Chem. Res. Toxicol. 1989,2, 51-56
51
Metabolism of the Nephrotoxin Dichloroacetylene by Glutathione Conjugat ion+ Wolfgang Kanhai, Wolfgang Dekant,* and Dietrich Henschler I n s t i t u t f u r Toxikologie, Universitat Wurzburg, Versbacherstrasse 9,0-8700 Wurzburg, Federal Republic of Germany Received August 23, 1988
Dichloroacetylene (DCA) is a potent nephrotoxin and nephrocarcinogen in rodents. T h e activation reactions responsible for this organ-specific toxicity are not known. We now report the identification of S-(1,2-dichlorovinyl)glutathione(DCVG) as a product of the glutathione (GSH) dependent metabolism of DCA in vitro and the identification of N-acetyl-S-(1,2-dichloroviny1)-L-cysteine (N-Ac-DCVC) as a urinary metabolite of DCA in rats. Formation of DCVG from DCA, used as 1:l complex with diethyl ether, in male rat liver and kidney subcellular fractions was dependent on time, native protein, and the presence of GSH. Initial reaction rates a t 23 "C were determined as 2923 nmol/(min.mg) for liver and 2838 nmol/(min.mg) for kidney microsomes. With cytosol, DCVG formation rates were 705 nmol/ (min-mg) (liver cytosol) and 129 nmol/(min-mg) (kidney cytosol). With liver microsomes, a KMof 7.5 mM and a V,, of 5464 nmol/(min.mg) for GSH were obtained. The product, DCVG, was definitively identified by 'H NMR spectrometry (400 MHz), mass spectrometry, and UV spectroscopy. N-Ac-DCVC was identified as a urinary metabolite from rats by GC/MS after esterification. Urine (collected for 24 h) from male rats exposed t o 36 f 5 ppm DCA (100 pmol of DCA introduced into the exposure system) for 1h contained 10.7 pmol of N-Ac-DCVC as determined by HPLC analysis. Formation of DCVG, renal processing to S-(1,2-dichlorovinyl)-~-cysteine, and cleavage of this cysteine S-conjugate by cysteine S-conjugate 6-lyase in the kidney with formation of reactive and mutagenic intermediates may account for DCA nephrotoxicity and nephrocarcinogenicity. N-Ac-DCVC is the end product of DCVG processing by the enzymes of mercapturic acid formation.
Introduction Dichloroacetylene' (DCAY is a product of the alkaline decomposition of trichloroethylene (Tri) and tetrachloroethane (1,2).DCA intoxications have been observed in humans inhaling DCA-contaminated Tri formed in closed recirculating systems fitted with alkali absorbers (2). Poisoning has also occurred after use of Tri for cleaning purposes (3). DCA is highly toxic in humans and causes irreversible degeneration of the trigeminal nerve ( 4 ) ;in rodents, it is highly nephrotoxic and is a potent nephrocarcinogen (5). No metabolism studies have been reported with DCA so far. DCA is highly reactive and may spontaneously decompose by a radical mechanism in the presence of oxygen (6). However, since in long-term exposure DCA did not damage the respiratory tract or any tissue other than the kidney, bioactivation reactions specifically causing nephrotoxicity and nephrocarcinogenicity are likely (5). The carbon-carbon triple bond in DCA easily adds nucleophiles to form more stable vinylic derivatives. Reaction of DCA with the nucleophile glutathione (GSH) present in mammalian cells in high concentrations was expected to yield the potent nephrotoxin and mutagen S-(l,a-dichloroviny1)glutathione (DCVG) whose formation during DCA metabolism may provide an explanation for the organotropic toxicity and carcinogenicity. GSH conjugation reactions have been demonstrated to occur during the metabolism of several nephrotoxic haloalkenes (7). Supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 172), and the Doktor-Robert-Pfleger-Stiftung, Bamberg. * Address correspondence to this author.
0893-228x/89/2702-0051$01.50/0
We have therefore investigated the GSH-dependent metabolism of DCA in rat liver and kidney subcellular fractions and the metabolism of DCA in rats to identify and quantify any metabolites formed by GSH conjugation.
Materials and Methods Syntheses. Dichloroacetylene was synthesized from Tri by the method of Pielichowski (8)and isolated as the diethyl ether complex by distillation. DCA content of the ether complex was determined as 1:l (c/c) by refractivity (9). The diethyl ether complex was further characterized by I3C NMR spectrometry (solvent C6D6;acS = 48.47 ppm). When injected into a gas chromatograph, the DCA-diethyl ether complex dissociates to give DCA and diethyl ether which are clearly separated (30 m X 0.32 mm fused silica column, 1-pm DB-624, J&W Scientific, Folsom, CA; oven temperature 30 "C). Electron impact mass spectrum of DCA: m / z (36Cl) = 94 (loo%, M+, 2C1), 59 (13%, Mf-C1),47 (12%,M+-CC1),35 (5%,M+-C&1),24(2%,M+ - 2C1). S-(1,2-Dichlorovinyl)glutathionewas synthesized and characterized as described in reference 10. Purity of synthetic DCVG exceeded 99% as determined by HPLC (UV absorption, 257 nm; for method, see reference 12). The synthesis of N-acety1-S(1,2-dichlorovinyl)-~-cysteine has been described previously (11). Dichloroacetylene is highly toxic and explosive. In aqueous solution, the dichloroacetylene-diethyl ether complex may violently decompose in the presence of oxygen. Therefore, all manipulations should be performed in a fume hood using a protective shield. Abbreviations: Tri, trichloroethene; DCA, dichloroacetylene; NMR, nuclear magnetic resonance spectrometry; GC/MS, gas chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; DCVG, S-(1,Z-dichloroviny1)glutathione; N-Ac-DCVC, N-acetylS-(1,Z-dichloroviny1)-L-cysteine; DCVC, S-( 1,Z-dichloroviny1)-L-cysteine; GSH, glutathione; AU, absorption units.
0 1989 American Chemical Society
52 Chem. Res. Toricol., Vol. 2, No. 1, 1989
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Figure 1. Three-dimensional plot of HPLC chromatograms (UV detection, 257 nm) showing the time-dependent formation (1-5 min) of the dichloroacetylene metabolite S-(1,2-dichlorovinyl)glutathioneby rat hepatic microsomes. Incubations contained GSH (10 mM), dichloroacetylene-diethylether complex (5 mM), and microsomal protein (0.01 mg/mg) in a total volume of 3 mL. Inset: UV spectrum of metabolite. Samples (100 pL) were taken through a gas-tight septum. Preparation of Subcellular Fractions from Rat Liver and Kidney. Male Wistar rats (200-250 g) were starved overnight and then anesthetized with an ip injection of pentobarbital sodium salt (40 mg/kg). After opening the peritoneal cavity, the abdominal aorta was ligated below the renal arteries, and a plastic cannula (1.5 mm 0.d.) was inserted into the aorta just above the iliac arteries; the inferior vena cava was severed below the renal veins. The liver and kidneys were perfused with 0.9% sodium chloride and 0.05% heparin and were uniformly blanched within 2-4 min. All further steps were performed at 4 "C. Livers and kidneys were removed and weighed, and a 30% homogenate was prepared in 0.154 M KC1 (adjusted to pH 7.4 with 0.1 M phosphate buffer) with a Potter-Elvehjem homogenizer. After low-speed (500g) centrifugation to precipitate cellular debris, the supernatant was centrifuged at llOOOg for 4 min. The supernatant from the 1lOOOg fraction was centrifuged a t 105000g for 40 min to yield the cytosolic and microsomal fractions. The upper fatty layer was removed from the supernatant fraction with a water aspirator, and the supernatant fraction was removed, diluted with an equal volume of phosphate buffer, and frozen. The microsomal pellet was suspended in buffer and centrifuged again a t 105000g for 40 min; the pellet was suspended in phosphate buffer and stored at -80 "C. Protein concentrations were determined with the Bio-Rad Protein Assay Kit with bovine serum albumin as standard (Bio-Rad Laboratories, Munchen, FRG). Enzymatic Assays. All incubations with DCA were carried out under nitrogen at 23 "C in 0.1 M phosphate buffer, pH 7.4, containing microsomal or cytosolic protein, 0.1 mM tetrasodium EDTA, and GSH (10 mM) in a final volume of 3 mL; closed flasks were used for all incubations. Samples were removed with a syringe through a gas-tight valve (modified Mininert valve, Pierce Chemical Co., Rockford, IL). Separation and Quantification of S-Conjugates. The reactions were stopped by addition of 0.1 mL of 30% trichloroacetic acid. Protein was precipitated by storage a t 4 "C for 24 h and removed by centrifugation. Samples of the supernatant (0.01-0.05 mL) were fractionated by HPLC (linear gradient over 40 min from 0 to 100% methanol in water, 0.1% trifluoroacetic acid; flow rate 1.0 mL/min; column: Supelco LC-l8S, 250 mm X 4 mm). The absorption of the eluate was monitored a t 257 nm (HewlettPackard 1040 diode array detector). Urine samples were filtered through HV filters (pore size 0.45 pm, Millipore, Eschborn, FRG) and aliquots (50 pL) separated by HPLC as described above. Peak areas were integrated with the software provided with the 1040 detection system, and the concentrations of the conjugates were determined by computerized comparison with standard curves. UV spectra of the eluates were recorded at 1.28-s intervals with a threshold setting of 5 mAU.
Instrumental Analyses. 'H NMR spectra were recorded in 5-mm tubes with a Bruker WH 400 spectrometer. Chemical shifts are reported in parts per million with (trimethylsily1)propionic acid-d, sodium salt (set a t 6 = 0.14 ppm) as internal reference. Chemical derivatization reactions and preparative HPLC were performed as described previously (12). GC/MS was performed with a Hewlett-Packard 5970 MSD with splitless injection (12 m X 0.2 mm column, 0.33 Wm HP-1) with a linear temperature gradient of 10°/min from 40 to 250 "C. Injector and transfer line temperatures were 250 and 260 "C, respectively. Exposure of Rats to DCA-Diethyl Ether Complex. The exposure chamber used was a modification of the system previously developed for DCA inhalation (5). The bodies of the animals were held in place in tubular, transparent PVC castings outside the actual exposure chamber, which consisted of glass, had dimensions of 50 mm X 50 mm X 250 mm, and permitted the exposure of two rats a t one time. Boreholders of appropriate size ensured that the animal tubes fitted into the gas space in such a way that only the noses of the animals were in contact with the exposure mixture. The exposure chamber was flushed with compressed air a t a flow rate of 60 L/h, and DCA-diethyl ether complex was constantly added into a mixing chamber at 60 "C with a microliter syringe attached to an infusor (Braun Melsungen, FRG). Concentrations of DCA-diethyl ether in the exposure mixture were monitored by gas chromatography (EC detection, H P 5790 GC, 30 m X 0.32 mm capillary column, DB-624,1.8-pm film thickness, oven temperature 30 "C). Samples (20 r L ) of the exposure mixture were taken through a septum with a gas-tight syringe.
Results Identification of DCVG as a Metabolite of DCA. To circumvent the problems of generating and handling pure DCA, we synthesized the diethyl e t h e r complex of DCA, which can be safely handled and stored for several months without decomposition. This diethyl ether complex is a 1:l complex between DCA and d i e t h y l ether (9); i t s chemistry is identical with that of DCA (8, 9, 13). In aqueous solution, the diethyl ether-DCA complex dissociates to yield DCA (9). The nephrotoxicity of the diethyl ether-DCA complex has been demonstrated (14). Incubations of D C A w i t h rat liver microsomes i n t h e presence of GSH resulted i n t i m e and protein concentration d e p e n d e n t consumption of GSH ( n o t shown) and i n t h e t i m e (Figure 1) and protein concentration d e p e n d e n t formation of a metabolite whose retention t i m e and elec-
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 53
Metabolism of Dichloroacetylene
7.0
5.0
60
2 .o
30
40
00
10
PPm
Figure 2. 'H NMR spectrum (400 MHz) of metabolite formed from glutathione and dichloroacetylene-diethyl ether complex by rat liver microsomes. Scan
579
(12.218
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o f DRTR: D E 5 7 .
I
D
100 2
7
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325
\
I
I
100
I50
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250
3 00
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Figure 3. Mass spectrum (electron impact, 70 eV; GC/MS) of dichloroacetylenemetabolite (after acid hydrolysis, trifluoroacetylation, and esterification)formed from GSH and dichloroacetylene-diethyl ether complex by rat liver microsomes.
tronic spectrum (Figure 1) were identical with those of synthetic DCVG. To substantiate the proposed structure, the metabolite was isolated by preparative HPLC and subjected to NMR and mass spectrometric analysis. The 'H NMR spectrum (Figure 2) showed several resonances closely corresponding to those reported for GSH S-conjugates (15, 16) and a singlet at 6 = 6.74 ppm which is attributed to the vinylic proton. Moreover, the NMR spectrum of the metabolite was identical with the 'H NMR obtained for DCVG prepared by chemical synthesis. For GC/MS, the isolated metabolite was hydrolyzed under conditions known to hydrolyze the peptide bonds in GSH S-conjugates (12). The dried residue obtained was treated with N,N-bis(trifluoroacety1)methylamineand boron trichloride/methanol (12). The mass spectrum (Figure 3) of the derivative obtained showed several fragments with the characteristic isotopic clusters of two chlorine atoms [ m / z (35Cl) = 325 (M+), 266 (M+ -
COOCHJ, 230 (M+ - COOCH:, - HCl), 212 (M' CFSCONHJ, 177 (M' - C1 - CF,CONHZ), 141 (Cl2C2HSCHZ+), 127 (CI,HC,S+)] in addition to fragments indicative of N-trifluoroacetylated cysteine S-conjugate methyl esters [m/z = 198 (M+- C12HC2S),69 (CF3+)]. By comparison with the mass spectrum of a synthetic reference compound (17) (spectrum not shown), the substance was unequivocally identified as N-(trifluoroacety1)-S(1,2-dichlorovinyl)-~-cysteine methyl ester. The identification of this cysteine S-conjugate after hydrolysis further substantiates the structure of the DCA metabolite as S(1,2-dichlorovinyl)glutathione. Quantification of DCVG Formation. The formation of DCVG from DCA in subcellular fractions was quantified by HPLC (Table I). Under the experimental conditions (12, 18) used with other haloalkenes (incubation temperature 37 "C, protein concentrations of more than 1mg/mL, and longer incubation times), the reaction of DCA with
54 Chem. Res. Toxicol., Vol. 2, No. I , 1989
Kanhai et al.
50 1 400
J
a E
*
Figure 4. HPLC chromatogram (UV detection, 257 nm) of the collected 6-h urine of rats (50-wL sample) exposed to 36 5 ppm DCA-diethyl ether complex for 1h. For separation conditions, see Materials and Methods. Inset: UV spectrum of metabolite with identical retention time as synthetic (E)-N-acetyl-S-(l,2-dichlorovinyl)-~-cysteine. Table I. Rate of GSH S-Conjugate Formation in Subcellular Fractions from Liver and Kidney of Male Rats' formation of DCVG from DCA [nmol/(min.mg)] rat liver microsomes 2923 f 335 cytosol 705 f 67 rat kidney microsomes 2838 f 271 cytosol 129 f 19 controls boiled liver microsomes 160 f 15 75 f 7 boiled liver cytosol a Incubation mixtures contained GSH (10 mM), dichloroacetylene-diethyl ether complex (5 mM), and microsomal or cytosolic protein at concentrations of 0.01 and 0.025 mg/mL, respectively. Samples (0.5 mL) were taken 3 and 6 min after the start of the incubation, DCVG formation was determined by HPLC (see Materials and Methods). Results are given as means f SE from four experiments.
GSH in subcellular fractions was time dependent for less than 1min due to the rapid consumption of reactants and possibly due to inhibition of GSH S-transferases by the DCVG formed. Inhibition of GSH conjugation reactions by the structurally similar S-(pentachlorobutadieny1)glutathione has been described (19). We therefore lowered the incubation temperature and the protein concentration. At 23 "C, DCVG formation was proportional to protein concentration at 0.01 and 0.025 mg of protein/mL and was dependent on the presence of GSH; an apparent KMof 7.5 mM and a V,, of 5464 nmol/(min.mg) for GSH were obtained for liver microsomes. Due to the limited solubility of the DCA-diethyl ether complex in aqueous solutions, K M and V,, for DCA could not be determined. Under these conditions, the reaction was linear with time for 6 min (Figure 1);the reaction rates started to decrease after 6-8 min in both liver and kidney microsomes. Microsomes exhibited a much higher activity for DCA than cytosol (Table I). In contrast to the haloalkenes hexachlorobutadiene (18)and tetrachloroethene (121, DCA was also conjugated with GSH in kidney microsomes a t a high rate. The nonenzymatic reaction rate between GSH and DCA was about of the rate determined for the reaction catalyzed by enzymatically competent microsomes
(Table I). Incubations in the presence of the competitive GSH S-transferase inhibitor l-chloro-2,4-dinitrobenzene could not be evaluated, since the presence of the inhibitor in the incubation mixtures instantly resulted in an explosive decomposition of the added DCA-ether complex. Identification of N-Acetyl-S-(l&dichlorovinyl)-Lcysteine as a Urinary Metabolite of Dichloroacetylene. Urine of rats exposed for 1 h to 36 f 5 ppm DCA-ether was collected during 6-h intervals over a period of 24 h. The total amount of DCA-diethyl ether complex added to the exposure chamber was 100 pmol. HPLC analysis (Figure 4) of the urine samples collected 6 h after the end of DCA exposure contained a peak (retention time 23.62 min) whose retention time and electronic spectrum (Figure 4) were identical with those of synthetic N-Ac-DCVC (11). This metabolite was isolated by preparative HPLC (12) and subjected to GC/MS after methylation with boron trichloride/methanol. The mass spectrum (Figure 5) contained several fragments indicative of the presence of chlorine atoms in the molecule [m/z (35Cl)= 271 (2C1, M+), 236 (lCl, M+ - Cl), 235 (1C1, M+ - HCl), 212 (M' - COOCHJ, 177 (M" - C1, -CH,CONH2), 170 (C4H6NCl2S)]and two fragments characteristic of the methyl esters of mercapturic acids [ m / z = 144 (C5H6N0 2 S ) ,88 (C3H6NO2)].Moreover, the spectrum was identical with that of synthetic N-Ac-DCVC ( I I ) , and the retention times of the synthetic reference compound in HPLC and GC under various separation conditions were identical with those of the metabolite. The mass spectrum of biosynthetic N-Ac-DCVC also gives information on the stereochemistry of the S-conjugates formed from DCA. The fragment m / z = 235 (35Cl) is formed by thermal HC1 elimination from the molecular ion (M+ - 36). This thermal reaction should proceed as syn elimination according to an Elelimination mechanism; a syn elimination is only possible when the vinylic proton in N-Ac-DCVC is attached in a trans position to the sulfur atom. These considerations identify trans-N-Ac-DCVC as a urinary metabolite of DCA in rats. Urinary excretion of N-Ac-DCVC amounted to 4.4 pmol in the first 6 h after DCA exposure and gradually decreased (urine collected from 6 to 12 h postexposure, 3.4 pmol of N-Ac-DCVC; 12-18 h, 2.1 pmol; and 18-24 h, 0.8 pmol). Total urinary excretion of N-Ac-DCVC within 24 h after exposure to
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 55
Metabolism of Dichloroacetylene S c a n 653 ( 1 4 . 5 6 3 m i n )
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Figure 5. Chromatographic separation and mass spectrum (electron impact, 70 eV) of N-acetyl-S-(1,2-dichlorovinyl)-~-cysteine (N-Ac-DCVC)present in rat urine after exposure to DCA-diethyl ether complex. N-Ac-DCVC was isolated from urine by preparative HPLC and transformed to the methyl ester with boron trichloride/methanol for gas chromatography.
DCA amounted to 10.7 pmol. Similar concentrations of N-Ac-DCVC were present in rat urine when DCA was generated from trichloroethylene inside the exposure chamber (results not shown) by previously published methods (4, 5).
Discussion Pure DCA is highly reactive and explosive in the presence of oxygen. In accidental human exposure, it is formed by alkaline decomposition of trichloroethene or 1,1,2,2tetrachloroethane; the excess haloalkane (alkene) present in these mixtures results in stabilization of DCA (20). DCA is conjugated with GSH at very high rates, which may be explained by the high reactivity of DCA with nucleophiles. DCA readily adds to nitrogen and sulfur nucleophiles to yield thermodynamically much more stable chlorovinyl derivatives (14). As observed with several haloalkenes (for review, see reference 7), microsomes catalyze the conjugation reaction much more efficiently than cytosol; this difference is most probably due to preferential distribution of the lipophilic haloalkenes and the haloalkyne DCA into lipid membranes. In rats, N-Ac-DCVC, formed by renal processing of DCVG by the enzymes of mercapturic acid formation, was definitively identified as a urinary metabolite of the DCA-diethyl ether complex. The amount of N-Ac-DCVC found in rat urine within 24 h after exposure to DCA indicates that about 10% of the DCA introduced into the exposure system is converted to N-Ac-DCVC. These data suggest that DCA is also metabolized through a GSH conjugate in vivo and that this represents a major pathway of biotransformation. On the basis of these results, we propose a bioactivation mechanism to account for the observed organotropism of DCA toxicity (Figure 6). In the initial step of the activation sequence, DCA (1) is conjugated with GSH to yield DCVG (2). Addition of GSH to the carbon-carbon triple bond may form either cis or trans DCVG (Figure 5). Trans additions of the nucleophile GSH to DCA are favored over cis addition (14) as shown by the mass spectral data obtained. DCVG is nephrotoxic in rats (10) and is toxic to rat renal tubular epithelial cells (21). The mechanism of DCVG nephrotoxicity has been elucidated (7,22). DCVG formed in the liver is concentrated in the kidney (23),where DCVG is metabolized to S-(1,2-dichlorovinyl)-~-cysteine (DCVC) (3) by y-glutamyltranspeptidase and dipeptidases. DCVC
cI-=-cl
-1
+
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1GSH-fronsferoses COCH, I
Cysteine conjugote Cn-iyose
6
Acylation o f cellular mocromolec ules
v
Toxic/ t y , mu togen/c/t y
Figure 6. Bioactivation mechanism of dichloroacetylene (1). Structures shown: S-(1,2-dichlorovinyl)glutathione(2);S-(1,2dichloroviny1)-L-cysteine (3); 1,2-dichloro-2-mercaptoethene (4);
chlorothioketene (5); chlorothioacetyl chloride (6); N-acetyl-S(1,2-dichlorovinyl)-~-cysteine (7).
is a substrate for renal &lyases (21,24,25), which cleave DCVC to pyruvate, ammonia, and 1,2-dichloroethenethiol (4). This enethiol is unstable and is rapidly converted to a thioacylating intermediate which may be either chlorothioketene ( 5 ) or chlorothioacetyl chloride (6) (26). Interaction of these highly reactive intermediates with lipids and proteins may cause mitochondrial toxicity, and interaction with DNA may cause the mutagenicity that is observed for DCVC (21,25,27). Alternatively, DCVC may be acetylated by renal acetyltransferase to yield N-Ac-DCVC (7),which is a urinary metabolite of DCA in rats. The reaction rates determined for the conjugation of DCA with GSH in vitro are more than 10-fold those observed for the GSH conjugation of tetrafluoroethene (28) and chlorotrifluoroethene (29) and several thousandfold those observed for the chlorinated alkenes tetrachloro-
56 Chem. Res. Toxicol., Vol. 2, No. 1, 1989
ethene (12) and hexachlorobutadiene (18). The high rates of DCVG formation from DCA offer a plausible explanation for the potent nephrotoxicity and nephrocarcinogenicity of this compound. Acknowledgment. We thank Dr. G. Scheutzow, Institut fur Organische Chemie, Universitat Wurzburg, for the recording of NMR spectra. Registry No. DCA, 7572-29-4; GSH, 70-18-8; DCVG, 9661459-4; N-Ac-DCVC, 2148-31-4.
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