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Cell Biology International (2006) 30, 665–671 (Printed in Great Britain)
Role of γ-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae
Vira M. Ubiyvovka, Oleksandra V. Blazhenkoa, Daniel Gigotb, Michel Penninckxc and Andriy A. Sibirnyad*
aInstitute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine
bUniversité Libre Bruxelles, Laboratory of Microbiology, Jean Wiame Institute, 1 Av E Gryson, B-1070 Brussels, Belgium
cUniversité Libre Bruxelles, Laboratory of Microbial Physiology and Ecology, 642 rue Engeland, B-1180 Brussels, Belgium
dRzeszów University, Department of Metabolic Engineering, Cwiklinskiej 2, 35-310 Rzeszów, Poland


GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding γ-glutamyltranspeptidase (γGT). This is confirmed by the absence of the corresponding activity of γGT in the mutant with disrupted GGT1 gene. It was shown that γGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a γGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.

Keywords: γ-glutamyltranspeptidase, Gene cloning, Yeast, Hansenula polymorpha, Saccharomyces cerevisiae, Xenobiotics, Detoxification.

*Corresponding author. Institute of Cell Biology, National Academy of Sciences of Ukraine, Molecular Genetics and Biotechnology Department, Drahomanov Street, 14/16, Lviv 79005, Ukraine. Tel.: +380 322 740 363; fax: +380 322 721 648.

1 Introduction

Glutathione (l-γ-glutamyl-l-cysteinylglycine, GSH) is a ubiquitous peptide participating in cell defense against oxidative stress, detoxification of xenobiotics and heavy metals (Meister and Anderson, 1983; Penninckx and Elskens, 1993; Penninckx, 2000, 2002; Pocsi et al., 2004). Mechanisms of GSH participation in xenobiotic detoxification have not yet been elucidated. It is known that GSH produces conjugates with electrophilic xenobiotics which can either be excreted from cells or further metabolized via the so-called mercapturic acid pathway in higher eukaryotes (Tsuchida, 1997; Ishikawa et al., 1997; Parkinson, 2001). N-acetylation of the glutamate residue of GSH conjugates can also occur in mammalian cells (Yin et al., 2004), as well as the coupling of GSH conjugates and products from the mercapturic acid pathway with glutamic acid (Mutlib et al., 2000). In unicellular eukaryotes, it has been found that GSH is important for electrophilic xenobiotic vacuolar accumulation and extrusion from baker's yeast Saccharomyces cerevisiae and methylotrophic yeast Hansenula polymorpha (Li et al., 1996; St-Pierre et al., 1994; Zadzinski et al., 1996; Ubiyvovk et al., 2003). However, processing of observed GSH conjugates in yeasts has not been studied. One may assume that γ-glutamyltranspeptidase (γGT), an enzyme of the first step of GSH catabolism, is involved in processing GSH-xenobiotic conjugates. In S. cerevisiae, mutant cis2 defective in γGT, manifested increased susceptibility towards oxidative stress, however, the effect of electrophilic xenobiotics on such mutants has not been studied in detail (Elskens et al., 1991; Elskens and Penninckx, 1997; Springael and Penninckx, 2003).

Metabolism of methanol in yeasts requires GSH-dependent detoxification of toxic methanol oxidation intermediates, such as formaldehyde, hydrogen peroxide and alkyl hydroperoxides, which occurs in formaldehyde dehydrogenase, formaldehyde reductase and GSH peroxidase reactions (Sahm, 1977; Sibirny et al., 1990; Sakai et al., 2001). Methanol-induced cells of methylotrophic yeast wild type strains, and especially of formaldehyde dehydrogenase- and formaldehyde reductase-deficient mutants, accumulate elevated levels of both formaldehyde and GSH (Ubiyvovk and Trotsenko, 1986; Sibirny and Ubiyvovk, 1988; Sibirny et al., 1990). It is known that GSH non-enzymatically binds formaldehyde producing non-toxic S-hydroxymethylglutathione (Sahm, 1977).

In this communication, we describe the isolation and characterization of the mutant of the methylotrophic yeast H. polymorpha defective in γGT. It was shown that γGT is important for detoxification of electrophilic xenobiotics (monobromobimane and N-[1-pyrene]maleimide) in both H. polymorpha and S. cerevisiae. We hypothesize that GSH conjugates with the studied xenobiotics are metabolized through the mercapturic acid pathway. The obtained data could have implications for the study of GSH-dependent multidrug resistance in some mammalian tumours and detoxification of herbicides in plants.

2 Materials and methods

2.1 Yeast strains and media

H. polymorpha strains used were CBS4732 leu2-2 ura3-20 (kindly provided by K. Lahtchev, Bulgaria) and originated from it: CBS4732 leu2-2∷LEU2 ura3-20 wild type strain and null mutant ggt1∷LEU2 ura3-20 (preparation described later). The S. cerevisiae strains were: wild type – Y1000 MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0 (BY4774) and null mutant Y15209 MATα, his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YLR299w::kanMX4 (cis2; ecm38). The strains were grown in YPD medium (2% glucose, 2% bactopeptone, 1% yeast extract with or without 2% agar). Synthetic medium 1 used for fluorescence microscopy and electrophile transport studies of H. polymorpha and S. cerevisiae contained the following compounds (per litre): 30g glucose, 0.7g MgSO4·7H2O, 1g KH2PO4, 0.4g CaCl2·2H2O, 0.5g NaCl, 1g K2SO4, 10.5g citric acid and 8.5g KOH, 26.4g (NH4)2SO4, pH 6.5, supplemented with vitamins and trace elements (Mehdi and Penninckx, 1997). Synthetic medium 2 for studying growth phenotype of H. polymorpha contained 10g/l glucose and was supplemented with vitamins and trace elements (Sibirny et al., 1990). Both types of synthetic medium were supplemented with the appropriate amounts of amino acids and nucleic bases.

2.2 Construction of HpGGT1 deleted cassette and null Hpggt mutant isolation

General DNA manipulation was performed as described in Sambrook et al. (1989). To create the Hpggt1∷LEU2 allele in which HpGGT1 gene was disrupted with ScLEU2 gene, the N′-end fragment (433bp upstream from nucleotide N9 within the HpGGT1 chromosomal gene numbered from the start codon) and C′-end fragment (836bp downstream from nucleotide N1440 within the HpGGT1 chromosomal gene numbered from the start codon) were amplified by PCR using primers (Table 1) (VU′1F and VU′2R for N′-end; VU′3F and VU′4R for C′-end), and genomic DNA of H. polymorpha CBS4732 leu2. The N′-end of HpGGT1 gene was cloned as HindIII/PstI fragment into the HindIII/PstI sites of pYT1 plasmid harboring ScLEU2 gene (Tan et al., 1995). The latter plasmid was digested by XbaI and BamHI and ligated with XbaI/BamHI C′-end fragment of HpGGT1 gene. The resulting plasmid, pYΔHpGGT1, was digested with HindIII and SacI releasing 3.47kb fragment comprised of ScLEU2 flanked by HpGGT1 5′ (N-end) and 3′ (C-end) sequences and transformed into H. polymorpha CBS4732 leu2-2 ura3-20 wild type cells by electroporation (Ubiyvovk et al., 2002a). γGT disruptants were picked up among Leu+ transformants. Correct replacement of wild type gene to disrupted gene was confirmed by PCR analysis with two sets of primers: VU′14F and VU′4R for the wild HpGGT1 gene and VU′32F and VU′31R for null Hpggt1 gene (Table 1).

Table 1.

Oligonucleotides used as primers for PCR amplification

Primers for construction of disruption cassette (Hpggt1two colonsScLEU2) from H. polymorpha CBS4732 leu2 genomic DNA
N-end primers

C-end primers

Primers for confirmation of null HpGGT1 mutation in H. polymorpha
Primers for identification of wild type HpGGT1 gene

Primers for identification of null Hpggt1 gene

2.3 Construction of H. polymorpha CBS4732 leu2-2∷LEU2 ura3-20 wild type strain

H. polymorpha CBS4732 leu2-2∷LEU2 ura3-20 wild type strain was obtained by the electroporation of H. polymorpha CBS4732 leu2-2 ura3-20 wild type cells with vector pYT1 linearized in PstI restriction site. Leu+ integrants were selected on glucose synthetic medium 2 without leucine.

2.4 Growth of H. polymorpha cells on various sulfur and nitrogen sources

The regulation of γGT activity in H. polymorpha was studied in nitrogen-deficient synthetic medium 2 without (NH4)2SO4 or sulfur-deficient media, with substitution of MgSO4 and (NH4)2SO4 by MgCl2·6H2O and NH4Cl (per litre: 0.4g and 2.8g, respectively). Sulfur-deficient media was supplemented with 0.1mM GSH or 26.5mM (NH4)2SO4; nitrogen-deficient media with 5mM glutamate or 1mM, 1.5mM, 2.5mM GSH or 26.5mM (NH4)2SO4. Yeast cells were grown overnight in synthetic medium 2 and transferred into 40ml of indicated media with initial OD590nm&007E;0.001. Cells from late logarithmic phase were harvested, washed twice with water and frozen at −20°C for measurement of γGT activity and protein content (Lowry et al., 1951).

2.5 γGT activity assay

The method of Payne and Payne (1984) was used with some modifications. Cell-free extracts (0.1–0.2mg of protein) were incubated in 0.5ml of reaction mixture, containing 0.1M Tris–HCl pH 7.5 and 2.5mM l-glutamic acid γ-(p-nitroanilide) for 3–4h at 37°C. The reaction was stopped by the addition of 0.3ml 3.5M acetic acid. Samples were centrifugated at 10,000×g for 5min. Formation of p-nitroaniline was measured in supernatant spectrofotometrically at 410nm (Mε 8800M−1cm−1). Enzyme activity was expressed in μmolh−1mgprotein−1.

2.6 Fluorescence microscopy of H. polymorpha and S. cerevisiae cells

Cells (wild type strains and null ggt mutants) grown in synthetic medium 1 to OD&007E;1.0–2.0 were washed and resuspended to OD&007E;1.0 in 10ml of the same fresh medium containing 100μM monobromobimane. After growth for 3h at 26°C, the cells were pelleted by centrifugation, washed twice with 0.1M Na-phosphate buffer pH 7.2. A portion of the cells grown for 3h (3h-0) was immediately resuspended with OD&007E;1.0 in 0.1M Na-phosphate buffer pH 7.2 containing 3% glucose and incubated for another 3h (3h-3) and 16h (3h-16) at 26°C. All cells – 3h-0, 3h-3 and 3h-16 were examined without fixation under a Nikon fluorescence microscope and phase-contrast attachment.

2.7 Transport of electrophiles out of H. polymorpha and S. cerevisiae cells

Cells (wild type strains and null ggt mutants) grown in synthetic medium 1 to OD&007E;1.0–2.0 were washed and resuspended to OD&007E;1.0 in the same fresh medium containing 100μM monobromobimane (cell I) or without electrophiles (cell II). After growth for 3h at 26°C, cells I and II were pelleted by centrifugation and washed twice with cold water. After that cells I and II were immediately resuspended in 1–2ml (OD&007E;50.0) of 0.1M Na-phosphate buffer pH 7.2 with 3% glucose and incubated for 1h at 26°C (buffer for cells II additionally contained 30μM N-[1-pyrene]maleimide). Supernatants of the centrifuged cells were stored frozen at −20°C and then applied to a C18μ column to study monobromobimane and N-[1-pyrene]maleimide derivatives in extracellular medium.

2.8 High-performance liquid chromatography (HPLC)

The derivatives were eluted from a C18μ Bondapak column (300×39mm) in a Waters HPLC apparatus with a linear gradient of acetonitrile (0–100%) containing 0.05% trifluoroacetic acid and were detected by measuring the absorbance at 340nm for N-[1-pyrene]maleimide derivatives, and at 400nm for monobromobimane derivatives. GSH-conjugated derivatives as well as cysteine and N-acetylcysteine-conjugated derivatives of N-[1-pyrene]maleimide and monobromobimane were identified using authentic compounds synthesized non-enzymatically as described earlier (Ubiyvovk et al., 2003). As N-[1-pyrene]maleimide-SH-containing conjugates could form dimers (G. Bartosz, personal communication), we identified two peaks of GSH-conjugated N-[1-pyrene]maleimide (retention time: 19.2; 19.8min); cysteine- N-[1-pyrene]maleimide (retention time: 19.8; 20.8min); N-acetylcysteine-N-[1-pyrene]maleimide (retention time: 19.9; 21.1min).

3 Results and discussion

3.1 Functional analysis of HpGGT1 gene

H. polymorpha genome was searched for the presence of the gene coding for γGT, involved in GSH catabolism. Two ORFs, HpGGT1 (putative product, γGT1) and HpGGT2 (putative product, γGT2), were identified based on their primary sequence homology to S. cerevisiae CIS2/ECM38/YLR299w gene.

Protein sequence analysis revealed that H. polymorpha putative γGT1 protein shares the strongest homology with S. cerevisiae Cis2p (42% identity and 59% similarity). H. polymorpha putative γGT2 protein possesses weaker homology to S. cerevisiae Cis2p (21% identity and 38% similarity) (Fig. 1).

Fig. 1

Alignment of the deduced amino acid sequences of the putative γGT1 and γGT2 proteins from H. polymorpha (H. pol.) with the homologous protein Cis2p from S. cerevisiae (S. cer.).

It is well known that yeast γGT catalyzes the transfer of the γ-glutamyl moiety of GSH and γ-glutamyl compounds to amino acids and also the hydrolytic release of l-glutamate from GSH, γ-glutamyl compounds and S-substituted derivatives of GSH (Penninckx and Elskens, 1993). In the present study, we have determined that in H. polymorpha hydrolytic activity to γ-glutamyl compounds (γ-glutamyl-p-nitroanilide) strongly increased in sulfur or nitrogen-deficient conditions (Table 2). Similar regulation of CIS2 gene expression by sulfur and nitrogen sources was demonstrated for S. cerevisiae (Springael and Penninckx, 2003). This activity was significantly reduced in H. polymorpha null ggt1 mutant in all studied media (Table 2). It is assumed that γGT has a specialized role for utilizing vacuolar stores of GSH during nitrogen or sulfur starvation conditions (Elskens et al., 1991; Mehdi and Penninckx, 1997), yet utilization of GSH as an exogenous sulfur source is thought to be independent of γGT in S. cerevisiae (Kumar et al., 2003).

Table 2.

Effect of sulfur (ammonium-containing medium) and nitrogen (sulfate-containing medium) on the activity of γGT (μmol h−1 mg protein−1) of the H. polymorpha wild type strain CBS4732 leu2-2 ura3-20 (WT) and Δggt1two colonsLEU2 ura3-20 (Δggt1) mutant ± SEM

Source of nutrientStrains
Sulfur−S0.23 ± 0.010.003 ± 0.001
GSH0.044 ± 0.0020.009 ± 0.002
SO42−0.048 ± 0.0030.011 ± 0.002

Nitrogen−N0.069 ± 0.0050.009 ± 0.002
GSH0.044 ± 0.0050.003 ± 0.001
NH4+0.010 ± 0.0040.004 ± 0.001
Glu0.043 ± 0.0040.015 ± 0.001

In our study we demonstrated that γGT activity did not initiate transformation of GSH to supply H. polymorpha cells with cysteine/sulfur when the GSH was added as the sole sulfur source (Fig. 2A, C). Yeast cells of null ggt1 mutant as well as the wild type strain were able to utilize exogenous GSH as the sole sulfur source and grow in two media with different nitrogen sources (ammonium or glutamate) (Fig. 2A, C data shown only for ammonium). It seemed that γGT of H. polymorpha was not implicated in supplying the cells with nitrogen from GSH, because both strains (wild type and mutant) demonstrated similarly weak growth in media with GSH as the sole nitrogen source compared to glutamate or ammonium-containing media (Fig. 2B, D). Weak growth of both strains on indicated concentrations of GSH could be explained by significantly higher yeast requirements in nitrogen than in sulfur sources and/or by GSH growth inhibitory effects in concentrations above 0.5mM for H. polymorpha (Ubiyvovk et al., 1999).

Fig. 2

Growth of H. polymorpha wild type strain (A, B) and Δggt1 mutant (C, D) depending on sulfur and nitrogen source: (A, C) sulfur-deficient NH4+-containing medium (circles) supplied with 0.1mM GSH (squares) or 26.5mM (NH4)2SO4 (triangles); (B, D) nitrogen-deficient medium (circles) supplied with 1.5mM GSH (full squares), 2.5mM GSH (open squares), 26.5mM (NH4)2SO4 (triangles) or 5mM glutamate (rhombi).

It was assumed that yeast H. polymorpha, similar to S. cerevisiae (Kumar et al., 2003), possesses an alternative γGT-independent GSH degradation pathway to supply it with sulfur and nitrogen from GSH. The second H. polymorpha gene GGT2 may be responsible for GSH breakdown. We plan to elucidate this in our further study with null ggt2 and double ggt1×ggt2 mutants.

H. polymorpha wild type strain and null ggt1 mutant were tested for growth in the media containing stress generating agents. It was shown that null ggt1 mutant was more sensitive to the cell surface polymer perturbing agent, calcofluor white, and tert-butyl hydroperoxide and more resistant to CdSO4 compared to the wild type strain (Fig. 3). No essential difference was observed between methanol, ethanol and formaldehyde action in growth spot tests of the H. polymorpha strains studied (data not shown).

Fig. 3

Growth sensitivity of H. polymorpha wild type strain CBS4732 leu2-2∷LEU2 ura3-20 (WT) and Δggt1∷LEU2 ura3-20 (Δggt1) mutant was determined by spotting the cells on synthetic medium 2 containing one of the following toxic compounds: 10mg/l calcofluor white (CW), 0.8mM tert-butyl hydroperoxide (t-BOOH) and 125μM CdSO4. Yeast cells were grown overnight in YPD tubes, washed once with water and adjusted to OD590 3.0, 0.3 and 0.03 before spotting of 4μl suspensions on the plates. Growth was estimated after (*) 3 days or (**) 5 days of incubation at 37°C.

3.2 Fluorescence microscopy of H. polymorpha and S. cerevisiae fluorogenic xenobiotic metabolism

Yeasts H. polymorpha and S. cerevisiae eliminate electrophilic xenobiotics (monobromobimane) from the cytosol after their spontaneous or GSH-transferase-mediated conjugation with GSH (GS-bimane) and transport them to vacuoles (Ubiyvovk et al., 2003; Zadzinski et al., 1996). Further fate of vacuolar GSH conjugates and possible detoxification pathways in yeast cells remains unknown (see, however, Elskens and Penninckx, 1997). It is well known that bimanes are almost nonfluorescent until they react with thiols. The fluorescence microscopy study of vacuolar accumulation of fluorescent GS-bimane conjugate (this thiol conjugate was identified earlier, Zadzinski et al., 1996; Ubiyvovk et al., 2003) and kinetics of the disappearance of vacuolar fluorescence by cells of wild type strains and mutants with null activity of γGT, null ggt, of H. polymorpha and S. cerevisiae were conducted. Initial GS-bimane vacuolar fluorescence was observed in both H. polymorpha and S. cerevisiae wild type strains and null ggt mutants after 3h growth in the media with electrophile monobromobimane (Fig. 4, 3h-0). Further, stable GS-bimane vacuolar fluorescence was observed for H. polymorpha and S. cerevisiae null ggt mutants after 3h (3h-3, data not shown) and 16h (3h-16) incubation, in contrast to the rapid disappearance of vacuolar fluorescence in H. polymorpha and S. cerevisiae wild type strains with diffuse distribution of fluorescence in the cytosol (Fig. 4). It was suggested that cytosol fluorescence could be represented mainly with monobromobimane conjugates of thiol intermediates of the mercapturic acid pathway: cysteinylglycine, cysteine and N-acetylcysteine. It was assumed that disappearance of vacuolar GS-bimane fluorescence is a γGT-dependent process based on degradation of xenobiotic derivatives and their elimination from vacuole to cytosol and probably outside the cell.

Fig. 4

Fluorescence (A) and phase-contrast (B) microscopy of the yeast H. polymorpha and S. cerevisiae wild type strains (HpWT, ScWT) and null ggt mutants (HpΔggt1, ScΔcis2) after 3h growth in the synthetic medium with 100μM monobromobimane (3h-0) and subsequent incubation for 16h (3h-16) in the Na-phosphate buffer pH 7.2 with 3% glucose.

3.3 Study of electrophile derivative transport out of H. polymorpha and S. cerevisiae cells

3.3.1 Extrusion of monobromobimane derivatives

HPLC study of extracellular extrusion of monobromobimane derivatives of the wild type and null ggt mutant cells of H. polymorpha and S. cerevisiae was performed with extracellular medium of yeast cells incubated for 1h in 0.1M sodium phosphate buffer previously loaded with monobromobimane for 3h. It was demonstrated that HPLC peak corresponding to cysteine-bimane in H. polymorpha and S. cerevisiae null ggt mutants was absent in the extracellular medium compared to the extracellular medium of the wild type strain (Fig. 5A). We assumed that the observed cysteine-bimane conjugate was the possible product of γGT-dependent GS-bimane degradation that was extruded to the extracellular medium.

Fig. 5

HPLC profiles of extracellular derivatives of monobromobimane (mBB) (A) and N-[1-pyrene]maleimide (N-Pyr) (B) exported by yeast H. polymorpha and S. cerevisiae wild type strains (WT) and null ggt (Δggt1,Δcis2) mutant cells. Cells were incubated with 100μM mBB or 30μM N-Pyr and extruded derivatives were analyzed by HPLC as described in Section 2. Conjugates of cysteine with mBB (Cys-mBB) and cysteine or N-acetylcysteine with N-Pyr (Cys/NAC–Npyr).

3.3.2 Extrusion of N-[1-pyrene]maleimide derivatives

We have also studied the γGT-dependent extrusion of another electrophilic compound N-[1-pyrene]maleimide that could form conjugates with GSH and be extruded from cells as different xenobiotic derivatives (Ubiyvovk et al., 2003). Extracellular medium of cells of the null ggt mutants of H. polymorpha and S. cerevisiae incubated for 1h with N-[1-pyrene]maleimide lacked cysteine-N-[1-pyrene]maleimide or/and N-acetylcysteine- N-[1-pyrene]maleimide derivatives (both compounds show very close HPLC retention time) compared to the extracellular medium of the wild type strain. Mutant cells appeared to extrude only conjugates of GSH with N-[1-pyrene]maleimide, in contrast to the wild type cells, which extrude both types of conjugate (Fig. 5B). It could be suggested that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a pathway similar to that known for mammalian cells – a γGT-dependent mercapturic acid pathway with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.

In conclusion, we have found for the first time that γGT is involved in detoxification of electrophilic xenobiotics in yeasts. In methylotrophic yeast, this enzyme can also fulfil some specific functions connected with methanol metabolism. In favor of this conclusion is the fact that methanol strongly induces γGT in gsh2 mutants of H. polymorpha defective in the first reaction of GSH synthesis (Ubiyvovk et al., 2002a,b). The specific role of γGT in methanol metabolism remains to be elucidated.


This study was supported by the Collaborative NATO Linkage Grant LST.CLG 979872. Access to H. polymorpha genome database provided by Rhein Biotech GmbH (Duesseldorf, Germany) is gratefully acknowledged. We are indebted to Dr. O.V. Stasyk for friendly discussion and help.


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Received 15 December 2005/24 March 2006; accepted 26 April 2006


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