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Studies on Metabolic Pathways of Cocaine and Its Metabolites Using Microsome Preparations from Rat Organs Stefan W. Toennes,*,† Markus Thiel,† Michael Walther,‡ and Gerold F. Kauert† Institute of Forensic Toxicology, Center of Legal Medicine, University of Frankfurt/Main, Kennedyallee 104, D-60596 Frankfurt/Main, Germany, and Institute of Pharmaceutical Chemistry, University of Frankfurt/Main, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany Received July 15, 2002
Cocaine metabolism has been studied previously with respect to the formation of predominant hydrolytic or hepatotoxic metabolites via oxidative pathways. In the present study, cocaine and eight of its metabolites (norcocaine, ecgonine methyl ester, benzoylecgonine, benzoylnorecgonine, 3-hydroxy-benzoylecgonine, cocaethylene, norcocaethylene, and ecgonine ethyl ester) were incubated with microsomes from rat liver, kidney, lung, and brain. Qualitative analysis of the metabolites produced was performed using solid phase extraction (SPE), trimethylsilylation, and GC/MS. It was found that the metabolites with a free carboxylic group (e.g., benzoylecgonine) were not further oxidized by microsomal enzymes and their presence in urine or blood may therefore be due to hydrolysis of the respective alkylated entities. Although microsomes from all organs exhibited oxidative metabolism, significant differences were noted. Kidney microsomes produced essentially the same results as liver, but aryl hydroxylated metabolites were not found in incubations with lung and brain microsomes. N-Hydroxy-norcocaine was found only in traces with brain microsomes. It appears that cocaine is converted to N-hydroxy-norcocaine (which is the precursor of toxic metabolites) not only in the liver but also in other organs of rat. This might be relevant in the development of lung toxicity observed in smokers of cocaine (“crack”).
Introduction The metabolism of COC1 has been studied for a long time; however, primary interest has focused only on the predominant hydrolytic metabolism to BZE and EME or on the specific oxidative pathway, which yields the hepatotoxic metabolites. COC is regarded as a potential human hepatotoxin due to the metabolic cascade leading to toxic metabolites, which was shown for liver (1) but also for brain (2, 3); other relevant organs containing metabolic enzymes such as kidney and lung have not been studied. Especially in case of the lungs, it can be supposed that the exposition to high COC doses might * To whom correspondence should be addressed. Tel.: +49 69 6301 7561. Fax: +49 69 6301 7531. E-mail:
[email protected]. † Institute of Forensic Toxicology, Center of Legal Medicine, University of Frankfurt/Main. ‡ Institute of Pharmaceutical Chemistry, University of Frankfurt/ Main. 1 Abbreviations: MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; COC, cocaine; 3/4-HO-COC, 3/4-hydroxy-cocaine; 3,4-diHOCOC, 3,4-dihydroxy-cocaine; HO-MeO-COC, hydroxy-methoxy-cocaine; NorCOC, norcocaine; 3/4-HO-NorCOC, 3/4-hydroxy-norcocaine; HOMeO-NorCOC, hydroxy-methoxy-norcocaine; N-HO-NorCOC, N-hydroxy-norcocaine; N-HO-3/4-HO-NorCOC, N-hydroxy-3/4-hydroxynorcocaine; EME, ecgonine methyl ester; NorEME, norecgonine methyl ester; CE, cocaethylene; 3/4-HO-CE, 3/4-hydroxy-cocaethylene; 3,4diHO-CE, 3,4-dihydroxy-cocaethylene; HO-MeO-CE, hydroxy-methoxycocaethylene; NorCE, norcocaethylene; 3/4-HO-NorCE, 3/4-hydroxynorcocaethylene; HO-MeO-NorCE, hydroxy-methoxy-norcocaethylene; N-HO-NorCE, N-hydroxy-norcocaethylene; N-HO-3/4-HO-NorCE, N-hydroxy-3/4-hydroxy-norcocaethylene; EEE, ecgonine ethyl ester; NorEEE, norecgonine ethyl ester; BZE, benzoylecgonine; 3/4-OH-BZE, 3/4hydroxy-benzoylecgonine; BNE, benzoylnorecgonine; N-HO-BNE, N-hydroxy-benzoylnorecgonine; ECG, ecgonine; AEME, anhydroecgonine methyl ester.
result in relevant amounts of pneumotoxic metabolites in smokers of COC. COC is degraded in vivo by three main routes: through enzymatic hydrolysis primarily in the liver but also in other organs and in plasma by butyrylcholinesterase or through oxidative metabolic processes (cytochrome P450 or FAD-containing monooxygenase) in liver and other organs. Chemical hydrolysis of the methyl ester at physiological pH plays only a minor role (4). Arylhydroxylated metabolites and N-desalkylated and Noxidated compounds are products of the oxidative metabolic pathways. Although metabolites of these oxidative pathways are present in blood or urine in only minor amounts, a number of compounds originating from the combination of different routes/pathways have been detected, mainly by analysis of authentic urine samples (5, 6). From these results, several metabolic pathways of COC have been postulated (7); however, particular pathways have still not been elucidated. The aim of the present study was to investigate whether COC and some of its metabolites are substrates for oxidizing microsomal enzymes in liver, kidney, lung, and brain to elucidate metabolic routes in the different organs. Especially the pathway from COC to NorCOC to N-HO-NorCOC received attention due to its importance for the cytotoxicity of COC. The experiments should further provide a basis for the interpretation of analytical findings in body fluids and tissues after administration of COC. Rat was chosen for the experiments because rat organs are easily available for microsome preparation.
10.1021/tx025580n CCC: $25.00 © 2003 American Chemical Society Published on Web 02/11/2003
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Experimental Procedures Chemicals and Reference Standards. Solutions (1 mg/ mL in methanol or acetonitrile) of the reference standards COC, NorCOC, CE, NorCE, EME, EEE, BZE, BNE, 3-HO-BZE and of the internal standards (0.1 mg/mL in methanol or acetonitrile) COC-d3, EME-d3, and BZE-d3 were purchased from Cerilliant (Promochem, Wesel, Germany). The derivatization reagent MSTFA was from Macherey & Nagel (Dueren, Germany), and biochemicals were from Sigma-Aldrich (Seelze, Germany) and of the highest grade available; all other reagents and organic solvents were of analytical grade and from Merck (Darmstadt, Germany). Incubation with Rat Organ Microsomes. Three male Sprague Dawley rats (3 months old) were anesthetized with enflurane, and the liver, kidneys, lungs, and brain were excised and immediately frozen in liquid nitrogen. The single use of enflurane is unlikely to have a relevant influence on microsomal enzyme activities (8, 9). After homogenization of the pooled organs in 3 vol of 0.1 M phosphate buffer, pH 7.4, microsomes were isolated by centrifugation at 10 000g and 100 000g, resuspended in 0.1 M phosphate buffer, pH 7.4, and again centrifuged at 100 000g; the pellets formed were stored at -80 °C until use. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Munich, Germany) and applying bovine serum albumin as standard. The microsome preparation from liver contained 7.9 mg protein/mL, from kidneys 4.7 mg/mL, from lungs 2.8 mg/mL, and from brain 2.1 mg/mL, respectively. Incubation of COC or its metabolites with microsomes was essentially performed as described by Kraemer et al. (10). The incubation mixture consisted of an amount of microsome preparation equivalent to 1 mg of protein, 150 µM substrate (COC, NorCOC, CE, NorCE, EME, EEE, BZE, BNE, or 3-HOBZE as 1 mg/mL solution in methanol or acetonitrile), 1.2 mM NADP, 2 U isocitrate dehydrogenase, 5 mM isocitrate, and 5 mM magnesium chloride in 0.1 M phosphate buffer (pH 7.4) up to a total volume of 500 µL. Incubation was carried out at 37 °C for 30 min, and the reaction was stopped by adding 500 µL of acetonitrile followed by centrifugation for 10 min at 14 000g. The supernatant was used for GC/MS analysis as described below. In preliminary studies, incubation time and substrate concentration were varied in order to optimize the experimental conditions with respect to the optimal yields of metabolites. The decreasing stability of the metabolizing enzymes was no concern as only qualitative studies were performed. Incubations without substrate or without microsomes were used as controls. GC/MS Analysis Procedure. After the microsome preparation was centrifuged, 4 mL of 0.1 M phosphate buffer, pH 6.0, and 100 ng of all internal standards (in 100 µL of acetonitrile) were added to the supernatant and the mixture was extracted using Bond Elut Certify HF (3 mL, 300 mg) solid phase extraction (SPE) cartridges (Varian, Darmstadt, Germany) with the extraction robot RapidTrace (Zymark, Idstein, Germany). The extraction protocol was as follows: conditioning with 2 mL of methanol and 3 mL of phosphate buffer, applying of the sample onto the column at 1 mL/min, rinsing with 2 mL of 0.25 M acetic acid and 3 mL of methanol at 1.5 mL/min, elution of the analytes with 3 mL of a freshly prepared solution of methylene chloride:2-propanol:concentrated aqueous ammonium hydroxide (80:20:2, v/v/v) at 1 mL/min. The extract was evaporated at room temperature to dryness, the residue was derivatized with 40 µL of MSTFA for 30 min at 100 °C, and 1 µL of the solution was analyzed by GC/MS. GC/MS analysis was performed on a Hewlett-Packard (Waldbronn, Germany) HP6890 GC equipped with an autosampler HP6890 ALS and interfaced to a HP5973 MSD. A Chrompack CP-Sil 5 CB lowbleed/MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) from Varian, which was protected by a guard column (1.5 m of deactivated (diphenyltetramethyldisiloxane) glass capillary (0.25 mm i.d.) from BGB Analytik
Toennes et al. AG (Anwil, Switzerland)) was used with 0.7 mL/min helium as carrier gas. Splitless injection was performed at 250 °C injection port temperature and a temperature program from 100 °C, which was held for 2 min and increased with 20 °C/min to 310 °C and held for 5 min. The MS transfer line was maintained at 280 °C, and the ion source was kept at 250 °C and operated with 70 eV of ionization energy. Mass spectra were recorded in full scan mode from m/z 40 to m/z 550. Data analysis was performed using HP ChemStation software (Rev. B.01.00). Screening for potential metabolites was carried out by MS: the total ion chromatogram was searched for selected fragments (cf. Table 1), which indicated the presence of COC-related compounds. These were essentially pyrrol residue fragments such as m/z 82, which indicates the intact N-methyl group; the fragment m/z 140, which indicates a trimethylsilylated Ndesalkylated metabolite; and the fragment m/z 156, which indicates the presence of a trimethylsilylated N-desalkylated and N-hydroxylated metabolite. Metabolite identification was performed by comparison of retention times and interpretation of the mass spectra according to Jindal and Lutz (5) and Jufer et al. (11). Limits of detection for the analytical procedure were determined by extraction of a series of samples containing 25-150 ng of the different reference substances. The reproducibility of the procedure was tested by analysis of five parallel microsome incubations of 150 µM NorCOC and expressed as the variation coefficient of the area ratios of the metabolites vs internal standards (relative responses) where COC-d3 was used for NorEME, N-OH-NorCOC, 3/4-OH-NorCOC, N-OH-3/4-OH-NorCOC and BZE-d3 for BNE and N-OH-BNE.
Results and Discussion Incubation of COC and Selected Metabolites with Microsomal Preparations from Rat Organs. The analytical procedure used in the present study was sensitive enough to detect less than 25 ng of each reference substance in the incubation mixture. It was therefore concluded that the formation of metabolites in an amount down to 0.1% of the precursor should be detectable. The reproducibility of the assay was found to be reliable for the purpose of a qualitative metabolism study as tested in five parallel incubations of NorCOC with rat liver microsomes. The variation coefficients of the GC/MS responses for NorEME, N-OH-NorCOC, and 3- and 4-OH-NorCOC were less than 10%, for BNE and N-OH-BNE less than 20%, and for N-OH-3/4-OH-NorCOC 22%. Incubation was stopped after 30 min, because in a preliminary study it was found that oxidative metabolic processes were detectable during 60 min only. Moreover, the yield of the N-hydroxy-N-desalkyl metabolite decreased after 30 min of incubation while the yields of the other metabolites did not decrease (Figure 1). This observation cannot be explained at the moment and indicates secondary metabolic or chemical reactions, which could not be studied further due to the lack of reference substance. A similar effect has been reported by Rauckman et al. (12) for the formation of NorCOC nitroxide from N-OH-NorCOC. However, under the conditions used, N-oxides decompose in the GC injection port (13, 14) and nitroxyl free radicals (nitroxides) were only detected by spin-trapping techniques (3), suggesting that a variety of N-oxidated metabolites have not been detected in the present study. Metabolic Pathways of COC and CE. A total of 30 metabolites of COC and CE were identified by MS (cf. Table 1) in microsome incubates. In the control experiments, no artificial oxidation products were found and
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Chem. Res. Toxicol., Vol. 16, No. 3, 2003 377
Table 1. Characteristic Fragments in the Mass Spectra of COC and Its Trimethylsilylated Derivatives (the Last Column Contains the Molecular Ion)a
COC 3/4-HO-COC 3,4-diHO-COC HO-MeO-COC NorCOC 3/4-HO-NorCOC HO-MeO-NorCOC N-HO-NorCOC N-HO-3/4-HO-NorCOC EME NorEME CE 3/4-HO-CE 3,4-diHO-CE HO-MeO-CE NorCE 3/4-HO-NorCE HO-MeO-NorCE N-HO-NorCE N-HO-3/4-HO-NorCE EEE NorEEE BZE 3/4-HO-BZE BNE 3/4-HO-BNE N-HO-BNE ECG
82 82 82 82 140 140 140 156 156 82 140 82 82 82 82 140 140 140 156 156 82 140 82 82 140 140 156 82
182 182 182 182 240 240 240 256 256 182 240 196 196 196 196 254 254 254 270 270 196 254 240 240 298 298 314 240
105 193 281 223 105 193 223 105 193 105 193 281 223 105 193 223 105 193 105 193 105 193 105
272 360 448 390 330 418 448 346 434 240 298 272 360 448 390 330 418 448 346 434 240 298
376 464 406 346 434 464 362 450 256 314 390 478 420 360 448 478 376 464 270 328 346 434 404 492 420 314
303 391 479 421 361 449 479 377 465 271 329 317 405 493 435 375 463 493 391 479 285 343 361 449 419 507 435 329
a The residue R1 may be methyl or ethyl; the residue R2 may be methyl, trimethylsilyl, or hydroxy-trimethylsilyl; and the residue R3 may be hydrogen or hydroxy trimethylsilyl.
Figure 1. Time course of metabolite yields after incubation of COC with rat liver microsomes: decrease of N-HO-NorCOC in contrast to all other metabolites (represented by NorCOC and 4-HO-COC as examples). Concentrations are given as analyte responses (area ratio analyte/internal standard).
only little hydrolysis was found. Table 2 summarizes the metabolites found in the different incubation mixtures of rat organ microsomes with the available reference substances, and Figure 2 shows the pathways of COC metabolism, which can be concluded from the results. As no differences were observed between the metabolism of COC and CE, NorCOC and NorCE, or EME and EEE, the nature of the alkylester group (methyl or ethyl) appears to have no effect on the metabolic pathways.
Therefore, the scheme in Figure 2 applies also to the metabolism of CE. For COC, CE, NorCOC, and NorCE, an enzymatic pathway for the cleavage of the alkyl ester was found with all organ microsomes where the peaks of the hydrolytic products (BZE and BNE) were much higher after microsome incubation than in the respective controls. This is in accordance with findings from Dean et al. (15) who reported the presence of a COC methyl esterase in tissue homogenates of rat liver, kidney, lung, and brain. This reaction is catalyzed in humans by the carboxylesterase hCE-1 (16), which is highly homologous to hydrolase A in rats (15). Further potential products of such a hydrolytic pathway were identified (4-HO-BZE from 4-HO-COC, N-HO-BNE from N-HO-NorCOC, and 4-HO-BNE from 4-HO-NorCOC), but the enzymatic nature of the reactions could not be established due to the lack of reference substance of the precursors. Also, an enzymatic pathway was found for the cleavage of the benzoyl residue in COC, CE, NorCOC, and NorCE yielding EME, EEE, NorEME, and NorEEE, respectively. This is in accordance with the finding that COC and NorCOC are debenzoylated by rat liver (17); in humans, this reaction is catalyzed by the carboxylesterase isoform 2 (hCE-2) (18). However, these hydrolytic products were present only in small amounts in liver incubates and the enzymatic nature of the hydrolysis could not be distin-
a
X
3-HOCE
X
X X
4-HOCE
X X
4-HOCOC
X
3,4-diHOCE
X
3,4-diHOCOC
X X X X
X*
EME
X X X X
X* X* X* X*
BZE X
4-HOBZE
X X X X
X X X X
ECG COC
3-HONorCOC
BZE
EME
NorCOC X X X X X
X X X X
NorCOC
X X
X
4-HONorCOC
X
HO-MeONorCOC
X X X X
X
HO-MeOCE
X X X X
X*
EEE X* X* X* X*
BZE X
4-HOBZE
X X X X
X* X* X* X*
ECG
EEE
NorCE X X X X
CE X X X X
NorCE
X
3-HONorCE
X X
X
4-HONorCE
X
HO-MeONorCE
X X X X
X X X X
X* X* X* X
X
BNE
X
X* X X X
NorEEE
X
X
N-HO-3/4HO-NorCE X
N-OHNorCE
3-HO-BZE no products were detected in the incubations with liver, kidney, lung, and brain microsomes
X X X X
X* X* X* X
X
BNE
X
X* X X X
NorEME X
X
N-HO-3/4HO-NorCOC
X
N-OHNorCOC
BNE no products were detected in the incubations with liver, kidney, lung, and brain microsomes
X
HO-MeOCOC
Some hydrolytic metabolites are marked with an asterisk where it was detected in excess of the control, which indicates enzymatic hydrolysis.
X X X X
CE
X X X X
3-HOCOC
X
4-HOBNE
X
4-HOBNE
X X X
X
X
N-HOBNE
X X X
X
X
N-HOBNE
Chem. Res. Toxicol., Vol. 16, No. 3, 2003
liver kidney lung brain
liver kidney lung brain
liver kidney lung brain
liver kidney lung brain
liver kidney lung brain
liver kidney lung brain
liver kidney lung brain
COC
Table 2. Substances Detected (“X”) after Incubation of Reference Substances of COC and Its Metabolites with Rat Organ Microsomesa
378 Toennes et al.
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Figure 2. Metabolic pathways of COC and its metabolites in rat organ microsomes. The metabolites on the left side were detected with all microsome preparations while the metabolites on the right side were only detected with liver and kidney microsome preparation. Only substances are shown that were detected in the present study.
guished for kidney, lung, and brain microsomes. Nothing is known up to date on the tissue distribution of the enzyme involved, but this pathway seems to play only a minor role in vivo as in blood samples from COC-treated male rats the EME concentrations were less than 10% of the BZE concentrations (19). Traces of ECG, the product of both pathways, were detected in incubates with EME and BZE; however, the assay was not sensitive enough to investigate this pathway further due to its low extraction recovery (less than 20%). All organs studied exhibited oxidative metabolic activity, and N-demethylation of COC or CE and N-hydroxylation of the resulting NorCOC or NorCE were found with all microsome preparations. However, in brain microsome incubations, only minor amounts of N-HO-NorCOC and N-HO-NorCE were detected, which is in accordance with the finding of Benuck et al. (2) that mouse brain microsomes were considerably less active than those from liver in converting NorCOC to N-HO-NorCOC. This observation may be explained by the much lower P450 content of rat brain (10% of liver (3)). N-HO-NorCOC has received special attention as it has been shown to be an important intermediate in the metabolic cascade leading to hepatocellular damage (reviewed by Boelsterli and Go¨ldlin (1)). High doses of COC can lead to massive hepatic necrosis in laboratory animals, and COC is therefore regarded as a potential human hepatotoxin. The present results indicate that metabolism to N-OHNorCOC and potential cytotoxicity can also affect kidney and lung. The latter might be of relevance for the development of lung toxicity (20) in smokers of COC (crack) whose lungs are repeatedly exposed to high doses of COC.
Kidney microsomes produced essentially the same qualitative results as those from liver, but the peaks of the metabolites were much smaller, which can be explained by the markedly lower P450 content of rat kidney in comparison to liver (10% (21)). With brain and lung microsomes, significant differences in the metabolite compositions were noted. When COC, CE, NorCOC, and NorCE were incubated with liver or kidney microsomes, the hydroxylation in the 4-position of the aryl residue was as important as the N-dealkylation. With lung or brain microsomes, no aryl hydroxylation was found. Additional metabolites of COC, CE, NorCOC, and NorCE with a hydroxyl group in the 3-position and with hydroxylation at both positions (3,4-dihydroxy metabolites of COC and CE and hydroxy-methoxy metabolites of all four precursors) were also identified but only in very small amounts and only with liver microsomes indicating that this is an insignificant pathway in vitro. The dihydroxylated derivatives of NorCOC and NorCE were not detected, but their formation can be inferred from the detection of the hydroxy-methoxy derivatives (see below). Additional aryl hydroxylation of N-HO-NorCOC and N-HO-NorCE was also detected but only with liver microsomes and not with kidney microsomes, which can most probably be explained by insensitivity due to the lower P450 content. For N-HO-3/4-HO-NorCOC and N-HO-3/4-HO-NorCE, it could not be established whether the aryl hydroxylated nor derivative and/or the Nhydroxylated nor derivative was a substrate of the metabolizing enzyme because the respective reference substances were not available. The preference of the 4over the 3-position might indicate that a regioselectivity of the enzyme involved exists, which was also observed
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by Srinivasan et al. (7) in blood samples from COCtreated living rats. In comparison to humans, a marked species difference exists where this marked regioselectivity is not observed (11, 22). In the incubation mixtures with COC, CE, NorCOC, and NorCE methyl, derivatives of the respective dihydroxy metabolites were found whose formation can only be explained by residual methyl transferase activity and a limited amount of cofactor associated with the microsomes. This finding can be explained by results from another study on COC metabolism using perfused whole rat liver (data presented at the 39th Annual International Meeting of the International Association of Forensic Toxicologists in Prague, Czech Republic, 2001). We found that COC was metabolized by whole rat liver to HO-MeO-COC whose peak was even higher than that of 4-HO-COC (data on file, publication in progress). This indicated that dihydroxylation followed by methylation is a major metabolic pathway in rat liver and can account for the detection of a small amount of that metabolite in microsome incubates in the present study. Ecgonine methyl and ethyl ester were found to be substrates for N-demethylation with the microsome preparations from all organs, but only small amounts of these metabolites and no N-hydroxylated norecgonine derivatives were observed. Misra et al. (23) reported N-hydroxy-NorEME as a major metabolite in rat urine after the application of N-OH-NorCOC; however, the present experiments indicate that such a metabolite is produced from NorCOC in negligible amounts only. In the incubation mixtures with the acidic metabolites BZE, BNE, or 3-HO-BZE, no oxidative metabolites exhibiting a free carboxylic acid group were found. This is in accordance with a study on the metabolism of AEME (24) where N-demethylation was found for the parent compound but not for the hydrolytic metabolite anhydroecgonine whose structure is similar to BZE. However, Thompson et al. (25) found that BZE and ECG were N-demethylated by liver microsomes at a very low rate, which suggests that compounds such as BZE or BNE may have only very low affinity to microsomal oxidizing enzymes. Therefore, the presence of acidic metabolites in blood or urine may be due to chemical or enzymatic hydrolysis of the corresponding alkylester entities. In early literature, AEME (also known as methylecgonidine) was thought to be a metabolite of COC (5) but currently it is believed that this compound is produced only by pyrolysis of COC during the smoking process (26) and is therefore regarded as a marker of smoked COC. In our study, small peaks of AEME were found in the GC/MS analyses of all COC-containing samples. Equal amounts were detected in the microsome incubation mixtures and in the controls as well; therefore, its presence must be attributed to artificial pyrolysis of COC in the injection port of the GC (27) and it can be concluded that COC is not metabolized to AEME by rat organ microsomes.
Acknowledgment. We thank Kirstin Hoffmann for her help with the microsome incubations.
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