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0090-9556/97/2502-0163-0167$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 2

Induction of Two UDP-Glucuronosyltransferase Isoforms Sensitive to Phenobarbital that are Involved in Morphine Glucuronidation
Production of Isoform-Selective Antipeptide Antibodies toward UGT1.1r and UGT2B1

Yuji Ishii, Atsuko Takami, Kazuoki Tsuruda, Akiko Kurogi, Hideyuki Yamada, and Kazuta Oguri

Faculty of Pharmaceutical Sciences, Kyushu University

    Abstract
Abstract
Introduction
Results
Discussion
References

We document here in that two UDP-glucuronosyltransferase (UGT) isoforms sensitive to phenobarbital are involved in morphine glucuronidation in Wistar and Sprague-Dawley rats. The hepatic microsomal morphine UGT activity in untreated Gunn rats was significantly less than that of untreated Wistar rats. Although the morphine UGT activity in the liver of Gunn rats was increased by phenobarbital (PB) treatment, this was significantly less than that in the liver of PB-treated Wistar rats. UGT1.1r is an isoform of morphine UGT in rat, and UGT2B1 is also considered an isoform of morphine UGT, because UGT2B1 (stably expressed in V79 cells) exhibited morphine UGT activity. We prepared specific antipeptide antibodies against UGT1.1r and UGT2B1. Using isoform-specific antipeptide antibodies, both UGT1.1r and UGT2B1 in Wistar and Sprague-Dawley rats were inducible by PB treatment. However, UGT1.1r is not present in the liver from Gunn rats. This study is the first demonstration that protein levels of two morphine UGT isoforms, UGT1.1r and UGT2B1, in the liver of Wistar and Sprague-Dawley rats are inducible by PB treatment.

    Introduction
Abstract
Introduction
Results
Discussion
References

Morphine is an important analgesic used in cardiac infarction and late stages of cancer. Morphine is metabolized predominantly to two glucuronide isomers: M-3-G1 and M-6-G (1). The major metabolite, M-3-G, does not possess analgesic activity, whereas the minor metabolite, M-6-G, is more potent than morphine (2). The formation of M-6-G is very low in rats and mice, but is comparatively high in guinea pigs (3) and humans (4).

UGTs(EC 2.7.1.17) capable of glucuronidating morphine have been purified and characterized from untreated- and PB-treated rat liver (5-8). These isoforms are quite specific for morphine. Although UGT2B12 (9) expressed in Chinese hamster lung fibroblast V79 cell also exhibits glucuronidation activity toward morphine, it also forms ether- and ester-type glucuronides of various compounds (10). Phenols and alcohols (morphine, 4-methylumbelliferone, 4-hydroxybiphenyl, chloramphenicol, and testosterone), as well as a series of both endogenous (medium-chain and long-chain polyunsaturated fatty acids and bile acids) and exogenous (nonsteroidal antiinflammatory drugs, fibrates, and sodium valproate) carboxylic acids are all substrates for UGT2B1 (10). However, purification of UGT2B1 has not been reported; we recently purified a counterpart of UGT2B1 from PB-treated dog liver (UGTDOG-PB) (11).

Morphine UGT activity in rat liver is inducible by PB treatment (5). We purified an isoform of morphine UGT (morphine UGTPB) (8) from PB treated Sprague-Dawley rat liver. Morphine UGTPB possesses an identical N-terminal to UGT1.1r (12), which is not present in Gunn rat liver. Coffman et al. (13) demonstrated that stably expressed UGT1.1r is capable of glucuronidating morphine. In this study, we prepared isoform-specific antipeptide antibodies toward two morphine UGT isoforms: UGT1.1r and UGT2B1. Protein expression levels of the two isoforms in rat liver were investigated using the isoform-selective antibodies with and without PB pretreatment.

We report here in that PB induces two morphine UGT isoforms (UGT1.1r and UGT2B1) in Wistar and Sprague-Dawley rats and that the former is not present in Gunn rat liver.

Materials and Methods

Morphine hydrochloride was purchased from Takeda Chemical Industries, Ltd. (Osaka, Japan). UDP-glucuronic acid was obtained from Seikagaku-Kogyo Co. (Tokyo, Japan). Sodium PB and epsilon -amino-n-caproic acid were purchased from Tokyo Kasei Industries Co., Ltd. (Tokyo, Japan). Egg yolk L-alpha -phosphatidylcholine was purchased from Sigma Chemical Co. (St. Louis, MO). Brij 58 and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Sepharose 4B was purchased from Pharmacia LKB (Uppsala, Sweden). N-Hydroxysuccinimide was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). M-3-G was prepared by the method described previously (14-16).

Animals and Treatments. Male Sprague-Dawley rats (80-90 g) were purchased from Charles River Japan (Yokohama, Japan). Male Wistar rats and male homozygous Gunn rats were obtained from Nippon SLC (Shizuoka, Japan). Animals were housed in stainless-steel cages for 1 week before treatment. Sodium PB (80 mg/kg/day in saline) was administered intraperitoneally for 4 days, whereas control rats received vehicle. Each group was composed of four animals. After the last injection, rats were starved for 20 hr and their livers were removed.

Preparation of Enzyme. Microsomes were prepared individually and suspended in buffer A [25 mM Tris-HCl buffer (pH 7.4) containing 20%(v/v) glycerol, 5 mM MgCl2, and 1 mM dithiothreitol] and stored at -80°C until used. Solubilization of microsomes was performed as described previously (8). Purification of morphine UGTPB was described previously (8).

Production of Antipeptide Antibody. To select suitable peptide sequences for the production of antipeptide antibodies specific for UGT1.1r and UGT2B1, the G-structure (17) and surface probability (18-20) of UGT1.1r and UGT2B1 were determined using the software GeneWorks (Teijin, Tokyo, Japan). According to Van Regenmortel and Daney de Marcillac (21), we chose candidate sequences with a high score of surface probability and without a rigid conformation such as the alpha -helix and beta -sheet. Those corresponded to residues 80-90 (KYPVPFQNENV) of the UGT1.1r precursor and residues 63-73 (LIEPTKESSIN) of the UGT2B1 precursor. None of the corresponding sequences of other rat liver UGTs registered on the GenBank has the same continuous sequence.

We synthesized the peptide sequences that possess the Cys SH group at the N-terminal using an Applied Biosystems fully automatic 431A peptide synthesizer. After cleavage from the resin, the peptide was purified using an Applied Biosystems 151 peptide separation system and lyophilized. The amino acid sequence of the purified peptide was confirmed using an Applied Biosystems 473A protein sequencer. Two milligrams of the synthesized peptide for UGT1.1r was mixed with 2 mg maleimide-activated KLH (Imject Immunogen Activated Immunogen Conjugation kits, Pierce, Rockford, IL). In the case of UGT2B1, the synthetic peptide was conjugated with maleimide-activated BSA (Pierce). The peptide-carrier conjugate was purified as described in the kit instructions. Two male New Zealand White rabbits were immunized with an emulsion of the peptide-carrier (100 µg) conjugate and BACTO complete Freund's adjuvant (Difco Lab, Detroit, MI). A booster was given 2 weeks after initial injection; blood was collected 1 week after the last booster, and serum was prepared.

Anti-UGT1.1r-peptide antibodies were observed in only 1 of the 2 rabbits used in this study. However, anti-UGT2B1-peptide antibodies were observed in both rabbits used in this study, but the titer differed markedly between the rabbits. The anti-UGT2B1-peptide serum obtained from the rabbit with high titer was further purified and used in the present study as described herein.

Purification of Antipeptide Antibody. Preparation of CH-Sepharose 4B. Activation of the Sepharose 4B gel with CNBr was performed according to Tukey and Tephly (22). epsilon -Amino-n-caproic acid was dissolved in 0.1 M NaCO3 buffer (pH 9.5) and mixed with CNBr-activated Sepharose 4B gel at a ratio of 20 µmol of epsilon -amino-n-caproic acid to 1 ml gel. The coupling reaction was performed at 4°C for 24 hr. Reactive groups that remained on the prepared CH-Sepharose 4B gel were blocked with 0.5 M Tris-HCl buffer (pH 7.4) for 1 hr. Coupling efficiency was determined by the usual fluorescamine method (23) and was higher than 18 µmol/ml gel. The gel was named CH-Sepharose 4B gel and used as described herein.

Conjugation of the Synthetic Peptide with the CH-Sepharose 4B Gel. CH-Sepharose 4B gel was activated with N-hydroxysuccinimide according to Cuatrecasas and Parikh (24), with slight modifications. A water-soluble carbodiimide was used instead of dicyclohexylcarbodiimide. Five milliliters of CH-Sepharose 4B gel was suspended in 0.1 N HCl for 1 hr, and then filtered through a glass-filter and washed with water until the washings were neutral. The gel was suspended in 15 ml of 0.1 M N-hydroxysuccinimide, and the pH was adjusted to 4.8. The suspension was generously mixed by magnetic stirrer, then 0.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added over a 5-min period. Mixing was continued for 30 min. The pH was maintained between 4.5 and 5.0, with 1 N HCl and 1 N NaOH during this period. Then, the activated CH-Sepharose 4B gel was filtered through a glass filter and washed with 10 volumes of water and coupling buffer [0.1 M Na2CO3 buffer (pH 8.0) containing 0.5 M NaCl]. Amounts of the synthesized peptide were coupled with activated CH-Sepharose 4B gel (ratio of 2.5:1) in the coupling buffer. The gel was then washed with 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl and 0.1 M sodium acetate buffer (pH 4.0) containing 0.5 M NaCl.

Purification of Specific IgG by Affinity Chromatography. Anti-UGT1.1r-peptide IgG was purified using a UGT1.1r-peptide-coupled CH-Sepharose 4B column according to Imajoh-Ohmi et al. (25). The purified anti-UGT1.1r-peptide IgG was supplemented with BSA to give a final concentration of 1 mg/ml and stored at -20°C until used.

For the purification of anti-UGT2B1-peptide antibodies, the IgG fraction was obtained by ammonium sulfate precipitation of the antiserum according to Nakamura and Onoue (26). The IgG fraction, dissolved in PBS was applied to the UGT2B1-peptide-coupled CH-Sepharose 4B column (0.9 × 4 cm, 2.5 ml) equilibrated with PBS. The column was washed with PBS, and the IgG specific for UGT2B1-peptide was eluted with 0.1 M glycine-HCl buffer (pH 3.0). Resulting fractions were immediately neutralized with 1 M glycine-NaOH buffer (pH 11.3). The purified anti-UGT2B1-peptide IgG was dialyzed against PBS and stored at -20°C until used.

Immunoblotting. SDS-PAGE was performed on a 7.5% (w/v) acrylamide gel according to Laemmli (27). After separation by SDS-PAGE, the proteins in the gel were transferred onto a PVDF membrane (Immobilon P, Millipore, Bedford, MA) according to the method of Towbin et al. (28), with slight modifications as follows. The transfer buffer that contained 0.02% (w/v) SDS was used for its good transfer efficiency. Immunochemical visualization was performed according to Guengerich et al. (29), with the modifications described herein. Double-blocking was performed according to Koga et al. (30). Blocking was conducted with 5% (w/v) skimmed milk (Snow Brand, Sapporo, Japan) in TBS-Triton [20 mM Tris-HCl buffer (pH 7.5): 0.15 M NaCl: 0.05% (w/v) Triton X-100] at 37°C for 30 min and subsequently with 20% (v/v) normal goat serum (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) in TBS-Triton at 37°C for 30 min. Then, the membrane was soaked and gently shaken in primary antibody solution consisting of purified anti-UGT1.1r-peptide IgG (5 µg/ml), 0.5% (v/v) normal goat serum, 0.5% (w/v) BSA, 3% (w/v) skimmed milk, and 0.1% (w/v) NaN3 in TBS-Triton at 4°C for overnight. The PVDF membrane was washed with washing buffer [10 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl, 0.2% (w/v) SDS, and 0.5% (w/v) Triton X-100]. Secondary antibody, goat anti-rabbit IgG, was purchased from Organon Teknika N. V.-Cappel Product (Durham, NC). A peroxidase-anti-peroxidase (PAP, soluble complex of horseradish peroxidase and antihorseradish peroxidase) was obtained from DAKO A/S (Glostrup, Denmark). The washing buffer was used to rinse the membrane during each step. Visualization was performed using ECL light detection on Hyperfilm-ECL (Amersham, Buckinghamshire, UK) in the dark.

Assays. Morphine UGT activity was determined as described previously (3, 4, 7). Protein was estimated according to Lowry et al. (31).

    Results
Abstract
Introduction
Results
Discussion
References

Comparison of Morphine UGT Activity between Homozygous Gunn Rats and Normal Wistar Rats. Figure 1 compares morphine UGT activity between Wistar rats and homozygous Gunn rats. The hepatic microsomal morphine UGT activity in the untreated Gunn rats was significantly less than that of untreated Wistar rats. The morphine UGT activity in Gunn rat liver was increased by PB treatment, but significantly less than that in Wistar rat liver.


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  		Fig. 1.   Comparison of morphine UGT activity in liver microsomes between Wistar and homozygous Gunn rats, and their inducibility by PB treatment.

Each group was composed of four animals. Values represent mean ± SE from three determinations. Microsomes were preincubated with Brij 58 at a final concentration of 0.05% (w/v) for 30 min at 4°C before incubation. aSignificantly different from Wistar control group (p < 0.001); bsignificantly different from Wistar PB group (p < 0.03); csignificantly different from Gunn control group (p < 0.001).

Immunoblotting. The UGT1.1r band was observed in liver microsomes of untreated Wistar and Sprague-Dawley rats by immunoblotting with anti-UGT1.1r-peptide antibody (fig. 2). UGT1.1r was inducible by PB treatment. However, no UGT1.1r was detectable in homozygous Gunn rat liver microsomes. This result supports the finding that the homozygous Gunn rat is deficient in liver microsomal UGT1.1r (12). The anti-UGT1.1r-peptide antibody recognized purified morphine UGTPB and the partially purified preparation (fig. 3). It seems reasonable to suppose that morphine UGTPB is identical to UGT1.1r.


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  		Fig. 2.   Immunoblotting of rat liver microsomes with purified anti-UGT1.1r-peptide antibody.

(A) Lanes 1 and 2, liver microsomes of untreated and PB-treated homozygous Gunn rats (10 µg protein); lanes 3 and 4, liver microsomes of untreated and PB-treated Wistar rats (10 µg protein); and lanes 5 and 6, liver microsomes of untreated and PB-treated Sprague-Dawley rats (10 µg protein), respectively. Typical data from one animal in each group are shown. (B) Area of the band of UGT1.1r in (A) was enlarged.


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  		Fig. 3.   Immunoblotting of morphine UGTPB and that in the preparations involving a purification procedure with purified anti-UGT1.1r-peptide antibody.

Lanes 1 and 2, liver microsomes of untreated and PB-treated Sprague-Dawley rats (10 µg protein); lane 3, Emulgen 911-solubilized liver microsomes of PB-treated Sprague-Dawley rats (30 µg protein); lane 4, fractions from peak II of omega -(beta -carboxypropionylamino)octyl Sepharose 4B column chromatography (2 µg protein) (8); and lane 5, purified morphine UGTPB (0.5 µg protein).

A minor band above the UGT1.1r is an unknown protein that was recognized with preimmune serum, as well as immune serum (data not shown). The unknown protein band shown in fig. 2 may be due to some contamination of the purified anti-UGT1.1r-peptide IgG preparation, because antibody toward the unknown band still has a high titer after several immunizations with emulsion composed of UGT1.1r-peptide-KLH conjugate and Freund's complete adjuvant.

UGT2B1 is considered a PB-inducible form, because the mRNA is raised by PB treatment (32). In this study, PB inducibility of UGT2B1 protein was confirmed by immunoblotting. Figure 4 shows that anti-UGT2B1-peptide antibody recognized a single protein in liver microsomes of the three strains of untreated rat used in this study. Because the band in those strains was similarly increased by PB treatment, UGT2B1 protein is normally expressed and inducible by PB treatment in the homozygous Gunn rat.


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  		Fig. 4.   Immunoblotting of rat liver microsomes with purified anti-UGT2B1-peptide antibody.

(A) Lanes 1 and 2, liver microsomes of untreated and PB-treated Sprague-Dawley rat (15 µg protein); lanes 3 and 4, liver microsomes of untreated and PB-treated Wistar rats (15 µg protein); and lanes 5 and 6, liver microsomes of untreated and PB-treated homozygous Gunn rats (15 µg protein). Typical data from one animal in each group are shown. (B) Band of UGT2B1 in (A) was enlarged.

    Discussion
Abstract
Introduction
Results
Discussion
References

Morphine UGT activity in homozygous Gunn rats was significantly lower than that of Wistar rats, irrespective of whether rats were PB-treated or not. Boutin et al. (33) also demonstrated that Gunn rats possess only 50% of the morphine UGT activity in Wistar rats. We have suggested that a PB-inducible morphine UGT isoform, morphine UGTPB, is identical to UGT1.1r that is not present in the Gunn rat liver (8). Coffman et al. (13) reported that stably expressed UGT1.1r catalyzes morphine glucuronidation. This fact was supported by the finding that morphine UGTPB was recognized with specific antipeptide antibody toward UGT1.1r (fig. 3). The present results support the hypothesis that two morphine UGT isoforms, UGT1.1r and UGT2B1, are PB-inducible in Wistar and Sprague-Dawley rat liver and that one of those is absent from Gunn rat liver. The morphine UGT activity of Emulgen 911-solubilized liver microsomes from the Wistar rat can be separated into two peaks, peak I and peak II, by omega -(beta -carboxypropionylamino)octyl Sepharose 4B column chromatography (8). Although the elution profile suggests that peak II is not present in Gunn rat liver, the morphine UGT in peak I also seems to be PB-inducible in the homozygous Gunn rat (8). This could be due to normally expressed UGT2B1, which is capable of glucuronidating morphine in homozygous Gunn rat liver. Recently, Coffman et al. have purified UGT1.1r and UGT2B1 from PB-treated female Wistar rats with low glucuronidating activity for 3alpha -hydroxysteroid (34). The two purified UGT isoforms are capable of glucuronidating opioids, including morphine; however, their sensitivities to PB have not been determined.

Emi et al. (35) recently investigated the mRNA level of UGTs by RT-PCR, with total RNA as a template. They concluded that the mRNA level of UGT1.1r in PB-treated rat liver was comparable with that in untreated rat liver, whereas the mRNA level of UGT2B1 was markedly increased by PB, but not MC treatment (35). However, an MC-mediated increase in UGT2B1 mRNA has been reported by another group (36). In addition, Hanioka (37) reported that MC induces morphine UGT activity in rat liver ~2-fold. Garbay et al. (38) suggested that care should be taken when semiquantitative gene expression is evaluated by RT-PCR, because pseudogenes of internal standards such as beta -actin and glyceraldehyde-3-phosphate dehydrogenase interfere with RT-PCR control. These authors suggest that it is uncertain whether the UGT1.1r mRNA level is sensitive to PB. Ikushiro et al. (39) reported that the protein expression level of UGT1.1r is not affected by PB treatment. They also reported that the p-nitrophenol UGT activity in liver microsomes of Wistar rats was not affected by PB treatment (39). On the contrary, p-nitrophenol UGT activity was reported to be inducible by PB treatment (5, 37, 40). The liver microsomal p-nitrophenol UGT activity in Wistar rats in the present study was also significantly induced 2.4-fold by PB treatment (p < 0.001, data not shown). It is uncertain whether such differences in the observations actually occurred; however, a difference in age of animals is a possible cause. Our preliminary results demonstrate that the transfer efficacy of UGT1.1r to PVDF membrane is low when the electroblotting procedure is performed according to Towbin et al. (28)(data not shown). In this study, we used the transfer buffer that contained 0.02% (w/v) SDS for its good transfer efficacy. Our efficient electroblotting procedure may be another cause for the difference in the conclusions. Recent work in Tephly's group demonstrated that purified (34) and stably expressed (13) UGT1.1r exhibits glucuronidation activity toward opioids and bilirubin and no bilirubin UGT activity was detectable in UGT2B1 (34). Administration of PB to animals and humans increases bilirubin clearance (41 and references therein). Bilirubin UGT activity in rat liver is also known to be PB-inducible (5, 40, 42), whereas that in guinea pig liver is coplanar polychlorinated biphenyl-inducible (43, 44). Inducibility of UGT activity toward bilirubin in liver microsomes of rat by PB treatment is less than that toward morphine (5, 42). The present results suggest that UGT1.1r and UGT2B1 protein are PB-inducible. It seems reasonable to suppose that the greater inducibility of UGT activity toward morphine than bilirubin is due to the two PB-inducible morphine UGT isoforms: UGT1.1r and UGT2B1.

The purified morphine UGTPB was recognized by antipeptide antibody specific toward UGT1.1r, whereas the migration on SDS-PAGE was slightly different from that of the fractions of peak II from which morphine UGTPB was purified. Matsui and Nagai (45) have described that androsterone UGT, which was purified using chromatofocusing and UDP-hexanolamine Sepharose 4B column chromatography, exhibited different migration behavior on SDS-PAGE compared with the sample purified using DEAE-cellulose and UDP-hexanolamine Sepharose 4B column chromatography. Although it is unclear why the migration on SDS-PAGE differes, the molecular mass on SDS-PAGE may be altered during the chromatofocusing process.

This study suggests that two morphine UGT isoforms, UGT1.1r and UGT2B1, are inducible by PB treatment and involved in morphine glucuronidation in Wistar and Sprague-Dawley rat livers.

Note Added in Proof. During the submission of this manuscript, Coffman et al. (34) reported the purification of two UGT isoforms in rat liver that are capable of catalyzing morphine glucuronidation.

    Acknowledgments

We thank Dr. Y. Tanaka in the Faculty of Pharmaceutical Sciences, Kyushu University, Japan, for his useful suggestions about efficient immunoblotting technique. We are indebted to Ms. S. Miura and Ms. N. Terashita for their excellent assistance.

    Footnotes

Received July 10, 1996; accepted November 13, 1996.

   This work was supported in part by the Sasakawa Scientific Research Grant from the Japan Science Society. Presented at the 10th annual meeting of the Japanese Society for Xenobiotics, Ohmiya, Japan, November 1995 [Y. Ishii, A. Takami, K. Tsuruda, A. Kurogi, H. Yamada, and K. Oguri: Xenobiot. Metab. Dispos. 10 (Suppl.), 341 (abstr.) (1995)].

2   A new nomenclature for the designation of UDP-glucuronosyltransferase (9) was used.

Send reprint requests to: Dr. Kazuta Oguri, Faculty of Pharmaceutical Sciences, Kyushu University-62, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-82, Japan.

    Abbreviations

Abbreviations used are: M-3-G, morphine-3-glucuronide; M-6-G, morphine-6-glucuronide; UGT, UDP-glucuronosyltransferase; PB, phenobarbital; UGT1.1r and UGT2B1, gene products of UGT1.1r and UGT2B1; KLH, Keyhole Limpet Hemocyanin; BSA, bovine serum albumin; CH-Sepharose 4B, omega -carboxypentylamino Sepharose 4B; CNBr, cyanide bromide; IgG, immunoglobulin G; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; TBS-Triton, Tris-buffered saline:Triton X-100; ECL, enhanced chemiluminescence; RT-PCR, reverse transcriptase-polymerase chain reaction; MC, 3-methylcholanthrene.

    References
Abstract
Introduction
Results
Discussion
References

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