Scientific, Folsom, CA). The GC injectorwas operated in pulsed splitless mode (1 µL injection) with aconstant flow (1.0 mL/min). The injector and detector temper-atures were 250 and 300 °C, respectively. The oven was held at35 °C for 5 min, increased at 20 °C/min to 250 °C, increased at10 °C/min to 280 °C, and held for 10 min. The diphenylmethylester derivatives of 3,3,3-trifluoro-2-(fluoromethoxy)propanoicacid (fluoropropanoic acid) (m/z 342, [M]+•, 19.8 min) and DCAA(m/z 294[M]+•, 21.2 min) were determined by selected ionmonitoring (18). Fluoropropanoic acid was quantified usingcalibration curves of the peak area ratio, obtained by analyzingblank rat urine containing 0.625, 1.25, 3.12, 6.25, 15.62, 31.25,62.5, 125, 250, and 375 µg/mL fluoropropanoic acid. Qualitycontrol samples contained 3.12, 31.2, and 125 µg/mL fluoropro-panoic acid. The standard curves were linear (r2 > 0.997) over0.625-375 µg/mL, the limit of quantification was 1 µg/mL, andthe intraday variability of quality control samples was 19, 10,and 8%.

Statistical Analysis. Data were analyzed using a two-wayrepeated measures analysis of variance or one-way analysis ofvariance, as appropriate, followed by the Student-Newman-Keuls test for multiple comparisons, using SigmaStat (SPSSScience, Chicago, IL). All results are reported as the mean (

Figure 2. LC-MS analysis of the FDVE-mercapturic acid conjugate N-Ac-DFEC excreted in rat urine. (A) Electrospray LC-MSchromatogram of a urine sample from a rat administered FDVE, monitored at m/z 344. The peak eluting at 12 min is N-Ac-DFEC.(B) Mass spectrum of synthetic N-Ac-DFEC. (C) Mass spectrum of N-Ac-DFEC detected in the urine of a DEX pretreated ratadministered FDVE. The ions at m/z 344, 366, 302, and 130 represent [M + H]+, [M + Na]+, [M + H - C2H2O]+, and [C5H8NO3]+,respectively.


standard deviation (SD). Statistical significance was assignedat p < 0.05.

Results

Time Course of FDVE Toxicity and MetabolitesExcretion in Urine. Rats received a single injection of0.25 mmol/kg FDVE (58 ( 3 µmol), and the urine wascollected daily for 1 week. The excretion of the mercap-turates N-Ac-DFEC and (E)- and (Z)-N-Ac-FFVC wasidentified by LC-MS. Chromatograms obtained by se-lected ion monitoring and mass spectra of syntheticmercapturates and those detected in urine of rats givenFDVE are shown in Figures 2 and 3, for N-Ac-DFEC and(E,Z)-N-Ac-FFVC, respectively. Previous experimentsidentified mercapturates excretion using GC-MS andNMR spectroscopy (12, 14, 15, 18). In contrast to GC-MS, in which (E)- and (Z)-N-Ac-FFVC eluted as a singlepeak, LC-MS permitted resolution and detection of bothpositional isomers (Figure 3A, peaks 2 and 3, respec-tively). The (E)/(Z) N-AC-FFVC formation ratio, basedon peak areas, was 0.6 ( 0.1. The excretion of inorganicfluoride, liberated by the addition-elimination reactionforming S-[1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)-vinyl]glutathione conjugates and by the renal â-lyase-catalyzed metabolism of FDVE-cysteine S-conjugates,

was identified, as was that of 3,3,3-trifluoro-2-(fluo-romethoxy)propanoic acid, reflecting â-lyase-catalyzedmetabolism of FDVE-cysteine S-conjugates, as describedpreviously (14, 15, 18). No unacetylated cysteine conju-gates were detected in urine.

The excretion of the mercapturic acid sulfoxide N-Ac-DFEC-SO was unambiguously established by comparingLC-MS spectra of urine to those of authentic compounds(Figure 4). N-Ac-DFEC-SO in urine eluted as two peakswith identical mass spectra, presumed to represent twodiastereomers, although further structural identificationof the diastereomers was not pursued. No urinary excre-tion of either (E)- or (Z)-N-Ac-FFVC-SO was observed,within the limits of detection (0.2 µg/mL).

The time course of urinary FDVE metabolites excretionis shown in Figure 5. The cumulative amounts (µmol)excreted over 7 days were 7.1 ( 0.8 N-Ac-DFEC, 3.5 (0.4 (E,Z)-N-Ac-FFVC, 0.10 ( 0.02 N-Ac-DFEC-SO, 1.1( 0.3 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid, and33.6 ( 4.5 inorganic fluoride (corrected for daily back-ground fluoride excretion), corresponding to 12.3, 6.1, 0.2,1.9, and 58% of the dose, respectively. The majority ofmetabolites (76-95%) were excreted within the first dayafter the FDVE injection. Approximately 79% of theFDVE dose was recovered as metabolites in urine over 7days.

Figure 3. LC-MS analysis of the FDVE-mercapturic acid conjugate (E,Z)-N-Ac-FFVC excreted in rat urine. (A) Electrospray LC-MS chromatogram of a urine sample from a rat administered FDVE, monitored at m/z 324. Peaks 2 and 3 correspond to (E)- and(Z)-N-Ac-FFVC, respectively, while peak 1 is a fragment ion from N-Ac-DFEC. (B) Mass spectrum of synthetic (Z)-N-Ac-FFVC elutingat 12.6 min (peak 3). (C) Mass spectrum of (Z)-N-Ac-FFVC (peak 3) detected in the urine of a DEX pretreated rat administeredFDVE. The spectra for (E)-N-Ac-FFVC (peak 2, 12.3 min) for panels B and C were similar. The ions at m/z 324, 346, 282, and 130represent [M + H]+, [M + Na]+, [M + H - C2H2O]+, and [C5H8NO3]+, respectively.


The time course of the biochemical indices of FDVEtoxicity is shown in Figure 6. FDVE caused diuresis,proteinuria, and increased BUN concentrations, as de-scribed previously (6). Decreased urine osmolality fol-lowing FDVE administration in rats has not previouslybeen reported. Diuresis, proteinuria, and excretion of ahypoosmolar urine were most prominent on day 1 butpersisted for a week, which was longer than that observedpreviously with inhaled FDVE (6). Renal necrosis isapparent for at least 2 weeks after FDVE exposure (6).Because metabolites excretion and biochemical indicesof toxicity were greatest on day 1, further experimentswere terminated after day 1, consistent with previousinvestigations (7, 8, 15).

Effect of Enzyme Induction and Inhibition onFDVE Metabolism in Vivo. Previous experimentsshowed that P4503A was the predominant catalyst ofFDVE mercapturates sulfoxidation in vitro (27); thus,animals were pretreated with P4503A inducers andinhibitors to determine the role of P4503A in FDVEmercapturates sulfoxidation in vivo (Table 1). DEX, PB,and TAO pretreatment had no consistently significanteffect on the excretion of either of the mercapturates,N-Ac-DFEC or (E,Z)-N-Ac-FFVC, and had no effect onthe relative excretion of (E)- and (Z)-N-Ac-FFVC. Incontrast, DEX and PB pretreatment increased 10-fold,and TAO abolished, respectively, the excretion of the

mercapturic acid sulfoxide N-Ac-DFEC-SO. Even in PBand DEX pretreated rats, no (E)- or (Z)-N-Ac-FFVC-SOwas detected by LC-MS. No unacetylated cysteine con-jugates were detected in the urine from control orpretreated rats. The fraction of total N-Ac-DFEC excretedas the sulfoxide (sulfoxidation index), 1.5% in controls,was significantly increased 5-7-fold by DEX and PB anddecreased to zero by TAO. The excretion of 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid and the fraction of cys-teine S-conjugate excreted as 3,3,3-trifluoro-2-(fluoro-methoxy)propanoic acid (â-lyase index) were diminishedin DEX pretreated rats and, overall, not significantly orconsistently affected by enzyme induction and inhibition.Fluoride excretion was increased by DEX and PB anddecreased by TAO, although the changes were muchsmaller than those for mercapturates sulfoxidation.

Effect of Enzyme Induction and Inhibition onFDVE Toxicity in Vivo. Histological lesions in alltreatment groups were characterized by tubular degen-eration and necrosis (Figure 7). These changes, in allgroups, most prominently involved proximal convolutedtubules within the juxtamedullary cortex but varied inseverity and, to a lesser degree, in distribution (Table2). In severely affected animals, the tubular necrosis wascharacterized by widespread loss or fragmentation ofnuclei with cytoplasmic condensation. There was no

Figure 4. LC-MS analysis of the FDVE-mercapturic acid sulfoxide conjugate N-Ac-DFEC-SO excreted in rat urine. (A) ElectrosprayLC-MS chromatogram of a urine sample from a rat administered FDVE, monitored at m/z 360. Peaks 1 and 2 are presumptivediastereomers, but further identification was not pursued. (B) Mass spectrum of synthetic N-Ac-DFEC-SO, eluting at 9.9 min (peak1). (C) Mass spectrum of N-Ac-DFEC-SO detected in the urine of a DEX pretreated rat administered FDVE, eluting at 9.9 min (peak1). The mass spectrum of peak 2 at 10.4 min was similar for both panel B and panel C. The ions at m/z 360, 382, 318, and 130represent [M + H]+, [M + Na]+, [M + H - C2H2O]+, and [C5H8NO3]+, respectively.


inflammatory cellular response accompanying the necro-sis.

DEX and PB pretreatment significantly increasedFDVE nephrotoxicity, based on increased proteinuria(Table 2), and numerically increased although not sta-tistically significantly, scores for proximal tubular ne-crosis. TAO partially ameliorated FDVE diuresis, pre-vented proteinuria, and significantly decreased proximal

tubular necrosis. Urine osmolality was decreased byFDVE, and this was unaffected by any pretreatment.BUN changes were not significantly affected by anypretreatment. Animals pretreated with DEX or PB hadthe most severe lesions. PB pretreated animals had thegreatest extent of necrosis and degeneration. DEX pre-treated animals showed the greatest amount of cortexinvolved, which in some animals tended to include theentire cortex, and those with the most severe necrosisalso had the greatest increase in BUN. TAO pretreatedrats were least affected and displayed qualitatively aslightly different distribution of lesions. There was mild,multifocal involvement of the tubules immediately be-neath the capsule, as well as those in the juxtamedullaryzone. The severity of the tubular lesions was generallyconsistent with the degree of proteinuria (Table 2).

Effect of Enzyme Induction and Inhibition onFDVE Metabolism in Vitro. After the rats treated withP4503A modulators and FDVE were sacrificed, the liverswere removed and microsomes were prepared. The influ-ence of enzyme modulation on hepatic microsomal sul-foxidation of FDVE-cysteine conjugates was assessed(Table 3). As compared with control rat liver microsomes,rat pretreatment with PB and DEX significantly in-

Figure 5. Excretion of FDVE metabolites in urine. Ratsreceived 0.25 mmol/kg FDVE, and urine was collected in 24 hintervals thereafter. Each data point is the mean ( SD (n ) 6).Separate standards for (E)- and (Z)-N-Ac-FFVC were notavailable; hence, both isomers were quantified together. The (E)/(Z) peak area ratio was constant throughout the 7 day period(0.57 ( 0.08). Separate standards for the N-Ac-DFEC-SOdiastereomers were not available; hence, both were quantifiedtogether. The area ratio for peak 1:2 (1.6) on day 1 wasunchanged on subsequent days.

Figure 6. Time course of FDVE renal effects. Rats received0.25 mmol/kg FDVE, and urine was collected in 24 h intervalsthereafter. Each data point is the mean ( SD (n ) 6).


creased the formation of both N-Ac-DFEC-SO and (Z)-N-Ac-FFVC-SO; the latter quantified as the sum of thetwo presumed diastereomers. Sulfoxidation of both N-Ac-DFEC and (Z)-N-Ac-FFVC in liver microsomes from TAOpretreated rats was not different from untreated rats. PB,but not DEX or TAO, slightly altered the formation ratioof the two N-Ac-DFEC-SO diastereomers. FDVE-mercapturic acid sulfoxides formation was not observedin the absence of NADPH or protein.

Discussion

The first hypothesis tested in this investigation wasthat FDVE S-conjugates undergo sulfoxidation in rats invivo. The excretion in urine of N-Ac-DFEC-SO confirmsthe hypothesis, establishing a new pathway of FDVEbiotransformation in rats. N-Ac-DFEC-SO may theoreti-cally arise from sulfoxidation of N-Ac-DFEC or by N-acetylation of DFEC-SO. The former has been shown tooccur in rat liver, but not kidney, microsomes in vitro(27). The latter reaction has not been evaluated, butDFEC sulfoxidation occurred avidly and spontaneouslyin the absence of microsomes (27), and N-acetylation ofDFEC-SO is conceivable. Thus, both pathways of N-Ac-DFEC-SO formation may occur in vivo.

This is apparently only the second report of haloalkylS-conjugate sulfoxide formation in rats in vivo and thefirst ever to quantify the urinary excretion of sulfoxidemetabolites. The results demonstrate that sulfoxidationis quantitatively small (0.2% of the dose) but appears tobe a toxicologically significant route of biotransformationof FDVE and its S-conjugates. Previously, the hexachlo-robutadiene metabolite N-acetyl-S-(1,2,3,4,4-pentachlo-robutadienyl)-L-cysteine sulfoxide was qualitatively iden-tified in rat urine after the administration of hexachloro-butadiene but not quantified (33). Although S-(1,2-dichlorovinyl)-L-cysteine sulfoxide was proposed to be ametabolite of S-(1,2-dichlorovinyl)-L-cysteine and trichlo-roethylene, shown to be formed by rabbit liver mi-crosomes (34) and to be a highly reactive and a proximaltubular cell nephrotoxin in rats in vitro and in vivo (22,35) and proposed as an important determinant of trichlo-roethylene and S-(1,2-dichlorovinyl)-L-cysteine nephro-toxicity (36, 37), there has not been evidence presentedfor its in vivo formation, from either trichloroethyleneor S-(1,2-dichlorovinyl)-L-cysteine. Similarly, althoughS-(1,2,2-trichlorovinyl)-L-cysteine undergoes sulfoxidationby rabbit liver microsomes and this sulfoxide has beensuggested to contribute to nephrotoxicity (34), its in vivoformation has also not been shown. The present qualita-

tive and quantitative results with FDVE amplify theseprevious suppositions, providing evidence that haloalkeneS-conjugates sulfoxidation may be quantitatively minorbut toxicologically significant.

It is notable that only N-Ac-DFEC-SO, but not (E)- or(Z)-N-Ac-FFVC-SO, was found in rat urine after FDVEadministration. Analysis of urine samples by multireac-tion monitoring LC-MS/MS indicated the presence of (E)-and (Z)-N-Ac-FFVC-SO, but even with the added selec-tivity and sensitivity of LC-MS/MS, (E,Z)-N-Ac-FFVC-SO concentrations were at the limits of detection andbelow the limit of quantification. This contrasts with thesulfoxidation of both N-Ac-DFEC and (Z)-N-Ac-FFVC byrat liver microsomes observed presently (Table 3) andpreviously (27). It remains presently unknown whetherthis in vivo difference reflects a high rate of spontaneousformation of DFEC-SO and its subsequent N-acetylationor differences between N-Ac-DFEC-SO and (E,Z)-N-Ac-FFVC-SO in their formation, renal handling, renal excre-tion, or reactivity with tissue macromolecules. A prelimi-nary evaluation showed that DFEC-SO and N-Ac-FFVC-SO, but not N-Ac-DFEC-SO, reacted directly with GSH,indicating their potential reactivity (not shown). Thismay explain the detection of N-Ac-DFEC-SO but notN-Ac-FFVC-SO in rat urine.

Excretion in rat urine of the FDVE mercapturates and3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid was re-ported previously by our laboratory (18). Excretion of themercapturates in the present investigation following 0.25mmol/kg intraperitoneal FDVE was approximately 2-3-fold greater than after inhalation of 220 ppm FDVE. Incontrast, F-propionic acid excretion in the present inves-tigation was 30-fold less. The exact reason for thisdifference is unclear but relates to the revised analyticalmethod used herein, possibly to the different method forsynthesizing the diphenyldiazomethane derivatizing re-agent. Archived urine samples from the previous inves-tigation were reanalyzed using the newer assay andfound to be correspondingly lower and similar to thevalues reported herein.

The second purpose of this investigation was to deter-mine the influence of P4503A induction and inhibitionon FDVE sulfoxidation. DEX pretreatment to induceP4503A1/2 caused a 10-fold increase in the absoluteexcretion of N-Ac-DFEC-SO and a 7-fold increase in thesulfoxidation index (excretion of N-Ac-DFEC-SO relativeto total N-acetylated DFEC), after FDVE injection in vivo.Microsomes from livers removed from those same ratsafter FDVE injection showed a 5-fold increase in the rates

Table 1. Influence of P450 Modulation on FDVE Metabolism in Vivoa

control (n ) 7) DEX (n ) 8) PB (n ) 5) TAO (n ) 7)

N-Ac-DFEC (µmol/24 h) 4.8 ( 1.3 6.9 ( 2.4 7.9 ( 3.1 2.6 ( 0.9(E,Z)-N-Ac-FFVC (µmol/24 h) 2.8 ( 0.7 4.6 ( 1.5 3.6 ( 1.5 1.6 ( 0.7N-Ac-FFVC (Z/E) ratio 1.7 ( 0.3 2.2 ( 0.2d 1.4 ( 0.2 1.7 ( 0.5N-Ac-DFEC-SO (µmol/24 h) 0.074 ( 0.024 0.79 ( 0.25d 0.66 ( 0.28d 0.0 ( 0.0N-Ac-DFEC-SO (peak 1/peak 2) 1.5 ( 0.2 1.9 ( 0.2d 1.5 ( 0.1F-propionic acid (µmol/24 h) 0.94 ( 0.37 0.50 ( 0.16d 1.03 ( 0.18 0.41 ( 0.18d

fluoride (µmol/24 h) 33.5 ( 7.5 42.4 ( 15.1d 43.7 ( 16.1d 12.8 ( 8.3e

sulfoxidation index (%)b 1.5 ( 0.4 10.5 ( 2.7d 7.8 ( 1.3d 0.0 ( 0.0e

â-lyase index (%)c 11.1 ( 4.9 3.9 ( 0.3d 8.5 ( 2.8 9.4 ( 3.8a Male rats were pretreated with DEX (50 mg/kg, ip, for 3 days), PB (0.1% in drinking water for 10 days), TAO (500 mg/kg 2 h before

FDVE), or corn oil (control, 2 mL/kg for 3 days) prior to FDVE (0.25 mmol/kg, ip, in corn oil). Urine was collected for 24 h after FDVEinjection. The results are the means ( SD. The predose urine fluoride excretion was 0.9 ( 0.3, 1.6 ( 0.4, 1.0 ( 0.3, and 0.9 ( 0.2 µmol/24h in control, DEX, PB, and TAO animals. b N-Ac-DFEC-SO/(N-Ac-DFEC-SO + N-Ac-DFEC) × 100%. c F-propionic acid/(F-propionic acid+ N-Ac-DFEC-SO + N-Ac-DFEC + (E,Z)-N-Ac-FFVC) × 100%. d Significantly different from control (p < 0.05). e Significantly differentfrom DEX and PB (p < 0.05).


Figure 7. Light micrographs of kidney sections removed from rats treated with FDVE.


of N-Ac-DFEC and (Z)-N-Ac-FFVC sulfoxidation. This isconsistent with the high activity of both expressedP4503A1 and P4503A2 toward DFEC and (Z)-N-Ac-FFVC sulfoxidation in vitro (27). TAO pretreatmentabolished N-Ac-DFEC-SO excretion and the sulfoxidationindex after FDVE injection in vivo, although N-Ac-DFECand (Z)-N-Ac-FFVC sulfoxidation by microsomes fromlivers removed from TAO- and FDVE-treated rats wasundiminished. The reason for this difference is notapparent but may relate to dissociation of the cytochromeP450-TAO metabolite complex during microsome prepa-ration (38). Nevertheless, previous results demonstratedTAO (and also ketoconazole) inhibition of liver microso-mal FDVE mercapturates sulfoxidation (27). PB pre-treatment caused a 9-fold increase in absolute N-Ac-DFEC-SO excretion, a 5-fold increase in the sulfoxidationindex after FDVE injection in vivo, and a 6-fold increasein N-Ac-DFEC and (Z)-N-Ac-FFVC sulfoxidation in thelivers removed from these animals. This is consistentwith the previous report of increased N-Ac-DFEC and(Z)-N-Ac-FFVC sulfoxidation by microsomes from PB-induced (but not FDVE-treated) rats (27). PB can induceP4502B as well as P4503A; thus, a partial contributionof P4502B to enhanced sulfoxidation in PB-treatedanimals cannot be eliminated (24). Together, therefore,these induction and inhibition results provide strongevidence for the role of P4503A1/2 in the sulfoxidationof FDVE S-conjugates in rats in vivo.

P4503A1/2-dependent FDVE S-conjugates sulfoxida-tion in rats in vivo is consistent with previous resultsshowing a role for this enzyme in the sulfoxidation ofother haloalkyl S-conjugates. Sulfoxidation of the mer-capturatic acid conjugates of tetrachloroethylene andtrichloroethylene, N-acetyl-S-(1,2,2-trichlorovinyl)-L-cys-teine, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine, was increased inliver microsomes from PB and DEX pretreated rats andinhibited by TAO (25). Similarly, sulfoxidation of thehexachlorobutadiene metabolite N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine was also increased inPB- and DEX-induced rat liver microsomes and inhibited

by TAO in untreated rat liver microsomes (24). Flavinmonooxygenase, as well as P450, participates in thesulfoxidation of S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine (34).

The third purpose of this investigation was to deter-mine the influence of P4503A induction and inhibitionon FDVE nephrotoxicity and its relation to P4503A-dependent sulfoxidation. PB and DEX increased and TAOdecreased biochemical and histological evidence of FDVEnephrotoxicity. The concordance between P4503A inducerand inhibitor effects on FDVE S-conjugates sulfoxidationand nephrotoxicity, the lack of concordance betweennephrotoxicity and P4503A inducer and inhibitor effectson â-lyase-mediated FDVE-cysteine S-conjugates me-tabolism, and previous results showing greater proximaltubular cell cytotoxicity of DFEC-SO and (Z)-N-Ac-FFVC-SO as compared with their corresponding cysteine S-conjugates (28) together suggest that FDVE S-conjugatessulfoxidation contributes to FDVE renal toxification inrats in vivo. Lower rates of (Z)-N-Ac-FFVC sulfoxidationin rat kidney as compared with liver microsomes andeven lower rates of N-Ac-DFEC sulfoxidation (27) andthe absence of TAO effects on DFEC and N-Ac-DFECrenal tubular cell cytotoxicity (28) suggest that FDVES-conjugates sulfoxidation in the liver may be moreimportant than in the kidney. Although proteinuria isconsistently the most reliable indicator of FDVE toxicityand was altered by P4503A modulation, not all measuresof FDVE renal effects were similarly altered. Neverthe-less, this is not unusual. For example, â-lyase inhibitiondecreased FDVE proteinuria but did not decrease diure-sis (7).

Sulfoxidation has been considered an alternative,â-lyase-independent bioactivation pathway of other cys-teine S-conjugates. The sulfoxide of the cysteine conju-gate of trichloroethylene, S-(1,2-dichlorovinyl)-L-cysteinesulfoxide, was more nephrotoxic in rats in vivo thanS-(1,2-dichlorovinyl)-L-cysteine at equivalent doses, al-though the S-conjugate was slightly toxic in isolated ratproximal tubular cells (22). In human renal proximaltubular cells, necrosis and apoptosis were caused by

Table 2. Influence of P450 Modulation on FDVE Toxicitya

predose postdose

control(n ) 7)

DEX(n ) 8)

PB(n ) 5)

TAO(n ) 8)

control DEX PB TAO

BUN(mg/dL)

11 ( 2 5 ( 3 13 ( 2 22 ( 4 25 ( 10 27 ( 9 32 ( 14 36 ( 19

urine volume(mL)

5.9 ( 1.5 9.7 ( 2.2 4.2 ( 0.5 6.7 ( 1.2 23.4 ( 7.5c 18.8 ( 6.5c 21.1 ( 5.7c 8.1 ( 5.7b,d

urine protein(mg/24 h)

20 ( 3 49 ( 8b 30 ( 3 24 ( 5 46 ( 16c 103 ( 34b,c 67 ( 15b,c 17 ( 5b,d

urine osmolality(mosm/kg)

2290 ( 520 1650 ( 350b 2740 ( 100b 2010 ( 320 615 ( 153c 918 ( 226c 676 ( 132c 877 ( 246c

necrosis 1 (low),4 (high) (median)

2.5 ( 1.2 (2.5) 3.3 ( 0.7 (3.5) 3.4 ( 0.8 (4.0) 1.3 ( 1.1d (1.5)

a Animal pretreatments are the same as described in the legend to Table 1. Blood and urine samples were collected for 24 h before andafter FDVE. The results are means ( SD. b Significantly different from control (p < 0.05). c Significantly different from predose value (p< 0.05). d Significantly different from DEX and PB (p < 0.05).

Table 3. Influence of P450 Modulation on Hepatic Microsomal Sulfoxidation of FDVE-N-Ac-cysteine Conjugatesa

control (n ) 5) DEX (n ) 4) PB (n ) 4) TAO (n ) 4)

N-Ac-DFEC-SO (P1 + P2) (pmol/min/mg) 12.7 ( 2.3 77.2 ( 23.3b 83.1 ( 3.5b 23.3 ( 9.3c

N-Ac-DFEC-SO (P1/P2 ratio) 1.42 ( 0.05 1.44 ( 0.05 1.30 ( 0.06b 1.43 ( 0.08(Z)-N-Ac-FFVC-SO (pmol/min/mg) 35.1 ( 5.5 186 ( 36b 202 ( 23b 65.6 ( 17.0c

a N-Ac-DFEC or (Z)-N-Ac-FFVC (2 mM) were incubated with microsomes from livers of rats administered FDVE following pretreatmentwith DEX, PB, or TAO. b Significantly different from control (p < 0.05). c Significantly different from DEX and PB (p < 0.05).


S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, although theeffects of the parent cysteine S-conjugate were slightlygreater (36). Nevertheless, S-(1,2-dichlorovinyl)-L-cys-teine sulfoxide caused greater and more rapid depletionof both ATP and GSH (36). Like cysteine S-conjugatesulfoxides, sulfoxides of the mercapturatic acid conjugatesof trichloroethylene, N-acetyl-S-(1,2-dichlorovinyl)-L-cys-teine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine, werealso significantly more cytotoxic than equivalent concen-trations of their corresponding mercapturic acids in ratrenal proximal tubular cells (25). This also occurred withthe sulfoxide of the mercapturate of tetrachloroethylene,N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (25). N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxidecaused significantly greater loss of isolated rat renaltubular cell viability than the parent mercapturate (33)and caused significant renal tubular necrosis in rats invivo (26). Whereas the â-lyase inhibitor aminooxyaceticacid partially protected against S-(1,2-dichlorovinyl)-L-cysteine renal toxicity in vitro and in vivo, it failed toprotect against S-(1,2-dichlorovinyl)-L-cysteine sulfoxidetoxicity in both settings, indicating that sulfoxide neph-rotoxicity was â-lyase-independent (22). Similarly, thetoxicity of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sul-foxide and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sul-foxide was also not blocked by aminooxyacetic acid (25).This was consistent with the ability of S-(1,2-dichlorovi-nyl)-L-cysteine sulfoxide and S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide to react directly with cellular nonpro-tein thiols as an electrophile and Michael acceptor (34,35). Similarly, N-acetyl-S-(1,2-dichlorovinyl)-L-cysteinesulfoxide also reacts nonenzymatically with GSH (39).The R-methyl analogue of N-acetyl-S-(1,2,3,4,4-pentachlo-robutadienyl)-L-cysteine sulfoxide, which is not a sub-strate for renal â-lyase, also caused renal tubular necrosisin rats in vivo (26). Thus, both â-lyase-dependent me-tabolism of cysteine S-conjugates and P450- or flavinmonooxygenase-dependent sulfoxidation of cysteine S-conjugates or their mercapturates can contribute to thebioactivation and renal toxicity of several haloakenes.

The exact mechanism(s) of toxicity of FDVE S-conju-gates and associated bioactivation pathways in ratsremains unknown. The relative contribution, of â-lyase-dependent metabolism of FDVE-cysteine S-conjugatesas compared with P4503A-catalyzed sulfoxidation ofFDVE-cysteine S-conjugates and FDVE-mercapturates,to the bioactivation and renal toxicity of FDVE has notbeen identified. The mechanism of toxicity of FDVES-conjugates sulfoxides, whether as a direct acting elec-trophile or via some other mechanism, is not known. Thereactivity of the various haloalkyl cysteine-S and mer-capturic acid sulfoxides differs. For example, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide, and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide react directly with GSH and non-protein thiols (39). In contrast, N-acetyl-S-(2,2-di-chlorovinyl)-L-cysteine sulfoxide, N-acetyl-S-(1,2,2-tri-chlorovinyl)-L-cysteine sulfoxide, and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine sulfoxide are relativelyless reactive, requiring GST for reaction with GSH (39,40). As described previously, a preliminary evaluation ofFDVE S-conjugate sulfoxides reactivity showed thatDFEC-SO and the vinyl sulfoxide N-Ac-FFVC-SO (butnot N-Ac-DFEC-SO) reacted directly with GSH, indicat-ing their potential reactivity as Michael acceptors (notshown). Should a similar reactivity occur with tissue

macromolecules, it might indicate an additional mecha-nism of FDVE toxicity.

Elucidation of whether P4503A catalyzes other routesof FDVE or FDVE S-conjugates metabolism leading tomercapturates sulfoxidation and toxification, or is specificto sulfoxidation of the mercapturates, awaits additionalstudies on the metabolism and toxification of FDVES-conjugates. Although the present data suggest thatDEX, PB, and TAO alter FDVE nephrotoxicity by modu-lating P4503A-dependent FDVE S-conjugates sulfoxida-tion, other mechanism(s) cannot be excluded. DEX, PB,and TAO may affect P4503A-dependent biotransforma-tion of other FDVE S-conjugates or FDVE itself, leadingto altered mercapturate sulfoxide excretion. Verificationawaits studies on DEX, PB, and TAO effects on themetabolism and toxification of FDVE S-conjugates. CYP3Amodulation did influence other routes of biotransforma-tion, evidenced by altered fluoride excretion, although theidentity of these pathways and the significance withrespect to toxicity are not known. DEX and PB upregu-lation of FDVE nephrotoxicity may not be restricted totheir effects on P450. Participation of the renal tubularcell organic anion transport system in the renal uptakeof FDVE S-conjugates was previously shown by probenecidabolition of FDVE nephrotoxicity (7, 8), similar to itseffect on the transport and nephrotoxicity of several otherhaloalkyl compounds and their S-conjugates (41). Themercapturate N-acetyl-S-dichlorovinyl-L-cysteine (but notN-acetyl-S-pentachlorobutadiene-L-cysteine) was recentlyshown to be a substrate for the rat kidney organic aniontransporter OAT1 (42), and S-dichlorovinyl-L-cysteineand S-chlorotrifluoroethyl-L-cysteine were substrates forhuman and rabbit OAT1 (43). OAT1 is induced by DEX(44), and the related transporter OAT2 is induced by PBand DEX (45). Mrp2, which mediates biliary excretionof organic anions conjugated by phase 2 enzymes, issubstantially induced by DEX but not by PB (30). Incontrast, PB significantly induces hepatic mrp3, but notmrp1 or mrp2, and has no effect on renal mrp1, -2, or -3,as measured by mRNA (46). The identification of DEXand PB effects on renal transport of FDVE S-conjugatesrequires further investigation.

Most importantly, the existence and toxicological sig-nificance of FDVE S-conjugates sulfoxidation in humansremain unknown. Human liver microsomal P4503A andexpressed P4503A4 catalyzed the sulfoxidation of N-Ac-DFEC and (Z)-N-Ac-FFVC, and human kidney mi-crosomes catalyzed (Z)-N-Ac-FFVC sulfoxidation, al-though the overall rate of FDVE mercapturate sulfoxi-dation was less than in rat liver microsomes (27).Whether FDVE S-conjugates sulfoxidation occurs inhumans who are exposed to FDVE remains unknown.

In summary, this investigation shows that FDVES-conjugates undergo sulfoxidation in rats in vivo, andinduction and inhibition of P4503A correspondinglyincrease and decrease FDVE S-conjugates sulfoxidationand FDVE nephrotoxicity. Thus, FDVE S-conjugatessulfoxidation constitutes a second pathway of FDVEbioactivation and renal toxification in rats in vivo, inaddition to the previously demonstrated â-lyase-depend-ent bioactivation of FDVE-cysteine S-conjugates.

Acknowledgment. This work was supported by NIHGrants R01DK53765 and P30ES07033.


References

(1) Hanaki, C., Fujii, K., Morio, M., and Tashima, T. (1987) Decom-position of sevoflurane by soda lime. Hiroshima J. Med. Sci. 36,61-67.

(2) Frink, E. J., Jr., Malan, T. P., Morgan, S. E., Brown, E. A.,Malcomson, M., Gandolfi, A. J., and Brown, B. R., Jr. (1992)Quantification of the degradation products of sevoflurane in twoCO2 absorbents during low-flow anesthesia in surgical patients.Anesthesiology 77, 1064-1069.

(3) Morio, M., Fujii, K., Satoh, N., Imai, M., Kawakami, U., Mizuno,T., Kawai, Y., Ogasawara, Y., Tamura, T., Negishi, A., Kumagai,Y., and Kawai, T. (1992) Reaction of sevoflurane and its degrada-tion products with soda lime. Toxicity of the byproducts. Anes-thesiology 77, 1155-1164.

(4) Gonsowski, C. T., Laster, M. J., Eger, E. I., II, Ferrell, L. D., andKerschmann, R. L. (1994) Toxicity of compound A in rats. Effectof increasing duration of administration. Anesthesiology 80, 566-573.

(5) Gonsowski, C. T., Laster, M. J., Eger, E. I., II, Ferrell, L. D., andKerschmann, R. L. (1994) Toxicity of compound A in rats. Effectof a 3-hour administration. Anesthesiology 80, 556-565.

(6) Keller, K. A., Callan, C., Prokocimer, P., Delgado-Herrera, M. S.,Friedman, M. B., Hoffman, G. M., Wooding, W. L., Cusick, P. K.,and Krasula, R. W. (1995) Inhalation toxicology study of ahaloalkene degradant of sevoflurane, Compound A (PIFE), inSprague-Dawley rats. Anesthesiology 83, 1220-1232.

(7) Kharasch, E. D., Thorning, D. T., Garton, K., Hankins, D. C., andKilty, C. G. (1997) Role of renal cysteine conjugate b-lyase in themechanism of compound A nephrotoxicity in rats. Anesthesiology86, 160-171.

(8) Kharasch, E. D., Hoffman, G. M., Thorning, D., Hankins, D. C.,and Kilty, C. G. (1998) Role of the renal cysteine conjugate b-lyasepathway in inhaled compound A nephrotoxicity in rats. Anesthe-siology 88, 1624-1633.

(9) Dekant, W., Martens, G., Vamvakas, S., Metzler, M., and Hen-schler, D. (1987) Bioactivation of tetrachloroethylene. Role ofglutathione S-transferase-catalyzed conjugation versus cyto-chrome P-450-dependent phospholipid alkylation. Drug Metab.Dispos. 15, 702-709.

(10) Dekant, W., Urban, G., Gorsmann, C., and Anders, M. W. (1991)Thioketene formation from a-haloalkenyl 2-nitrophenyl disul-fides: Models for biological reactive intermediates of cytotoxicS-conjugates. J. Am. Chem. Soc. 113, 5120-5122.

(11) Pahler, A., Parker, J., and Dekant, W. (1999) Dose-dependentprotein adduct formation in kidney, liver, and blood of rats andin human blood after perchloroethene inhalation. Toxicol. Sci. 48,5-13.

(12) Jin, L., Baillie, T. A., Davis, M. R., and Kharasch, E. D. (1995)Nephrotoxicity of sevoflurane compound A [fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether) in rats: Evidence forglutathione and cysteine conjugate formation and the role of renalcysteine conjugate b-lyase. Biochem. Biophys. Res Commun. 210,498-506.

(13) Jin, L., Davis, M. R., Kharasch, E. D., Doss, G. A., and Baillie, T.A. (1996) Identification in rat bile of glutathione conjugates offluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a neph-rotoxic degradate of the anesthetic agent sevoflurane. Chem. Res.Toxicol. 9, 555-561.

(14) Spracklin, D., and Kharasch, E. D. (1996) Evidence for themetabolism of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinylether (compound A), by cysteine conjugate â-lyase in rats. Chem.Res. Toxicol. 9, 696-702.

(15) Iyer, R. A., Frink, E. J., Jr., Ebert, T. J., and Anders, M. W. (1998)Cysteine conjugate â-lyase-dependent metabolism of compoundA (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in humansubjects anesthetized with sevoflurane and in rats given com-pound A. Anesthesiology 88, 611-618.

(16) Iyer, R. A., and Anders, M. W. (1997) Cysteine conjugate â-lyase-dependent biotransformation of the cysteine S-conjugates of thesevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pen-tafluoro-1-propene (compound A). Chem. Res. Toxicol. 10, 811-819.

(17) Iyer, R. A., and Anders, M. W. (1996) Cysteine conjugate â-lyase-dependent biotransformation of the cysteine S-conjugates of thesevoflurane degradation product compound A in human, non-human primate, and rat kidney cytosol and mitochondria. Anes-thesiology 85, 1454-1461.

(18) Kharasch, E. D., Jubert, C., Spracklin, D., and Hoffmann, G.(1999) Dose-dependent metabolism of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A), an anesthetic deg-

radation product, to mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid in rats. Toxicol. Appl. Pharmacol.160, 49-59.

(19) Tong, Z., and Anders, M. W. (2002) Reactive intermediateformation from the 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived cysteine S-conjugate S-[2-(fluo-romethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine in pyridoxalmodel systems. Chem. Res. Toxicol. 15, 623-628.

(20) Iyer, R. A., Baggs, R. B., and Anders, M. W. (1997) Nephrotoxicityof the glutathione and cysteine S-conjugates of the sevofluranedegradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) in male Fischer 344 rats. J. Pharmacol.Exp. Ther. 283, 1544-1551.

(21) Park, S. B., Osterloh, J. D., Vamvakas, S., Hashmi, M., Anders,M. W., and Cashman, J. R. (1992) Flavin-containing monooxy-genase-dependent stereoselective S-oxygenation and cytotoxicityof cysteine S-conjugates and mercapturates. Chem. Res. Toxicol.5, 193-201.

(22) Lash, L. H., Sausen, P. J., Duescher, R. J., Cooley, A. J., andElfarra, A. A. (1994) Roles of cysteine conjugate b-lyase andS-oxidase in nephrotoxicity: Studies with S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine sulfoxide. J. Phar-macol. Exp. Ther. 269, 374-383.

(23) Werner, M., Guo, Z., Birner, G., Dekant, W., and Guengerich, F.P. (1995) The sulfoxidation of the hexachlorobutadiene metaboliteN-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine is cata-lyzed by human cytochrome P450 3A enzymes. Chem. Res.Toxicol. 8, 917-923.

(24) Werner, M., Birner, G., and Dekant, W. (1995) The role ofcytochrome P4503A1/2 in the sex-specific sulfoxidation of thehexachlorobutadiene metabolite, N-acetyl-S-(pentachlorobutadi-enyl)-L-cysteine in rats. Drug Metab. Dispos. 23, 861-868.

(25) Werner, M., Birner, G., and Dekant, W. (1996) Sulfoxidation ofmercapturic acids derived from tri- and tetrachloroethene bycytochromes P450 3A: A bioactivation reaction in addition todeacetylation and cysteine conjugate â-lyase mediated cleavage.Chem. Res. Toxicol. 9, 41-49.

(26) Birner, G., Werner, M., Rosner, E., Mehler, C., and Dekant, W.(1998) Biotransformation, excretion, and nephrotoxicity of thehexachlorobutadiene metabolite (E)-N-acetyl-S-(1,2,3,4,4-pen-tachlorobutadienyl)-L-cysteine sulfoxide. Chem. Res. Toxicol. 11,750-757.

(27) Altuntas, T. G., Park, B. S., and Kharasch, E. D. (2004) Sulfoxi-dation of cysteine and mercapturic acid conjugates of the sevo-flurane degradation product fluoromethyl-2,2-difluoro-1-(trifluo-romethyl)vinyl ether (compund A). Chem. Res. Toxicol. 17, 435-445.

(28) Altuntas, T. G., Zager, R. A., and Kharasch, E. D. (2003)Cytotoxicity of S-conjugates of the sevoflurane degradationproduct fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether(compound A) in a human proximal tubular cell line. Toxicol.Appl. Pharmacol. 193, 55-65.

(29) Ko, K.-Y., and Kim, J.-Y. (1999) Generation of diphenyldiazo-methane by oxidation of benzophenone hydrazone with Magtrieve.Bull. Korean Chem. Soc. 20, 771-772.

(30) Johnson, D. R., and Klaassen, C. D. (2002) Regulation of ratmultidrug resistance protein 2 by classes of prototypical microso-mal enzyme inducers that activate distinct transcription path-ways. Toxicol. Sci. 67, 182-189.

(31) Kostrubsky, V., Szakacs, J. G., Jeffery, E. H., Woods, S. G.,Bement, W. J., Wrighton, S. A., Sinclair, P. R., and Sinclair, J. F.(1997) Protection of ethanol-mediated acetaminophen hepato-toxicity by triacetyloleandomycin, a specific inhibitor of CYP3A.Ann. Clin. Lab. Sci. 27, 57-62.

(32) Yu, L. J., Drewes, P., Gustafsson, K., Brain, E. G., Hecht, J. E.,and Waxman, D. J. (1999) In vivo modulation of alternativepathways of P-450-catalyzed cyclophosphamide metabolism: im-pact on pharmacokinetics and antitumor activity. J. Pharmacol.Exp. Ther. 288, 928-937.

(33) Birner, G., Werner, M., Ott, M. M., and Dekant, W. (1995) Sexdifferences in hexachlorobutadiene biotransformation andnephrotoxicity. Toxicol. Appl. Pharmacol. 132, 203-212.

(34) Ripp, S. L., Overby, L. H., Philpot, R. M., and Elfarra, A. A. (1997)Oxidation of cysteine S-conjugates by rabbit liver microsomes andcDNA-expressed flavin-containing mono-oxygenases: Studieswith S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine. Mol. Phar-macol. 51, 507-515.

(35) Sausen, P. J., and Elfarra, A. A. (1991) Reactivity of cysteineS-conjugate sulfoxides: Formation of S-[1-chloro-2-(S-glutathio-nyl)vinyl]-L-cysteine sulfoxide by the reaction of S-(1,2-dichlo-rovinyl)-L-cysteine sulfoxide with glutathione. Chem. Res. Toxicol.4, 655-660.


(36) Lash, L. H., Putt, D. A., Hueni, S. E., Krause, R. J., and Elfarra,A. A. (2003) Roles of necrosis, apoptosis, and mitochondrialdysfunction in S-(1,2-dichlorovinyl)-L-cysteine sulfoxide-inducedcytotoxicity in primary cultures of human renal proximal tubularcells. J. Pharmacol. Exp. Ther. 305, 1163-1172.

(37) Krause, R. J., Lash, L. H., and Elfarra, A. A. (2003) Human kidneyflavin-containing monooxygenases and their potential roles incysteine S-conjugate metabolism and nephrotoxicity. J. Pharma-col. Exp. Ther. 304, 185-191.

(38) Pessayre, D., Larrey, D., Vitaux, J., Breil, P., Belghiti, J., andBenhamou, J. P. (1982) Formation of an inactive cytochromeP-450 Fe(II)-metabolite complex after administration of trolean-domycin in humans. Biochem. Pharmacol. 31, 1699-1704.

(39) Rosner, E., and Dekant, W. (1999) Stereoselective formation ofglutathione S-conjugates from halovinylmercapturate sulphox-ides. Xenobiotica 29, 327-340.

(40) Rosner, E., Muller, M., and Dekant, W. (1998) Stereo- andregioselective conjugation of S-halovinyl mercapturic acid sulfox-ides by glutathione S-transferases. Chem. Res. Toxicol. 11, 12-18.

(41) Dekant, W., Vamvakas, S., and Anders, M. W. (1994) Formationand fate of nephrotoxic and cytotoxic glutathione-S-conjugates:Cysteine conjugate â-lyase pathway. Adv. Pharmacol. 27, 115-162.

(42) Pombrio, J. M., Giangreco, A., Li, L., Wempe, M. F., Anders, M.W., Sweet, D. H., Pritchard, J. B., and Ballatori, N. (2001)

Mercapturic acids (N-acetylcysteine S-conjugates) as endogenoussubstrates for the renal organic anion transporter-1. Mol. Phar-macol. 60, 1091-1099.

(43) Groves, C. E., Munoz, L., Bahn, A., Burckhardt, G., and Wright,S. H. (2003) Interaction of cysteine conjugates with human andrabbit organic anion transporter 1. J. Pharmacol. Exp. Ther. 304,560-566.

(44) Bahn, A., Hauss, A., Appenroth, D., Ebbinghaus, D., Hagos, Y.,Steinmetzer, P., Burckhardt, G., and Fleck, C. (2003) RT-PCR-based evidence for the in vivo stimulation of renal tubularp-aminohippurate (PAH) transport by triiodothyronine (T3) ordexamethasone (DEXA) in kidney tissue of immature and adultrats. Exp. Toxicol. Pathol. 54, 367-373.

(45) Guo, G. L., Choudhuri, S., and Klaassen, C. D. (2002) Inductionprofile of rat organic anion transporting polypeptide 2 (oatp2) byprototypical drug-metabolizing enzyme inducers that activategene expression through ligand-activated transcription factorpathways. J. Pharmacol. Exp. Ther. 300, 206-212.

(46) Cherrington, N. J., Hartley, D. P., Li, N., Johnson, D. R., andKlaassen, C. D. (2002) Organ distribution of multidrug resistanceproteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic inductionof Mrp3 by constitutive androstane receptor activators in rats.J. Pharmacol. Exp. Ther. 300, 97-104.

TX049899E


role of cytochrome p4503a in cysteine s-conjugates sulfoxidatıon and the nephrotoxicity of the...

Documents