1 Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY and
2 MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK
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Abstract |
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Abbreviations: AFB1, aflatoxin B1; AP-1, activator protein 1; ARE, antioxidant-responsive element; BHA, butylated hydroxyanisole; GGT, -glutamyltranspeptidase; GLCL, glutamate-cysteine ligase; GLCLC, glutamate-cysteine ligase catalytic subunit; GLCLR, glutamate-cysteine ligase regulatory subunit; GS, glutathione synthetase; GST, glutathione S-transferase; I3C, indole-3-carbinol; NQO, NAD(P)H:quinone oxidoreductase; TNF-
, tumour necrosis factor
.
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Introduction |
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GSTs are a major group of detoxification enzymes conjugating glutathione to a vast spectrum of reactive metabolites and multiple cytosolic and membrane-bound isoenzymes exist. To date, six distinct gene families encoding the cytosolic GSTs have been identified in mammals, namely , µ,
,
,
and
(2). It has been shown previously that the
class GST subunit A5 can be induced in rat liver between 10- and 20-fold by the synthetic antioxidant ethoxyquin, as well as by other chemopreventive agents, including coumarin and indole-3-carbinol (I3C) (36). This is of particular interest because of the association between ethoxyquin, coumarin or I3C treatment and protection against aflatoxin B1 (AFB1)-induced hepatic neoplasia (4,6,7). GST A5-5 has been shown to possess a high specific activity towards AFB1 8,9-epoxide, a mutagenic metabolite of AFB1 (6,8). Furthermore, treatment of rats with the glutathione-depleting agents buthionine sulphoximine and diethylmaleate prior to AFB1 treatment increases AFB1 DNA binding in liver and kidney (9), supporting the hypothesis that glutathione is of critical importance in protection from certain mutagens.
Glutathione is synthesized from its constituent amino acids by glutamate-cysteine ligase (GLCL) and glutathione synthetase (GS) (10). Evidence suggests that GLCL catalyses the rate limiting step in glutathione synthesis, as it is subject to feedback inhibition by glutathione (10,11). GLCL is a heterodimer composed of a catalytic subunit (73 kDa, heavy subunit, GLCLC) and a regulatory subunit (30 kDa, light subunit, GLCLR). Both hydrophobic and disulphide interactions between GLCLC and GLCLR exist and it has been proposed that variation in intracellular glutathione concentrations may modulate disulphide bridge formation between the subunits causing conformational changes in the enzyme (1214). This regulates the Km of GLCLC for glutamate and feedback inhibition by glutathione.
GLCLC and GLCLR are encoded by separate genes, but under certain conditions can be regulated in an apparently coordinate manner. It has been shown previously that both subunits are induced by t-butylhydroquinone or ß-naphthoflavone in human HepG2 cells (15,16). The involvement of antioxidant-responsive elements (ARE) in the promoters of both subunits as a mediator of transcriptional activation of the human GLCL genes by these inducing agents has been predicted (1618). Recent studies in our laboratory, however, have provided evidence that induction of GLCLR may occur by an ARE-independent mechanism (19). Separate studies have shown that an activator protein 1 (AP-1) site is important in the induction of GLCLC by oxidants or tumour necrosis factor (TNF-
) (2023). Furthermore, the involvement of an AP-1 site in the overexpression of GLCLC in certain drug-resistant cancer cell lines has been proposed (24). Other studies have demonstrated differential regulation of GLCLR and GLCLC by hormones and plating density in cultured rat hepatocytes (25). The promoters of the rat GLCL subunits have not been characterized and it is unknown whether they may be subject to transcriptional control by mechanisms similar to the human GLCL subunits. The regulation of both subunits in vivo has received little attention, although it has been shown that GLCLC mRNA is increased during rat liver regeneration, whereas GLCLR mRNA levels remain unchanged (26).
Treatment of mice with the synthetic antioxidant butylated hydroxyanisole (BHA) causes an increase in hepatic glutathione levels and this has been shown to be associated with an increase in GLCLC mRNA; levels of GLCLR were not determined (27,28). Whilst several agents that protect against cancer increase hepatic glutathione levels in rodents, the effect of the majority of chemopreventive agents on GLCL levels has not been examined. In particular, it is unknown whether both GLCLC and GLCLR can be induced by chemopreventive agents in vivo.
-Glutamyltranspeptidase (GGT), another component of the
-glutamyl cycle, is the only known enzyme to break the
-glutamyl bond of glutathione and, as such, is important for the recycling of glutathione and the metabolism of glutathionexenobiotic conjugates (29). This enzyme is induced, mainly in the periportal areas of the liver, by a wide range of chemopreventive agents, including ethoxyquin (30) and coumarin (unpublished data). It has been shown previously that chronic low level feeding of rats with AFB1 gives rise to sub-populations of cells that have increased GST, GGT and glutathione levels; these sub-populations of cells are resistant to the cytotoxicity of AFB1 (31). The mechanism for the increase in glutathione levels in these cells could be, in part, a consequence of the increased GGT levels. Alternatively, or in addition, it is possible that levels of glutathione synthesizing enzymes are increased in AFB1-induced preneoplastic foci, as overexpression of GLCLC in cisplatin-resistant ovarian cancer cells has been predicted to be responsible for increased glutathione levels and drug resistance (32). GGT has also been found to be a mediator of cisplatin resistance in tumour cells in vivo (33).
The regulation of glutathione synthesizing enzymes is likely to be of critical importance in cancer prevention and in resistance to environmental carcinogens and oxidative stress. In the present study we have examined the induction of GLCLC and GLCLR in the rat by naturally occurring and synthetic chemopreventive agents and in AFB1-induced preneoplasia. We show that induction of GLCL is likely to contribute to a mechanism for enhanced detoxification and resistance to carcinogens.
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Materials and methods |
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Animals
Adult male Wistar rats (200250 g), bred and maintained at the Imperial Cancer Research Fund Animal Unit, Clare Hall, London, were used to examine the effect of chemopreventive agents on levels of GLCL and glutathione-metabolizing enzymes. Each group of rats (3 animals/group) was fed the same basic powdered diet (Standard Mouse/Rat Food; Special Diet Services, Essex, UK) to which the following compounds were added: no additives; 0.5% w/w coumarin; 0.5% w/w -angelicalactone; 1% w/w ellagic acid; 0.1% w/w phenobarbital; 0.5% w/w ethoxyquin. Levels of dietary additives were similar to those used in previous studies where chemoprevention and induction of xenobiotic-metabolizing enzymes have been shown (1,34,35) and were administered for 14 days, after which the animals were fasted overnight and killed the following day. Adult male Fischer 344 rats, purchased from Harlan Olac UK and maintained at the MRC Toxicology Unit, Leicester, were used to examine GLCL expression in hepatic preneoplasia. Rats were fed a diet containing AFB1 (2 p.p.m.) for 13 weeks to induce preneoplastic foci in liver (4).
Tissue and cytosol preparation
Livers and kidneys from rats treated with chemopreventive agents were stored at 80°C for up to 2 months. Cytosols were prepared from frozen tissue as described previously (36). Slices of tissue for immunohistochemistry were fixed immediately after excision in ice-cold acetone (37).
Analytical
GLCL activity, GST activity towards 1-chloro-2,4-dinitrobenzene and total glutathione concentrations were determined as described previously (36,38). Glutathione peroxidase activity was measured as described by Howie et al. (39). Glutathione reductase activity was measured using the method of Carlberg and Mannervik (40) adapted for use on a Cobas Fara centrifugal analyser. Statistical analyses were performed using a one-way Anova, with post hoc analyses carried out using Scheffe's test. Protein determination was by the method of Bradford (41) adapted for use on a Cobas Fara Centrifugal analyser (38).
Antibodies
The antibodies raised against peptides corresponding to regions of GLCLC or GLCLR have been described previously (15). Antibodies raised against rat GST A1-1, A3-3, A4-4, A5-5, M1-1 and P1-1 were a generous gift from Professor John Hayes (University of Dundee) and have been characterized previously (5). GS was purified from rat kidney using a modification of the method of Oppenheimer et al. (42) and antibodies raised in rabbits using standard protocols.
Immunoblotting
Western blot analysis was performed by the method of Towbin et al. (43). The relative intensities of bands on autoradiographs were estimated by scanning densitometry. Loading and transfer of samples was assessed by staining the nitrocellulose membranes after transfer with Ponceau S (38).
Immunohistochemistry and histochemistry
Antisera raised against the GLCL peptides or purified rat GST P1-1 were used at 1:200 dilutions with anti-rabbit alkaline phosphatase-conjugated secondary antibody as described previously (44). GGT activity was located histochemically as described by Manson et al. (37).
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Results |
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To determine whether the chemopreventive agents used in the present study induce the GLCL subunits in kidney as well as liver, kidney cytosols from rats treated with the different agents were examined by western blotting. Figure 2 shows that very little change in levels of expression of GLCLC occurred in kidney after treatment with any of the compounds studied. A low level of induction of GLCLR was observed in kidneys from rats treated with all agents.
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Liver cytosol from rats treated with coumarin, -angelicalactone, ellagic acid, phenobarbital and ethoxyquin were subject to western blotting using antibodies raised against the rat
class GSTs A1-1, A3-3, A4-4 or A5-5, µ class GST M1-1 or
class GST P1-1. Figure 3
shows that coumarin,
-angelicalactone and ellagic acid are the most effective inducers of
class GST, with coumarin causing the greatest level of induction. Coumarin caused an increase in the GST P1 subunit of ~20-fold, whereas
-angelicalactone and ellagic acid treatment resulted in ~10- and 6-fold induction, respectively. The synthetic antioxidant ethoxyquin caused lower levels of induction of the P1 subunit (4-fold) and phenobarbital had no effect on P1 expression. Of all the compounds studied, coumarin was also found to be the most effective inducer of the AFB1-metabolizing
class GST A5-5, causing a 7-fold induction (the A5 subunit is the lower of the two cross-reacting subunits shown in Figure 4c
, indicated by the arrow; the polyclonal antibodies raised against the recombinant A5 subunit cross-react with the constitutively expressed A3 subunit due to the high level of amino acid sequence identity between the two polypeptides).
-Angelicalactone and ethoxyquin were both found to cause similar levels of induction of ~6-fold and phenobarbital and ellagic acid resulted in induction of A5 by 4- and 3-fold, respectively. The
class GST subunits A1 and A3, which have a higher basal expression than A5, showed less pronounced levels of induction. Figure 3
shows that the A1 subunit was induced ~2-fold by coumarin and
-angelicalactone and ~1.5-fold by the other compounds examined. The A3 subunit was induced 2-fold by coumarin,
-angelicalactone and ethoxyquin and ~1.5-fold by the remaining compounds. The A4 subunit was also found to be inducible by coumarin (4-fold),
-angelicalactone (3-fold), ethoxyquin (3-fold), ellagic acid (2.5-fold) and phenobarbital (2-fold). It is interesting to note that the antibody raised against A4-4 also cross-reacts with a higher molecular weight subunit that was apparent in cytosols from coumarin-,
-angelicalactone- and ethoxyquin-treated rats. The identity of this band is, however, unknown. The µ class GST M1-1 was inducible to a similar extent (~2-fold) by all of the compounds used in the present study.
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In addition to determining the effect of chemopreventive agents on GST isoenzyme expression levels, it was important to establish whether the activities of GST, glutathione peroxidase or glutathione reductase were altered. Table I shows that coumarin caused the greatest increase in GST activity towards the model substrate1-chloro-2,4-dinitrobenzene (2-fold), with ethoxyquin and
-angelicalactone each causing an increase of 1.6- and 1.7-fold, respectively. Phenobarbital and ellagic acid were found to increase GST activity by 1.5- and 1.3-fold, respectively. It is important to note, however, that not all GST isoforms have high activity towards1-chloro-2,4-dinitrobenzene and that the increases in GST activity towards this substrate will not reflect the levels of induction of several of the different GST subunits.
Glutathione reductase activity was also increased by treatment with all of the chemopreventive agents used in this study. Ethoxyquin had the greatest effect, causing an induction of 3.3-fold. Coumarin, -angelicalactone, phenobarbital and ellagic acid induced glutathione reductase activity by 3-, 2.9-, 2.7- and 1.9-fold, respectively. In contrast, the chemopreventive agents were not found to increase glutathione peroxidase activity towards H2O2 in rat liver (Table I
). Coumarin was found to decrease glutathione peroxidase activity to approximately half that of the control activity. Ethoxyquin was also found to cause a modest decrease in glutathione peroxidase activity, whereas none of the other agents had any significant effect. A decrease in hepatic glutathione peroxidase activity towards H2O2 following ethoxyquin treatment has been observed previously, although the mechanism for this is unknown (3). Glutathione peroxidase activity towards cumene hydroperoxide was not changed significantly following treatment with any of the chemopreventive agents.
Regulation of GLCL subunits in preneoplastic foci and co-localization with GGT and GST P1-1
AFB1-induced preneoplastic foci are resistant to AFB1 cytotoxicity. This has been shown to be associated with increased glutathione levels and GST activity in the resistant cells (31). Because there is an intimate association between glutathione metabolism and resistance to environmental carcinogens, it is important to determine whether the increased glutathione levels in AFB1-induced hepatic foci could be a consequence of increased de novo synthesis of glutathione. Male Fischer 344 rats were treated with dietary AFB1 for 13 weeks to induce the appearance of focal preneoplastic lesions. The livers were subjected to immunohistochemistry using antibodies raised against peptides corresponding to regions of GLCLC or GLCLR, as well as antibodies raised against purified GST P1-1. GGT activity was examined histochemically. Induction of GGT and GST P1-1 in altered cell foci are classical markers of preneoplastic changes in initiated rat liver.
Treatment with AFB1 resulted in the appearance of focal lesions which stained positively for GST P1-1. Whilst not all of these contained increased levels of GGT, the majority of GGT-positive foci also contained increased levels of GLCLC (Figure 4). GLCLR was also found to be up-regulated in most (although not all) of the GLCLC-positive foci, but the level of staining was consistently less than that seen for GLCLC relative to the surrounding tissue (Figure 4
). The GLCL subunits therefore appear to be subject to regulation in preneoplastic foci, although it is apparent that this is not always a coordinated response, as several of the GLCLC-positive foci did not contain a detectable increase in GLCLR.
Co-localization of GLCL subunits with GGT and GST P-1-1 in rat kidney
Having found that GLCLC and GLCLR are induced in rat liver by chemopreventive agents and by carcinogens in preneoplastic foci, it was therefore of interest to determine whether each of the GLCL subunits is expressed in the same cells in normal rat kidney or whether differential regulation could occur in a cell-specific manner. Normal rat kidney was subject to immunohistochemistry with the antibodies raised against the GLCLC or GLCLR peptides and GST P1-1 and to histochemistry for GGT activity. Figure 5 shows that GLCLC and GLCLR were both present in the highest concentrations in the proximal tubules in the inner cortex where staining for GGT and GST P1-1 staining were strongest. Both GLCLC and GLCLR are present in high concentrations in the same cells, suggesting coordinate regulation by tissue-specific factors. In the outer cortex, staining for GGT was much more widespread than for any of the other enzyme subunits (Figure 6
).
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Discussion |
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Several studies have demonstrated that, in addition to inducing phase II enzyme activity, certain chemopreventive agents cause an increase in acid-soluble thiol or glutathione levels (27,4749). In livers from BHA-treated mice an increase in the mRNA encoding GLCLC was demonstrated and it was proposed that this could be a mechanism by which BHA increases hepatic glutathione levels (28). A separate study showed that BHA and ethoxyquin caused an increase in GLCL activity and GLCLC mRNA levels in rat liver, whereas Oltipraz had no effect (50). The effect of other naturally occurring chemopreventive agents on GLCL activity has received little attention, but induction of GLCL activity could potentially make an important contribution to their anticancer properties.
In the present study we have demonstrated that certain naturally occurring phytochemicals, which have been shown previously to protect against cancer, cause induction of GLCL activity in rat liver. Of particular interest is the finding that the increase in GLCL activity is associated with induction of both the catalytic and regulatory subunits. This co-induction is likely to be of critical importance in vivo as GLCLC has a low affinity for glutamate (Km = 18.2 mM) in the absence of GLCLR (14). GLCLC is also sensitive to competitive inhibition by glutathione unless associated with GLCLR (14). Furthermore, it has been demonstrated recently that overexpression of human GLCLR in HeLa cells, without an increase in GLCLC expression, is sufficient to increase GLCL activity and generate an enhanced ability to resynthesize glutathione following its depletion by diethylmaleate (51). These studies demonstrate that both GLCLC and GLCLR are likely to be important in regulating glutathione homeostasis.
Despite induction of the GLCL subunits by the chemopreventive agents used in the present study, only ellagic acid was found to cause an increase in the apparent steady-state glutathione levels. Nevertheless, the capacity to resynthesize glutathione following its depletion is probably more important for cellular protection than the absolute levels of glutathione in the cell (51,52). It is likely that glutathione concentrations are subject to tight control mechanisms and, whilst temporary fluctuations could occur in response to different stimuli, it is possible that in a longer term feeding study such as ours, glutathione levels have been subject to homeostatic regulation. This could, in part, be due to feedback inhibition by glutathione of GLCL activity. The fact that the animals were fasted overnight before the glutathione measurements were performed may also have been a contributory factor, with substrate availability being rate limiting for glutathione synthesis.
To our knowledge this is the first demonstration that both the GLCLC and GLCLR subunits are induced by chemopreventive agents in vivo. It has, however, been reported that t-butylhydroquinone, which is the metabolite of BHA believed to be responsible for induction of gene expression, causes induction of both subunits in human cell lines (15,38), as does ß-naphthoflavone (16,17). AREs have been described in the 5'-flanking regions of the respective human genes and it has been proposed that induction by ß-naphthoflavone is mediated through these (16,17). A separate 42 bp region of DNA which is responsive to t-butylhydroquinone has also been identified in the 5'-flanking region of the gene encoding GLCLR (19). In addition, AP-1 sites have been shown to be involved in the induction of human GLCLC by oxidants and cigarette smoke condensates, as well as by TNF- (20,21,23,53). AP-1 has also been shown to be important in the basal expression of the genes encoding both the human GLCLR and GLCLC subunits (16,17,19). It is, however, unknown whether the rat GLCL genes contain AREs or AP-1 sites in their promoter regions and it remains to be determined whether the chemopreventive agents shown in the present study to induce levels of the GLCL polypeptides involve ARE-dependent mechanisms.
Recent studies have shown that induction of gene expression mediated by an ARE is influenced by the transcription factor Nrf2 (54,55). Mice with deletions in the gene encoding Nrf2 were found to have no overt abnormalities, but are defective in their ability to induce NQO or certain GSTs in response to treatment with BHA. The effect of BHA on the GLCL subunits was not determined in these experiments and Nrf2/ mice should provide an important in vivo model to examine the role of the ARE and Nrf2 in regulating GLCL expression in the mouse. Furthermore, as it has not been demonstrated unequivocally that inducers of drug-metabolizing enzymes such as -angelicalactone, coumarin and ellagic acid mediate induction of gene expression via an ARE, the Nrf2/ mouse model should allow this hypothesis to be explored in greater depth.
The most effective inducers of GLCL expression and activity were coumarin, -angelicalactone and ellagic acid; these were also found to be good inducers of the GST P1 and A5 subunits. These three naturally occurring phytochemicals have been shown to be effective chemopreventive agents in rodents (1,7). This has been demonstrated previously to be associated with increases in activity of glutathione S-transferases, and enhanced detoxification of carcinogens has been proposed to be a major mechanism of action by which many chemopreventive agents act (1). It is interesting to note, however, that induction of glutathione-related enzymes by chemopreventive agents does not necessarily constitute a coordinated response. For example, in contrast to the marked induction of GST P1 and GST A5 specifically by coumarin and
-angelicalactone, the µ class GST M1 subunit seemed to be induced equally as well (~2-fold) by all the inducing agents used in the present study. Other studies have also demonstrated that the
class GST T1 is inducible by chemopreventive agents, but is not coordinately induced with
, µ and
class GSTs (5). Ethoxyquin was found to be equally as effective an inducer of the GST T1 subunit as coumarin. This is in contrast to the induction of GST P1 and GST A5, where coumarin caused a substantially greater induction than ethoxyquin.
The fact that coordinate up-regulation of enzymes participating in glutathione metabolism does not occur in response to treatment with chemopreventive agents is highlighted by the fact that glutathione peroxidase activity towards H2O2 was decreased in response to coumarin and to a lesser extent by ethoxyquin. Other studies have also demonstrated that certain antioxidants cause a decrease in glutathione peroxidase activity in rat liver (3). The mechanism for this decrease is unclear, although it is possible that administration of coumarin or ethoxyquin results in an altered disposition of selenium compounds, causing a reduced availability of selenocysteine, which could be rate limiting in glutathione peroxidase 1 synthesis. Alternatively, it is feasible that the chemopreventive agents or their metabolites could result in inactivation of glutathione peroxidase enzyme activity. This area certainly warrants further investigation.
GLCL and GGT are clearly both important participants in the -glutamyl cycle and in influencing glutathione homeostasis. GST P1 is also likely to have a protective role and it is apparent from the expression of GGT, GLCL and GST P1 in AFB1-induced preneoplastic lesions that an intrinsic link in the regulation of these enzymes exists. It is notable that although expression patterns of each of the four polypeptides were similar in the AFB1-treated rat liver, a tightly coordinated regulation of the GLCL subunits, GGT and GST P1 does not appear to exist. The expression of GST P1 in the focal lesions was more frequent than GGT or the GLCL subunits. Most, but not all, of the GGT-positive foci contained increased levels of the GLCL catalytic subunit, while the GLCL regulatory subunit appeared to be overexpressed to a lesser extent than the catalytic subunit. The frequent overexpression of the GLCL subunits in preneoplastic cells does, however, suggest that GLCL activity is likely to contribute to the increased glutathione levels observed in these cells (31).
A more tightly linked regulation of GLCLC, GLCLR, GGT and GST P1 was observed in the inner cortex of the kidney. Here, each of the glutathione-related enzymes was found to be most highly expressed in the proximal tubules (Figure 5). We have also found that GS is highly expressed in the same cells as GGT and GLCL (data not shown). This suggests that in cells with a high capacity for glutathione synthesis and turnover, each of these components of the
-glutamyl cycle would be required to be expressed at high levels in the same cell, including GS, which is not generally believed to be rate limiting in glutathione synthesis. In the outer cortex, however, this co-regulation is less apparent and GGT is more widely expressed than the other enzymes (Figure 6
).
In conclusion, we have demonstrated that GLCL can be added to the list of enzymes induced by chemopreventive agents and by carcinogens in preneoplastic foci. This is of critical importance, as GLCL activity is a principal determinant of the ability to synthesize glutathione. Hence, the induction of GLCL contributes to the glutathione-dependent battery of defences utilized by cells to protect against reactive electrophiles and peroxides. In addition to induction of GST and other phase II enzymes, part of the chemopreventive action of many antioxidants and phytochemicals may also be by their ability to regulate GLCL activity.
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Notes |
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4 To whom correspondence should be addressed Email: mclellan{at}icrf.icnet.uk
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Acknowledgments |
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References |
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