Chem. Res. Toxicol. 1989,2, 429-435
429
Enzymatic Defluorination and Metabolism of Fluoroacetate, Fluoroacetamide, Fluoroethanol, and (-)-erythro-Fluorocitrate in Rats and Mice Examined by '@Fand I3C NMR Berhane Teclet and John E. Casida* Pesticide Chemistry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, California 94720 Received May 23, 1989
Fluoroacetate administered intraperitoneally (ip) to rats and mice is defluorinated t o give fluoride ion evident in urine and kidney by 19F NMR. The use of [2-13C]-, [1,2-13C]-,and [ 1,2-14C]fluoroacetate,prepared from isotopically labeled glycine, combined with 13C NMR and TLC radioautography, respectively, reveals a complex mixture of urinary metabolites including an S-(carboxymethyl) conjugate complex in rats and mice and sulfoxidation products thereof in rats. Direct I3C NMR examination of the bile following treatment with [2-13C]fluoroacetate shows the presence of S-(carboxymethy1)glutathioneor a related conjugate and an 0-conjugate of fluoroacetate. Incubation of [13C]fluoroacetate with rat and mouse liver cytosol involves formation of S-([13C]carboxymethy1)glutathioneand fluoride ion. Fluorocitrate is also detected by 19FNMR examination of fluoroacetate incubations with mouse liver cytosol. Fluoroacetamide administered ip to rats and mice yields urinary fluoride ion formed via fluoroacetate which is liberated on hydrolysis by an organophosphate-sensitive amidase. 19FNMR chemical shifts of other metabolites of fluoroacetamide are consistent with fluoroacetohydroxamic acid in the liver of mice and fluorocitrate in the urine of rats. Fluoroethanol gives urinary fluoroacetate and fluoride ion in rats and mice and is converted to fluoroacetaldehyde by mouse and rat liver microsomes. (-)- and (+)-erythro-fluorocitrates administered ip to rats yield mostly the parent compounds in urine at 6 h with increasing amounts of fluoride ion thereafter. 19FNMR establishes that rat and mouse liver cytosols defluorinate (-)- but not (+)-erythro-fluorocitrate and pig heart aconitase also defluorinates (-)-erythro-fluorocitrate. Metabolic defluorination of fluoroacetate and its progenitors, fluoroacetamide and fluoroethanol, is therefore attributable to both conjugation of fluoroacetate with glutathione and conversion to (-)-erythro-fluorocitrate, which is both an inhibitor of and a substrate for aconitase. 13C NMR spectra of urine of rats and mice poisoned with fluoroacetate or (-)-erythro-fluorocitrate show elevated citrate and glucose and diminished urea consistent with disruptions in the tricarboxylic acid cycle and ammonia metabolism.
Introduction Fluoroacetate is of toxicological interest as a natural product, pesticide, and environmental pollutant. Fluoroacetamide and fluoroethanol, possible fluoroacetate precursors, are also highly toxic (1-3). Metabolic activation of fluoroacetate involves the "lethal synthesis" of 6)erythro-fluorocitrate ( 4 ) which in turn inhibits citrate metabolism by blocking its transport into mitochondria (5) and its breakdown by aconitase (6). The biochemical mechanisms by which fluoroacetate and/or fluorocitrate disrupt the central nervous system and heart are not adequately defined ( 1 , 2, 7). Fluoroacetate is readily metabolized in rats ( 2 , 4 , 8 )and undergoes defluorination by liver enzymes in the presence of GSH' (9-13). The present study makes novel use of '9F and 13CNMR spectroscopy to examine the metabolism of fluoroacetate and its progenitors and derivatives in rats and mice and in in vitro systems. Spectra of urine, bile, and tissue extracts are obtained and interpreted directly without iso*Towhom correspondence should be addressed at 114 Wellman Hall, University of California, Berkeley, CA 94720. t Present address: Agricultural Research Division, American Cyanamid Co., P.O. Box 400,Princeton, NJ 08540. 0893-228x/89/2702-0429$01.50/0
lation of individual metabolites, negating losses that would otherwise occur during separation or purification procedures. I3C NMR spectra also reveal changes in endogenous metabolites on poisoning with fluoroacetate and (4erythro-fluorocitrate.
Materials and Methods Spectroscopy. 'H,13C, and 19F NMR spectra were recorded with a Bruker WM-300 spectrometer at 300.14 ('H), 75.47 ('%), and 282.4 ('?I?) MHz with D20as the lock signal. Chemical shifts are referenced to internal 1% 3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropanoic acid (0 ppm) for 'H, internal 1% acetone (29.8 ppm) for lSC, and upfield from external 2,2,2-trifluoroethanol(O ppm) in D20for l9F. 13C NMR spectra were recorded with IH decoupling. 'H and 13C spectra were determined in 5-mm tubes and '?I? spectra in 10" tubes. '9 NMR spectra were recorded with a sweep width of 55.5 KHz, a pulse width of 15 ps (90°), and
* Abbreviations: FAB-MS, fast-atom-bombardment mass spectrometry; GLC-MS, gas/liquid chromatography-mass spectrometry; GSH, glutathione; ip, intraperitoneal(1y); NMR, nuclear magnetic resonance spectroscopy; PSCP, phenyl saligenin cyclic phosphonate; TCA, trichloroaceticacid; TLC, thin-layer chromatography. Compounds referred to as RSCH2CO2-are probably the GSH conjugate in bile and a mixture of cysteine, N-acetylcysteine, and related conjugates in urine. The nomenclature of the fluorocitrate stereoisomers is discussed in ref 7. 0 1989 American Chemical Society
430 Chem. Res. Toxicol., Vol. 2, No. 6, 1989 a repetition time of 2.3 s and processed with a line broadening of 3 Hz. The number of scans collected in 1qand 13Cexperiments was normally about 1000 for enzyme studies and 9000-24 OOO for in vivo metabolite mixtures. The 19Fand 13Cchemical shifts of reference compounds and metabolitesare pH dependent and vary by 6 f0.05 to f1.9 over the pH range encountered with urine and tissue extracts. Relative proportions of urinary metabolites in the 19Fspectra were estimated by integration relative to extemal trifluoroethanol (7 pg) (14);because of low signal to noise ratios in some samples and the slight variation in Tl of the metabolites, these data should be considered only as approximate. FAB-MS was accomplished from a thioglycerol/glycerol matrix with a Kratos MS-50 magnetic sector spectrometer in the electron impact mode in the Chemistry Department of the University of California, Berkeley. Chemicals. The organofluorine compounds used were free of fluoride ion as determined by 'gF NMR. Sodium fluoroacetate, fluoroacetamide, fluoroethanol,and barium (f)-fluorocitrate were from Sigma Chemical Co. (St. Louis, MO). Barium fluorocitrate was converted to sodium fluorocitrate free of fluoride ion before use. (-)- and (+)-erythro-fluorocitrate (tris salt with cyclohexylamine) and epoxy-cis-aconitate were kindly provided by Ernest Kun and Jerome McLick of the Laboratory for Environmental Toxicology and Chemistry at San Francisco State University. [2-'%]- and [1,2-'%]glycine and [1,2-13C]bromoaceticacid (each 99.5 atom % 13C)were obtained from Isotec, Inc. (Miamisburg, OH), and [ 1,2-"C]glycine hydrochloride (99% radiochemical purity, 55 mCi/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Pyridinium poly(hydrogen fluoride) (70%) was from Aldrich Chemical Co. (Milwaukee, WI). PSCP, an amidase inhibitor, was prepared as previously described (15). Fluoroacetaldehyde. Treatment of fluoroethanol with equimolar pyridinium chlorochromate in dichloromethane for 2 h at 25 OC gave 3-10% fluoroacetic acid and 1-3% fluoroacetaldehyde ['qNMR 6 154.1 (dt, J = 48 and 10 Hz)], which were examined as a mixture and not individually isolated. In this procedure and all those indicated below, care must be exercised to avoid any possible exposure to fluoroacetic acid and its derivatives by using suitable hoods and trapping procedures. [2-'%]-, [1,2-'%]-, and [1,2-"C]Fluoroacetate. Appropriately labeled glycine as above was converted to fluoroacetate via diazotization with NaN0, and then treatment with pyridinium poly(hydrogen fluoride) reagent at room temperature (16). The procedure is illustrated for [13C]glycinebecause of difficulties in obtaining pure product. [13C]Glycine(500 mg; dried a t 100 "C in a vacuum oven) was dissolved in pyridinum poly(hydrogen fluoride) (70%, 13 mL) in a polyolefin bottle and stirred at 0-5 "C under N2 for 0.5 h. NaNO, (10 mmol, dried as above) was added in three portions over 20 min. The mixture was stirred for an additional 1h, quenched with ice-cold water, and extracted with diethyl ether in a polyolefin separatory funnel. The ether was washed with saturated brine, dried (MgSO,), and distilled off at reduced pressure. The residue was taken up in deionized water and distilled at reduced pressure (5 mmHg) by use of a Kugelrohr microdistillationapparatus (oven temperature 100 "C) with the receiver bulb cooled (dry ice/acetone). The distillate thus obtained was dissolved in water, adjusted to pH 11 with aqueous NaOH, and extracted with ether to remove the pyridine. The solution was then treated with saturated aqueous MgS04 and the precipitate of MgF, removed by centrifugation. The supernatant was acidified with HC1 to pH 1, saturated brine added, and the solution extracted with ether. The combined ether extract was washed with brine, dried (MgSOJ, and fiitered and the solvent removed. The residue was taken up in deionized water and neutralized to pH 7 with 5% NaOH and the solvent removed on the rotary evaporator to give [2-13C]fluoroacetate(70 mg, 10%): lgF NMR 6 -140.3 (dt, J = 176 and 46 Hz); 13CNMR 6 79.0 (d, J = 178 Hz). [1,2-13C]Fluoroacetate(100 mg, 15%): 13CNMR 6 78.7 (dd, J = 178 and 57 Hz), 175.7 (dd, J = 58 and 19 Hz). The method described for [13C]glycinewas reduced proportionately in scale for [ 1,2-14C]glycine(1 mCi, 75 mg after adding unlabeled compound) in 0.01 M HC1 which was lyophilized to dryness. [1,2-14C]Flu~r~acetate (6 mg, 670, 1.0 mCi/mmol) was obtained with a radiochemical purity of >95% based on TLC on 0.25-mm silica gel chromatoplates developed with 1-propanol-30%
Tecle and Casida aqueous ammonia-water (6:3:1) (Rf = 0.46). Fluoroacetohydroxamic Acid. The hydroxamic acid was prepared from fluoroacetic acid, which was in turn obtained by acidifying an aqueous solution of sodium fluoroacetate with H#04, extracting into ether, and drying (MgSO,). The fluoroacetic acid was refluxed for 1h with excess SOCl, which was subsequently distilled off and the residue then taken up in dry hexane to which was added excess N,N,O-tris(trimethylsily1)hydroxylamine(17). After the solution was stirred for 20 min, the solvent was removed and the resulting product taken up and held in hexane overnight, and following evaporation the residue was dissolved in water and filtered. 19FNMR 6 -148.3 (t, J = 48 H). S-(Carboxymethy1)glutathione.A solution of GSH in water was adjusted to pH 9 with NH40H and then treated with an aqueous solution of equimolar sodium iodoacetate or sodium [1,2-13C]bromoacetateand stirred overnight at room temperature. The pH was then adjusted to 7 with 5% NaOH, and the spectra were recorded. S-(Carboxymethyl)-GSH: FAB-MS 366 (MH+), 432 (MH+ + 3 Na+);'H NMR 6 2.36 (m, 2 H), 2.71 (m, 2 H), 3.10 and 3.20 (d of AB quartet, 2 H), 3.35 and 3.45 (AB quartet, 2 H), 3.94 (m, 3 H), 4.75 (dd, 1 H); 13C NMR 6 25.6, 30.8, 33.2, 36.6, 42.9, 52.4, 53.5, 171.5, 173.5, 174.2, 175.7, 177.0. S-([1,2-13C]Carboxymethyl)-GSH: 13CNMR 6 36.5 (d, J = 52 Hz), 176.8 (d, J = 52 Hz). Treatment of this compound in water with excess magnesium monohydroperoxyphthalate for 30 min at 25 OC yields two products with very similar chemical shifts for the methylene carbons [6 58.3 (d, J = 52 Hz) and 58.2 (d, J = 52 Hz)] and identical values for the carbonyl carbons [6 170.5 (d, J = 52 Hz)] as appropriate for diastereomeric sulfoxides plus an additional product with the S-methylene carbon at 6 48.0 (d, J = 52 Hz). 19F Metabolites of Fluoroacetate, Fluoroacetamide, Fluoroethanol, and Fluorocitrate. Male albino rats (180-200 g) and male albino mice (25-30 g) were obtained from Simonsen Laboratories, Inc. (Gilroy, CA). Aqueous solutions of the test compounds were administered either ip or orally to rats but only ip to mice. The treated animals were held in metabolism cages which allowed the separate collection of urine and feces, and they were given free access to food (Purina Rodent Chow) and water during the holding period. Special studies with rats involved pretreatment with antibiotics or candidate antidotes. For antibiotic treatment, the rats were fed 15 mg of kanamycin sulfate and 15 mg of lincomycin hydrochloride daily with their food for 4 days and an additional 1mg of each of the antibiotics/mL of their drinking water ad libitum (18). On the fifth day 50 mg of each of the antibiotics was administered orally followed by either oral or ip administrationof (f)-fluorwitrate. Candidate antidotes coadministered ip with fluoroacetate (2-5 mg/kg) were sodium acetate and ethanol (each at 2 g/kg) (I) and GSH (60-200 mg/kg). In antidote studies with fluoroacetamide the rats received ip treatments of PSCP (20 mg/kg with 500 p L of methoxytriglycol as the carrier vehicle) or GSH (160 mg/kg) 1h before the toxicant. For urinalysis, pooled samples from three rats or four mice (0.5-5 mL) were lyophilized to semidryness and the residue was dissolved in deionized water (3 mL). The supernatant from centrifugation was transferred to the NMR tube. Liver, kidney, or brain for analysis was homogenized in water-acetone (51) (5 mL/g) with a Polytron (Kinematica GmbH, Luceme, Switzerland) and the supernatant from centrifugation (30 min, 35000g) treated as with the urine samples. Labeled Metabolites from [2-13C]-, [ 1,2-13C]-, and [ 1,2'%]Fluoroacetate and S-([1,2-13C]Carboxymethyl)-GSH in Rats and Mice. Rats and mice were treated ip at 2 mg/kg with [1,2-'3C]fluoroacetate or rats with S-([1,2-lsC]carboxymethyl)-GSH at 2 mg/kg for collection of their urine a t 6 h. For %-labeled biliary metabolites, rats were anesthetized with sodium phenobarbital (200 mg/kg, ip), and the bile ducts were cannulated. [2-13C]Fluoroacetateor unlabeled fluoroacetatewas administered ip at 2 mg/kg, and the bile was collected for 3 h with anesthesia throughout. The urine or bile was lyophilized to semidryness and the residue dissolved in 1mL of DzO,filtered, and subjected to 13CNMR analysis. In studies with [1,2-14C]fluoroacetatethe rats were treated ip at 0.25,2, or 4 mg/kg (by using a mixture of 14C and unlabeled fluoroacetate at the two highest doses). Radiocarbon in urine and the tissue extracts (prepared as above) was determined by liquid scintillation counting. Urine from the 0-6-h period was analyzed by silica gel TLC with 1-butanol-30%
I9Fand 13C NMR Studies on Fluoroacetate Metabolism
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 431
Table I. "F NMR Analyses of the Urine, Liver, and Kidney of Rats 4-6 h after Intraperitoneal Administration of Fluoride, Fluoroacetate, Fluoroacetamide, Fluoroethanol, and Fluorocitrate .. urinary ISF, Z of admincompound' 19FNMR chemical shifts, administered, mg/kg* detected urine liver kidney istered dosec sodium fluoride (0.73) fluoride -41.1 (9) 37 -139.8 (t) -139.7 (br) 1 sodium fluoroacetate .(5) -140.0 (t) fluoroacetate -45.1 (s) 2 -43.9 (8) fluoride -147.7 (t) -147.8 (t) 9 -147.6 (br) fluoroacetamide fluoroacetamide (14) -147.8 (br) fluoroacetohydroxamic acid -140.8 (t) -140.1 (br) 1 fluoroacetate -139.8 (t) -43.9 (8) -42.0 (8) 1 fluoride -39.2 (8) -141.3 (t) -140.8 (t) 10 -139.9 (t) fluoroacetate fluoroethanol (8) -41.4 ( 8 ) 3 fluoride 32 -113.7 (d) fluorocitrate (-)- and (+)-erythro-fluorocitrate( 5 ) 1 -42.0 ( 8 ) fluoride '9'6
a %' NMR chemical shifts (6) for the standard compounds in water relative to trifluoroethanol external standard are (d and t, J = 48 Hz) as follows: sodium fluoride, -45.5 ( 8 ) ; sodium fluoroacetate, -140.3 (t); fluoroacetamide, -148.2 (t);fluoroacetohydroxamicacid, -148.3 (t); fluoroethanol, -147.8 (m); (-)- and (+)-erythro-fluorocitrateas tris salta with cyclohexylamine,-113.2 (d) and -112.8 (d), respectively; sodium (&)-fluorwitrate,-113.3 (d). *Sodiumfluoride and (+)-erythro-fluorocitrateat asymptomatic doses. Fluoroacetate,fluoroacetamide, fluoroethanol, and (-)-erythro-fluorocitrate at approximate LD, doses: the first three compounds give severe convulsions initiated at about 0.5 h and progressive muscular weakness thereafter; the last one produces severe convulsions repeatedly from 0.5 to 6 h. 'Detection and quantitation are based on two or three replicates in each case. Urine collected for 6 h after treatment with sodium fluoride and (+)erythro-fluorocitrateand for 4-6 h after treatment with the other more toxic compounds. Tissues are examined on death or sacrifice. The same compounds are detected in rat and mouse urine and tissues at 6 h except for liver products, in mice but not rata, as follows: unknowns 6 -29.4 (br s) and -30.8 (br s) from fluoroacetate treatment; unknown 6 0.9 (br, s) and unmetabolized fluoroethanol 6 -148.6 (m) from fluoroethanol treatment.
aqueous NH,OH-water (5l:l)and radioautography. Hepatic Cytosol and Microsomes. Rat and mouse liver microsomes and cytosol were prepared by centrifugation of 20% (w/v) liver homogenate5 in phosphate buffer (0.1 M, pH 7.4)at 12000g for 10 min. The supernatant was then centrifuged a t 105000g for 1 h. The microsomal pellet was washed once by resuspension and recentrifugation in the same cold buffer. The cytosol fraction was used fresh or following storage (-80 "C) at 7 mg of protein/mL. Protein was determined by the method of Bradford (19). Incubation mixtures with the microsomal fraction consisted of 12 mg of protein, 3 pmol of organofluorinesubstrate, and 0 or 20 pmol of NAD+, NADH, NADP+, or NADPH in 2.5 mL of buffer. Following incubation for 3 h at 37 "C, the mixture was treated with 50% TCA (100 pL) or 25% perchloric acid (100 pL), and after centrifugationthe supernatant was neutralizedwith NaOH and transferred to the NMR tube for analysis. Incubation mixtures with the cytosol consisted of 16 mg of protein, 3 rmol of organofluorine substrate, and GSH (10 mM unless indicated otherwise) in 3 mL of buffer. Cytosol incubations carried out for 3 h a t 37 "C were terminated by adding TCA or perchloric acid and analyzed as above. Aconitase Incubations. Aconitase from pig heart (Sigma, 15 mg, 0.45 unit) was activated by dissolving in 0.1 M Tris buffer (2 mL, pH 7.4) containing 1mM ferrous ammonium sulfate and 50 mM L-cysteine hydrochloride and incubating at 0 "C for 1h. (-)-erythro-Fluorocitrate (100 pg as tris salt with cyclohexylamine) was then added with incubation for 1 h at 25 "C prior to direct lgF NMR spectroscopy. The possible contribution of ferrous ion in influencing fluoride ion solubility and NMR sensitivity was not examined.
C
4
d F%H2''CO;
'
I
180 X0.25
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160
f
I
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I
I
120
140
I
100
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I
80
I
60
I
~
~
I
20
40
PPm rat control
I
mouse F'3CH,'3CO;
U
1
180
160
140
1
120
1
1
100
1
1
80
1
60
1
t
40
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1
~
1
20
ppm
Results Fluoroacetate Metabolites in Rats and Mice. Fluoride ion is n o t evident b y 19FNMR in the urine of untreated rats and mice. On ip administration of sodium fluoride to rats, about 37% appears in the urine as fluoride within 6 h (Table I). Fluoroacetate treatment gives small
Figure 1. NMR spectra of urine of rata and mice 0-6 h after ip administration of [ 1,2-13C]fluoroacetateat 2 mg/kg showing the presence of lSC-enrichedthiol conjugate(s) in rats and mice and fluoroacetate-inducedeffects on endogenous metabolites,i.e., elevation of citrate (c) and decrease of urea (u) in rata and mice and increase in glucose (g) in rata. Control urine is from untreated rats and mice.
amounts of both the parent compound and fluoride ion but no other detectable fluorine-containing compounds in the urine of rats and mice (Table I). Fluoroacetate is detected in the liver and kidney (Table I) and the brain of rats and mice 4-6 h after treatment. Two unidentified fluoroacetate metabolites are observable from the liver of mice but not rats (Table I). Pretreatment of rats with antidotal levels of sodium acetate and ethanol greatly increases the amount of unmetabolized fluoroacetate in urine
(8% at 6 h) whereas GSH coadministered at 60-200 mg/kg is not antidotal and does not change t h e urinary products detected b y 19FNMR. Fluoroacetate greatly alters the 13C NMR pattern of urinary metabolites primarily due to metabolic changes discussed later. In addition, there are 13C-enriched metabolites of fluoroacetate itself. [ 1,2-13C]Fluoroacetate yields two defluorinated metabolites in r a t urine evident
~
~
I
432 Chem. Res. Toxicol., Vol. 2, No. 6,1989 F'3CH2CO;
-
GS''CH,CO; 37.4 FCH2C02gIuc
I
1
FCH2CO; + GSH c d 140.1 81.5 179.5 a
-
FCH,CO; 'F NMR
Tecle and Casida GSCH&O; e f 37.4 179.5
+
Fb -42.8
al
no GSH
GSH control
I
F"CH,"CO; 13C NMR
no GSH
Figure 2. 13C NMR spectra of bile of rata showing 0-and Sconjugates of [2-18C]fluoroacetate+3 h following ip administration at 2 mg/kg. Control bile is from a rat treated similarly with
unlabeled fluoroacetate.
by their characteristic 13C-13Ccoupling [6 37.4 (d, J = 52 Hz) and 58.4 (d, J = 52 Hz)] and one in the urine of mice [6 36.8 (d, J = 54 Hz)] (Figure 1). These 13C-labeled metabolites were compared to those excreted 0-6 h following ip administration to rats of S-([1,2-13C]carboxymethyl)-GSH (2 mg/kg) (prepared from [ 1,2-13C]bromoacetate and GSH). The latter GSH conjugate yields five observable S-(carboxymethyl) conjugates in urine, three of which are major [6 37.3 (d, J = 54 Hz), 37.6 (d, J = 52 Hz), and 37.7 (d, J = 54 Hz)] and two of which are minor [6 48.8 (d, J = 54 Hz) and 58.4 (d, J = 52 Hz)]. These minor metabolites appear to be sulfoxidized derivatives since products of similar chemical shifts are formed on peracid oxidation of S-(carboxymethyl)-GSH. The two urinary metabolites of [1,2-13C]fluoroacetatein rata and one in mice are therefore probably an RSCHzCOz-conjugate complex (6 37.4) and oxidation product(s) thereof (6 58.4). The C-C coupling constant for the RSCH2CO2conjugate complex in the mouse spectrum appears to differ slightly from that in the rat spectrum possibly because of variations in the media or overlapping signals of endogenous compounds. The bile of rats treated ip with [2-13C]fluoroacetate(2 mg/kg) compared with that of control rats receiving the same dose of unlabeled fluoroacetate shows two 13CNMR signals attributable to two new 13C-enrichedmetabolites (Figure 2). The major metabolite at 6 37.4 ppm is tentatively assigned as S-(carboxymethyl)-GSH because it gives a singlet resonance with a chemical shift identical with that obtained on addition of the synthetic standard to bile. The other metabolite at 6 78.5 ppm contains fluorine since it appears as a doublet with a characteristic l9F-l3C coupling (J= 176 Hz). As biliary products these compounds are considered to be conjugates and are most likely S-(carboxymethy1)-GSH and the O-glucuronide of fluoroacetate, respectively (Figure 2). Minor metabolites may be obscured by the small amount of toxicant administered and the large number of endogenous signals. Although not illustrated and in contrast to the urine, fluoroacetate does not alter the balance of endogenous metabolites in bile. The cumulative percentage of urinary radiocarbon following ip administration of [ 1,2-14C]fluoroacetate(0.25 mg/kg) to rats is 6% at 4 h, 32% at 24 h, and 45% at 72 h. TLC of the 0-24-h urine in the butanol-NH40H/water system revealed at least nine metabolites, with the major ones in the R,range of 0.5-0.7. Radiocarbon in the tissues of rata was determined as a percentage of the administered dose on sacrifice at 4 h after the 4 mg/ kg dose, 24 h after
. dif
F"CH,CO;
=A
Figure 3. IDFand
NMR spectra of incubation mixtures of
fluoroacetate,GSH, and liver cytosol showing fluoride ion and S-(carboxymethyl)-GSHformed on conjugation of fluoroacetate with GSH. Fluoroacetate is unlabeled or is [2-13C]- or [1,2'3c]fluoroacetate. Rat cytosol is used for the ?F studies and mouse cytosol for the '3c investigationswith fluoroacetate at 1mM, GSH at 10 mM, and incubations for 3 h at 37 "C. Protein is precipitated with TCA for the '9 studies and perchloric acid for the I3C investigations.
the 2 mg/kg injection, and 72 h after the 0.25 mg/kg treatment. Percentage values obtained at 4,24, and 72 h, respectively, are 1.7,0.8, and 0.6 for brain, 1.6, 1.2, and 0.9 for heart, 2.5,3.0, and 0.7 for kidney, and 8,4, and 0.5 for liver. Fluoroacetate Metabolites in Liver Cytosol. Fluoroacetate forms a conjugate with GSH releasing fluoride ion on incubation with rat or mouse liver cytosol on the basis of both 19F NMR analysis and 13C NMR identification of the defluorination product (Figure 3). The 13C NMR spectrum of the incubation mixture with mouse liver cytosol and GSH shows an S-methylene signal at 6 37.4, appearing as a singlet for the metabolite of [213C]fluoroacetateand a doublet (J = 53 Hz) for the metabolite of [ 1,2-13C]fluoroacetate(Figure 3). This metabolite is formed only on fortification with GSH and is tentatively assigned as the GSN or cysteine conjugate since its chemical shift is similar to that of S-([1,2-13C]carboxymethyl)-GSH. The same metabolite is observed with rat liver cytosol. Fluoride ion liberation from fluoroacetate by rat and mouse liver cytosol increases with both the incubation time (0, 1, 3, 6, 9, and 24 h) at 10 mM GSH and the GSH concentration (0, 1,3,6, and 9 but not the saturating level of 20 mM) with 3-h incubation. Mouse liver cytosol has detectable defluorinase activity even without added GSH and is in general more active than rat liver cytosol, giving complete defluorination of 3 mM fluoroacetate within 24 h. Mouse but not rat liver cytosol retains its defluorinase activity when stored at -80 "C for 3 weeks, without stabilization by added thiol compounds. GSH cannot be replaced by 10 mM cysteine, 2-mercaptoethanol, or dithiothreitol in the rat and mouse cytosol systems.
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 433
lgF and 13C NMR Studies on Fluoroacetate Metabolism
FCH,CH,OH a -147.6
FCH~CO;
b
.-t
-
FCH~CHO -+ FCH,CO; b -153.8 c -139.9
Fd -41.4
microsomes NADPH
(-147.8)
urine C
Figure 4. NMR spectra of the urine and liver of rata 0-4 and 6 h, respectively, after ip administration of fluoroacetamide at 14 mg/kg. Chemical shifts for urinary samples are followed in parentheses by those for liver samples.
Fluoroacetate defluorination by rat liver cytosol is not inhibited by 2 mM (-)-erythro-fluorocitrate or 10 mM epoxy-cis-aconitate, suggesting that they do not compete for the same enzymatic site. GSH S-transferase from rat liver (300 units, Sigma) or equine liver (600 units, Sigma) does not detectably defluorinate fluoroacetate in pH 7.4 phosphate buffer with GSH at 10 mM and incubation for 3 h at 37 "C. Enzymes in mouse liver cytosol convert a small portion of the fluoroacetate to a product with a '9F chemical shift [6 -113.5 (d, J = 48 Hz)] indicative of fluorocitrate, presumably (-)-erythro-fluorocitrate. Fluoroacetamide Metabolites in Rats and Mice and in Liver Cytosol or Microsome Incubations. Fluoroacetamide administered ip to rats at 14 mg/kg gives unmetabolized fluoroacetamide and trace amounts of fluoroacetate and fluoride ion in the urine, liver, and kidney (Figure 4 and Table I). An additional metabolite, detected in organic extracts of the liver from rats but not mice, has the same 19F chemical shift as that of synthetic fluoroacetohydroxamic acid (Figure 4 and Table I) and is enhanced in admixture. On pretreatment of rats with PSCP (20 mg/kg), fluoroacetamide is less toxic and is the only excretion product within up to 48 h. A trace of fluoride ion and fluoroacetic acid are noted between 48 and 72 h. Coadministration (ip) of GSH (180 mg/kg) with fluoroacetamide (60 mg/kg) prolongs the survival time of the rats, greatly lowers the conversion to urinary fluoroacetate and fluoride ion, and leads to a new trace urinary metabolite at 6 -114.1, tentatively assigned as fluorocitrate. Fluoroacetamide is not detectably hydrolyzed or defluorinated by rat or mouse liver cytosol or microsomes. Fluoroethanol Metabolites in Rats and Mice and in Liver Cytosol or Microsome Incubations. The major urinary metabolites of fluoroethanol(8 mg/ kg, ip) in rats are fluoroacetate and fluoride ion, accounting for 10 and 3%, respectively, of the dose at 6 h (Figure 5 and Table I). Fluoroacetate is readily evident by 19FNMR in extracts of the liver and kidney of treated rats and mice (Table I), and fluoroethanol is also detected in trace amounts in the liver of mice. In addition, mouse but not rat liver contains a trace of unidentified metabolite (Table I). Fluoroethanol is oxidized to fluoroacetaldehyde by rat liver microsomes with or without added NADPH (Figure 5), NAD+, or NADP+. Cofactor requirement is more evident with the less active mouse microsomes in which case the highest fluoroacetaldehyde yields are with NADPH or NAD+ compared with NADP+, NADH, or no cofactor. Mouse and rat liver cytosol (alone or with any of the
Figure 5. 19F NMR spectra of urine of rata 6 h after ip administration of fluoroethanol at 8 mg/kg and of a mixture of fluoroethanol(1.2 mM), microsomes (12 mg of protein),and NADPH (8 mM) incubated for 3 h at 37 "C. (-)-erythrofluorocitrate a 113.5
urine
Ib
6-24h
-
Fb -42
cytosol
cytosol
+ GSH
a
a
Figure 6. IgF NMR spectra of urine of rata up to 48 h after ip administration of (-)-erythro-fluorocitrateat 5 mg/kg and of a mixture of (-)-erythro-fluorocitrate with rat cytosol (16 mg of protein alone or with 10 mM GSH) incubated for 9 h at 37 "C or with 0.45 unit of pig heart aconitase incubated for 1 h at 25 "C. The insert in the aconitase spectrum is a 5X expansion. No fluoride ion is released within 24 h in control incubations lacking cytosol or aconitase. aforementioned cofactors) are not active in oxidizing fluoroethanol. NADP+-dependent alcohol dehydrogenase from Thermoanaerobium brockii (60 units with 1 mM substrate and 1 mM NADP+) also failed to convert any fluoroethanol to fluoroacetaldehyde in 2 h a t 40 "C. Fluorocitrate Metabolites in Rats and in Liver Cytosol and Aconitase Incubations. (-)- and (+)erythro-fluorocitrates administered ip at 5 mg/ kg give mostly the parent compound (32%) in the 4-6-h urine, but the major 19Fsignal in the 6-48-h urine is due to fluoride ion (Figure 6 and Table I). Similar results are obtained with (&)-fluorocitrate administered ip or orally a t 16 mg/ kg. No fluorine-containing metabolite other than fluoride ion is detected with (-)- and (+)-erythro-fluorocitrate. In contrast, (&)-fluorocitrate yields two minor fluorine-containing metabolites at 6 -108.1 (d, J = 48 Hz) and -115.3 (d, J = 48 Hz). The antibiotic treatment blocks the conversion of both orally and ip administered (&)fluorocitrate to fluoride ion and the other urinary metabolites.
434 Chem. Res. Toxicol., Vol. 2, No. 6, 1989 RSO,CH,CO; r-
[FCH,CONHOH]
Tecle and Casida Fluoroacetate is defluorinated by a GSH-dependent enzyme(s) in mouse and rat liver cytosol as shown here by NMR techniques and previously by other analytical methods (10-13). The defluorinase(s) involved appears (appear) to differ between rats and mice in activity, GSH requirement, and stability. S-(Carboxymethyl)-GSH formed on fluoroacetate conjugation probably undergoes further reactions to give the cysteine conjugate [previously identified by GLC-MS (9)] and mercapturic acid derivative which undergo some sulfoxidation prior to excretion (22).
Figure 7. Partial metabolic pathways for fluoroacetic acid, fluoroacetamide, fluoroethanol, and (-)-erythro-fluorocitratein rats and mice and in in vitro systems that involve (a) enzymes condensing fluoroacetyl-CoAwith oxaloacetak, (b) aconitase, liver cytosol, and possibly intestinal microflora; (c) GSH-dependent defluorinase of liver cytosol; (d)further cleavage and sulfoxidation of GSH conjugate (n = 0, 1, or 2); (e) amidase(s) sensitive to organophosphorus esters; (f) oxidative metabolism as tentative pathway; (g) liver microsomes with NADPH.
(-)-erythro-Fluorocitrate is extensively defluorinated on incubation with rat liver cytosol for 9 h at 37 "C independent of GSH (Figure 6). Similar results are obtained with mouse liver cytosol. (+)-erythro-Fluorocitrateis not defluorinated with either rat or mouse liver cytosol under these conditions. Pig heart aconitase also defluorinates (-)-erythro-fluorocitrate (Figure 6). Alterations in Levels of Endogenous Metabolites on Poisoning with Fluoroacetate and (-)-erythroFluorocitrate. The '3c NMR spectrum of the 0-6-h urine of rats treated with [1,2-13C]fluoroacetate(2 mg/kg) when compared with controls shows dramatic differences in the relative intensity of signals. A greatly increased excretion of glucose and citrate and a decreased level of urea are indicated (Figure 1);these assignments are substantiated by peak enhancement on fortification with authentic standards. Unlabeled fluoroacetate, [2-13C]fluoroacetate (2 mg/kg), or (-)-erythro-fluorocitrate (5 mg/kg) elicits the same responses in rats. These differences are maintained in the 6-24-h urine, but after 24 h the NMR spectra of the urine of treated rats and controls are essentially the same. Under similar conditions mouse urine shows the elevation in citrate and drastic drop in urea levels, but the effect on glucose is less pronounced than with rats (Figure 1). 13C NMR does not show significant changes in composition of the brain, liver, and kidney from fluoroacetate-treated animals.
Discussion Partial metabolic pathways for fluoroacetate, fluoroacetamide, fluoroethanol, and (-)-erythro-fluorocitrate in rats and mice are given in Figure 7. Fluoride ion from metabolic defluorination of fluoroacetate is readily evident by lgF NMR of urine and kidney extracts. Some of the liberated fluoride ion is probably deposited in bone (20, 21). A major portion of administered sodium fluoride or (-)-erythro-fluorocitrate is accounted for in urine 4-6 h following treatment but not with fluoroacetate, fluoroacetamide, or fluoroethanol. No attempt was made to further quantitate the distribution of the organofluorine compounds and fluoride ion. The detectability limits for the various metabolites in the variety of samples used may differ due to variations in TIand spin coupling. The wide variation in the observed chemical shift of fluoride ion is presumably due to differences in pH, protein content, and ionic composition of the media, but in each case the fluoride ion was unambiguously assigned by admixture experiments.
Most of the fluoroacetate metabolites in urine are defluorinated compounds. Thus?there is little excretion of 19F-containingmetabolites, yet there is a high percentage excretion of radiocarbon from [1,2-'4C]fluoroacetate, confirming findings with [2-'4C]fluoroacetate (8). 19FNMR reveals only small amounts of urinary fluoroacetate and fluoride ion with no detectable fluorocitrate whereas paper chromatographic analyses following [2-14C]fluoroacetate administration are reported to show 3% excretion of ['4C]fluorocitrate (8). Fluoroacetamide is metabolized slowly in rats with much more urinary excretion of the parent compound than of fluoroacetate and fluoride ion. The rate-limiting reaction in the toxic action appears to be enzymatic hydrolysis by an organophosphate-sensitive amidase on the basis of in vivo rather than in vitro observations (see also ref 12). An additional metabolite in liver may be fluoroacetohydroxamic acid or a conjugate thereof; this N-hydroxy derivative is also toxic (2). Fluoroethanol undergoes rapid in vivo oxidation to fluoroacetate which is detected in urine, liver, and kidney. Microsomal enzymes oxidize fluoroethanol to fluoroacetaldehyde without a clear cofactor requirement, indicating the possible involvement of more than one enzyme component. Liver cytosol is inactive in carrying out this oxidation, even with added candidate cofactors. The present findings therefore are in contrast to earlier reports (2,23) of a liver alcohol dehydrogenase(s)which oxidizes (oxidize) fluoroethanol. Entry of fluoroacetate into the citric acid cycle is minimized by coadministration of ethanol and sodium acetate, consistent with the reduced toxicity of the mixture ( I ) , but not by coadministered GSH (60-200 mg/kg). Fluoroacetamide poisoning is partially alleviated by 1-h pretreatment with PSCP (20 mg/kg) or GSH (180 mg/kg), apparently by slowing the rate of fluoroacetate liberation. Aconitase defluorinates (-)-erythro-fluorocitrate as evident by 19FNMR in the present study, confirming earlier investigations using a fluoride ion selective electrode (7, 24, 25). The poor signal to noise ratio of the 19FNMR signal for fluoride ion in the aconitase reactions may be due in part to formation of insoluble fluoride salts. Cytosol defluorinates (-)- but not (+)-erythro-fluorocitrate, and this stereoselectivity suggests the involvement of aconitase. Two minor unidentified urinary metabolites of (*)fluorocitrate which are not observed in analogous experiments with (-)- or (+)-erythro-fluorocitrate probably originate from the threo-fluorocitrate isomers and may be conjugates. Defluorination of (-)- and (+)-erythro-fluorocitrate and (*)-fluorocitrate in normal rats treated ip or orally and the lack of defluorination of (A)-fluorocitrate in antibiotic-treated rats may be explained by fluorwitrate excretion with bile into the intestine where it is metabolized without isomeric specificity by intestinal microorganisms (26, 27). Poisoning by fluoroacetate and (-)-erythro-fluorocitrate is accompanied by decreased excretion of urea and in-
19F and 13C N M R Studies on Fluoroacetate Metabolism
creased excretion of citrate and glucose. The major mechanism for ammonium ion removal in the liver is as urea, and in the brain it is as glutamine (28). Fluoroacetate or fluorocitrate poisoning is associated with increased ammonium ion and decreased glutamine in the brain (29-32). Fluoroacetate induces hyperglycemia and ketosis in rata (33,34).The glucosuria noted here with toxic doses of fluoroacetate or (-)-erythro-fluorocitrate in rata may be the result of glycogen breakdown due to adrenal stimulation associated with the convulsion. The amenability of fluoroacetate and its progenitors to NMR spectroscopy as an analytical method allows a further dimension in probing the toxicology of these compounds. The l9Fnucleus is sufficiently sensitive for enzyme investigations and for in vivo studies at sublethal doses. Further, use of 13C-enriched fluoro compounds labeled at either single or multiple sites provides in essence dual labeling whereby the integrity of the administered compound in relation to ita metabolites may be unequivocally assigned.
Acknowledgment. This study was supported in part by the Health Effects Component of the University of California Toxic Substances Research and Teaching Program and by National Institutes of Health Grant PO1 ES00049. We thank our Berkeley colleagues J. L. Engel for technical assistance, I. Hincenbergs for performing the bile cannulations, and V. V. Krishnamurthy and R. F. Toia for helpful suggestions.
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