The relative importance of glutathione and metallothionein on protection of hepatotoxicity of menadione in rats

Chem.-Biol. Interactions, 84 (1992) 113-124

113

Elsevier Scientific Publishers Ireland Ltd.

THE RELATIVE IMPORTANCE OF GLUTATHIONE AND METALLOTHIONEIN ON PROTECTION OF HEPATOTOXICITY OF MENADIONE IN RATS HING MAN CHAN, REYHANEH TABARROK, YUKIHIKO TAMURA and M. GEORGE CHERIAN

Department of Pathology, The University of Western Ontario, London, Ontario, N6A 5C1 (Canada) (Received January 30th, 1992) (Revision received July 1st, 1992) (Accepted July 1st, 1992)

SUMMARY

The effects of induction of metallothionein (MT) on the toxicity of menadione were investigated in rat liver slices. The protective role of hepatic glutathione (GSH) was also studied and compared to that of MT. A 3-h incubation of rat liver slices with menadione (100-300 #M) containing medium (37°C, pH 7.4, 95%02:5%CO2) resulted in cellular toxicity, as shown by changes in cytosolic K, Ca and GSH concentrations and lactate dehydrogenase (LDH) leakage. A dosedependent decrease in cytosolic K and GSH was observed concomitant with an increase in cytosolic Ca and LDH leakage after incubation with menadione. Pretreatment of rats with zinc sulphate (ZnS04) (30 mg/kg body wt.) increased MT levels in liver slices and suppressed the toxicity of menadione. Intracellular GSH concentrations in liver slices were either depleted or increased by injection of rats with buthionine sulfoximine (BSO), (4 mmol/kg body wt.) and Noacetyl-Lcysteine (NAC) (1.6 g/kg body wt.), respectively. Intracellular GSH was found to be crucial in protection against menadione toxicity. Menadione toxicity was increased when the rats were injected with sodium phenobarbital (PB) (4 x 80 mg/kg body wt.). Pretreatment with Zn provided partial protection against menadione toxicity in liver slices from both BSO- and PB-injected rats. These findings suggest that induction of MT synthesis does protect against quinoneinduced toxicity, but the role may be secondary to that of GSH. The mechanisms by which MT protect against menadione toxicity are still unclear but may involve protection of both redox cycling and sulphydryl arylation.

Key words: Menadione -- Liver slices -- Metallothionein -- Glutathione

Correspondence to: M.G. Cherian, Department of Pathology, The University of Western Ontario, London, Ontario, Canada N6A 5C1. 0009-2797/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

114 INTRODUCTION

Quinones represent a clinically important category of chemotherapeutic agents with applications in both antitumor and antimicrobial therapy [1]. The cytotoxic effects of quinone compounds are thought to be mediated through two principle mechanisms: oxidative stress and arylation of cellular thiols. Oxidative stress results from the metabolism of quinones via one electron 'redox cycling' reactions which occur in the presence of certain flavoenzymes to semiquinone free radicals [2]. In the presence of oxygen the unstable semiquinone radicals undergo rapid autoxidation with the formation of the superoxide anion radical and regeneration of the parent quinone. Dismutation of superoxide anions results in the generation of hydrogen peroxide which ultimately lead to irreversible cell injury [3,4]. The ability of quinones to arylate cellular thiols has also been reported [5,6]. Inhibition of critical thiol-dependent enzymes may also lead to the initiation of irreversible cell injury. Menadione (2-methyl-l,4-naphthoquinone) is a representative quinone which has toxic effects in hepatocytes, isolated mitochondria and in microsomes [7-10]. It increases oxygen uptake, depletes glutathione and NADPH and increases cytosolic calcium in isolated hepatocytes [111. Glutathione (GSH), a ubiquitous tripeptide, is believed to represent a major part of cellular defense against quinone cytotoxicity. The reduced form of GSH provides protection against toxicity of free radicals from the metabolism of oxidants. It can also conjugate directly with menadione, thus, protecting more critical nucleophilic sites [6,12]. Metallothionein (MT), a low molecular weight metal binding protein is also thought to play a role in cellular defense against reactive free radicals. The highly nucleophilic thiol group of MT exerts reactivity towards electrophiles including free radicals. MT was found to scavenge and decrease hydroxyl and superoxide radicals produced by xanthine/xanthine oxidase reaction [13]. Elevated MT levels in cadmium resistant Chinese hamster V79 cells were associated with concomitant resistance to oxidative stress induced by generation of hydrogen peroxide and superoxide anions [14]. The purpose of this study was to evaluate the extent of protection inferred by MT against menadione toxicity in relation to that of GSH using an in vitro liver slice incubation system. MATERIALS AND METHODS

Chemicals All chemicals were of reagent grade obtained from BDH Inc. (Ontario) unless specified otherwise. Treatment of animals Three adult male Sprague-Dawley rats (350-400 g) were treated in one of the following regimes. (A) Control: rats were injected with 0.85% saline 24 h prior to sacrifice; (B) Zn injected: MT synthesis was induced in rats by injection

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(s.c.) with 10 mg/kg and 20 mg/kg body weight ZnS04 at 48 and 24 h, respectively, prior to sacrifice; (C) BSO injected: rats were injected (i.p.) with 4 mmol/kg body weight buthionine sulfoximine (BSO) (Chemical Dynamics Corp., New Jersey) 4 h prior to sacrifice to decrease GSH concentrations; (D) Zn + BSO (induction of MT synthesis together with reduction of GSH levels): treatments B + C; (E) NAC injected: rats were injected (i.p.) with 800 mg/kg body weight N-acetyl-L-cysteine (NAC) at 16 and 4 h prior to sacrifice to increase GSH concentrations; (F) PB injected: rats were injected (i.p.) with 80 mg/kg body weight sodium phenobarbital daily for 4 days prior to sacrifice to induce NADPH cytochrome P-450; (G) PB + Zn: treatments B + F.

Preparation of rat liver slices Rats were sacrificed by decapitation and the liver was excised and placed in ice-cold Krebs-Henseleit buffer. Different areas of the liver lobes were cored with a 1-cm diameter stainless steel core [15] and slices (0.5-ram thickness and 20-30 mg wet wt.) were prepared using a Stadie-Riggs tissue slicer (Arthur H. Thomas Co, Philadelphia). The slicing procedure was completed within 30 min after sacrifice. About 80 slices were obtained from each rat and used for one set of experiments. Incubation of slices The liver slices were incubated under standardized conditions [16,17] in incubation medium (Krebs-Henseleit buffer supplemented with 1% BMEvitamins, 1% BME amino acids and 0.5% glutamine (Gibco Lab., New York); pH 7.4). To the incubation media, 100,200 and 300 #M menadione (Sigma Chem. Co., St. Louis) was added. Dimethylsulphoxide (DMSO) was used as a drug vehicle. The final DMSO concentration in all incubation media was 35 ~M. Incubation of the slices lasted for 3 h at 37°C under constant aeration with 95%O2:5%CO2 in a shaking water bath. The control experiment (no menadione added) contained 35 ~M of DMSO. Chemical analysis After incubation, slices were removed from the flasks, blotted dry on filter papers and weighed. Three slices were homogenized in 6% trichloroacetic acid and centrifuged at 8000 × g for 10 rain. K and Ca concentrations in the supernatant were measured by atomic absorption spectrophotometry using airacetylene. Three slices were homogenized in 0.25 M sucrose solution, centrifuged at 8000 × g for 10 min and MT levels in the supernatant were measured using an ELISA developed for MT quantification [18l. Three slices were homogenized in 2.5% perchloric acid, centrifuged at 8000 × g for 10 min and the GSH levels in the supernatant were measured by the Tietze enzymatic method [19]. Three slices were homogenized in 154 mM potassium chloride and the malondialdehyde concentrations were measured as an indicator of lipid peroxidation by coupling to thiobarbituric acid [20]. Lactic dehydrogenase (LDH) leakage from the liver slices was measured by measuring the LDH activities in the media after incubation by a diagnostic kit (Sigma) followed by UV absorbance spectrophotometry.

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Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) and means of different groups were compared by Student-Newman-Keuls test according to Sokal and Rohlf [21]. Five percent was considered as the level of significance unless stated otherwise. RESULTS

Effect of different treatments on liver slices without menadione The concentrations of K, Ca, GSH and MT in rat liver slices and LDH leakage from the liver slices to the media after the 3-h incubation period (no menadione dosed) are summarized in Table I. Cytosolic K concentrations in the liver slices of rats injected with BSO alone and PB alone were significantly lower whereas those injected with Zn alone were significantly higher than those of the control. No significant change was observed in cytosolic Ca concentrations and LDH leakage. Intracellular GSH concentrations were significantly decreased in both groups injected with BSO and increased in the group injected with NAC. Intracellular MT concentrations were significantly increased in all groups injected with Zn and injection of BSO and PB did not affect MT induction when compared with the Zn injection alone group. Toxicity of menadione The relative changes in cytosolic K, Ca and GSH concentrations and LDH leakage expressed as percentages of the values at '0' ~M menadione exposure

TABLEI CYTOSOLIC P O T A S S I U M , C A L C I U M , G L U T A T H I O N E A N D M E T A L L O T H I O N E I N C O N C E N T R A T I O N S IN L I V E R S L I C E S A F T E R I N C U B A T I O N IN M E D I U M W I T H NO M E N A D I O N E FOR 3 H O U R S Lactic d e h y d r o g e n a s e (LDH) leakage is e x p r e s s e d as L D H activities in the m e d i u m after incubation over total tissue weight of slices. Mean + S.D. (n = 3). Treatment

Potassium (tLg/g tissue)

Calcium (t~g/g tissue)

Glutathione MetaLloth(t~mol/g tissue) ionein (t~g/g tissue)

Lactic dehydrogenase (units/g tissue)

Control Zn injected BSO injected BSO + Zn NAC injected PB injected PB + Zn

1795 2244 1426 2041 1824 1270 1454

275 219 263 290 316 280 233

2.5 2.7 0.9 0.9 4.5 2.2 2.1

190 186 216 191 243 228 214

+ 199 ± 240* ± 100" -~ 232 ± 195 + 186" ± 181

+ 46 ± 58 ± 50 ± 124 ± 33 ± 95 ± 166

+ ± ± ± ± ± +

0.2 0.1 0.1" 0.1" 0.2* 0.5 0.3

15 350 10 401 15 11 310

+ 2 + 34* + 6 + 62* ± 5 ± 6 ± 48*

+ ± ± + ± ± ±

31 58 83 26 59 71 38

* Significantly different t h a n control values (P < 0.05). Nine liver slices from 3 r a t s were used in each treatment.

117 1 O0

80 v

60

(J E

o o

4-0

20 0

0

I

~

I

1O0

200

300

Menadione

conc

(~M)

Fig. 1. Relationship between cytosolic K concentration in liver slices exposed to menadione, y axis = cytosolic K concentration as a p e r c e n t a g e of the value of the slices exposed to vehicle added only medium (Table I), x axis = menadione exposure concentration. Rats were treated in one of the following regimes before sacrifice (see Materials and Methods) ; control (O), Zn injected (O), BSO injected (h), BSO and Zn injected (A), NAC injected (1"1),PB injected (11), PB + Zn injected (V). Slices were incubated in media containing menadione for 3 h at 37°C. The results are mean values of at least 9 slices from 3 animals. *Significantly different from control at the same menadione exposure level; P < 0.05.

concentration (Table I) were used as indicators for toxicity (Figs. 1 - 4). Menadione incubation resulted in a dose-dependent toxicity to rat liver slices (represented by the data of the control group). Increasing concentrations of menadione (100-300 M) caused significant decreases in cytosolic K (Fig. 1) and GSH concentrations (Fig. 2) and concomitant increases in cytosolic Ca concentrations (Fig. 3) and LDH leakage (Fig. 4). 1O0 8C o

•

60-

E

o t

40'

O 20 0 0

I 1O0 Menodione

I 200

t 300

conc (~M)

Fig. 2. Relationship between eytosolic GSH concentration in liver slices exposed to menadione, y axis = eytosolie GSH concentration as a percentage of the value of the slices exposed to vehicle added only medium (Table I), x axis = menadione exposure concentration. Symbols used are the same as those in the legend to Fig. 1. The results are mean values of at least 9 slices from 3 animals. *Significantly different from control at the same menadione exposure level; P < 0.05.

118 220 1

2°°I o

o 160

•

,3

:* •

0

100

,

200

300

Mencldione conc (/.~M) Fig. 3. Relationship between cytosolic Ca concentration in liver slices exposed to menadione, y axis = cytosolic Ca concentration as a percentage of the value of the slices exposed to vehicle added only medium (Table I), x axis = menadione exposure concentration. Symbols used are the same as those in the legend to Fig. 1. The results are mean values of at least 9 slices from 3 animals. * Significantly different from control at the same menadione exposure level; P < 0.05.

Effect of GSH on menadione toxicity Depletion of GSH by injection of BSO enhanced menadione toxicity. In comparison to the values of the control group, there were significantly higher decreases in cytosolic K concentrations at all 3 menadione exposure concentrations (Fig. 1). Enhanced toxicity was also shown by significantly higher increases in cytosolic Ca concentrations at all 3 menadione exposure concentrations (Fig. 3) and LDH leakage (at 200 and 300 ~M) (Fig. 4). There was, however, a 220200.

0

~

100 Menodione

A

,

,

200

300

c o n c (/zM)

Fig. 4. Relationship between L D H leakage from liver slices exposed to menadione, y axis = L D H leakage as a percentage of the value of the slices exposed to vehicle added only medium (Table I), x axis = menadione exposure concentration. Symbols used are the same as those in the legend to Fig. 1. The results are mean values of at least 9 slices from 3 animals. *Significantly different from control at the same menadione exposure level; P < 0.05.

119 lesser extent of decrease in intracellular GSH concentrations (at 200 and 300 ~M) (Fig. 2). This is probably a result of the low initial GSH concentrations (Table I). The absolute mean GSH concentration in slices exposed to 300 ~M menadione was only 0.3 ~mol/g tissue which is approaching the detection limit of the assay. Increase in hepatic GSH concentration by injection of NAC, on the other hand, showed some protection against menadione toxicity. There were significantly less increases in cytosolic Ca concentrations and LDH leakage at all 3 menadione exposure levels (Figs. 3 and 4). No significant change, however, was observed in cytosolic K concentrations. A significantly higher decrease in GSH was observed at 100 ~M (Fig. 2).

Effect of PB on menadione toxicity Injection of PB increased menadione toxicity. There were significantly higher decreases in cytosolic K concentrations at all 3 menadione exposure concentrations (Fig. 1) and in intracellular GSH exposed to 100 t~M menadione (Fig. 2). The increase of LDH leakage at 200 ~M was also significantly higher (Fig. 4). Cytosolic Ca concentrations, however, showed no significant change. Protective effect of M T on menadione toxicity Injection of Zn resulted in at least 30-fold increase in MT concentrations in liver slices (Table I). The induced MT showed protection against menadione toxicity, as shown by a lesser extent of decrease in cytosolic K concentrations (Fig. 1) and increase in cytosolic Ca concentrations and LDH leakage (Figs. 3 and 4) than controls at all 3 menadione exposure concentrations. However, no significant change was observed in intracellular GSH concentrations (Fig. 2). Induction of MT synthesis was found to provide partial protection to the enhanced toxicity caused by depletion of GSH by injection of BSO. The relative increases in cytosolic Ca concentrations and LDH leakage found in the BSO injection alone group were not observed in the BSO + Zn group (Figs. 3 and 4). There were, however, no significant differences in the relative decrease of cytosolic K and GSH concentrations between the BSO alone and BSO + Zn groups (Figs. 1 and 2). The increase in menadione toxicity inferred by PB injection was also partially reduced by induction of MT. The relative decreases in cytosolic K concentrations and LDH leakage found in the PB injected alone group were absent in the PB + Zn group (Figs. i and 4). The PB + Zn group also showed a lesser degree of increase in cytosolic Ca concentrations. No significant difference was found in the relative decrease of intracellular GSH concentrations in both PB-injected groups, irrespective of Zn pretreatment. Intracellular MT concentrations were found to decrease with increase in menadione concentrations in all cases (Fig. 5). There was a significantly higher decrease of MT in the BSO injected group as compared with that of the control. The relative decrease of MT concentrations of both the BSO + Zn and PB + Zn groups were also significantly higher than that of the Zn injected alone group when exposed to 200 and 300 ~M menadione.

120 lO0

80 "-I

60-

(3 C

°,oI--

*

20-

0 O

I 1 O0 Menadione

I 200 conc

I 300

(¢zM)

Fig. 5. Relationship between eytosolie MT concentration in liver slices exposed to menadione, y axis = eytosolie MT concentration as a percentage of the value of the slices exposed to vehicle added only medium (Table I), x axis = menadione exposure concentration. Symbols used are the same as those in the legend to Fig. 1. The results are mean values of at least 9 slices from 3 animals. *Significantly different from control at the same menadione exposure level; P < 0.05.

DISCUSSION

The use of the liver slice system for short term in vitro toxicity study has proved to be useful for studying the transport, metabolism and toxicity of various chemicals [17,22]. The role of MT in the protection against toxicity of menadione were investigated in this report using liver slices from rats after various treatments to manipulate their intrinsic hepatic MT, GSH and cytochrome P-450 levels. Cytosolic K, Ca and GSH levels in the liver slices and LDH leakage were used as indicators of the relative hepatotoxicity. Under our experimental conditions (i.e. 5°70 C O 2 and 95% O2), menadione showed a dose-dependent (100-300 ~M) hepatotoxicity. Cytotoxic effects of menadione are mediated by a one electron redox cycling production of semiquinone free radicals concomitant with the release of hydrogen peroxide and superoxide anion which in turn leads to GSH depletion, pyridine nucleotide oxidation, oxidation of protein thiols, alterations of calcium homeostasis and of the cytoskeleton [11,23-26]. These toxic effects are demonstrated in the present study by decreases of cytosolic K and GSH and increases in cytosolic Ca concentrations in the liver slices and LDH leakage. The availability of GSH plays an important protective role in menadione metabolism [6,27,28]. GSH reduces the hydroperoxides generated by the metabolism of menadione, in the presence of glutathione peroxidase, to corresponding alcohols. We have shown that depletion of intracellular GSH by BSO injection potentiated menadione toxicity as reported by previous studies [29- 31]. Moreover, the protective role of GSH was further demonstrated by the suppression of menadione toxicity by increasing intracellular GSH with injection of NAC.

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The metabolism of quinones in liver occurs either through a one electron or a two electron reduction pathway [11]. The one electron reduction resulting in the formation of semiquinone radicals, which can enter redox cycles with molecular oxygen to produce active oxygen species and oxidative stress, is mainly catalyzed by flavoenzymes such as NADPH-cytochrome P-450 reductase. Alternatively, the flavoprotein DT-diaphorase, catalyzes the two electron reduction reactions, forming hydroquinone without production of free semiquinone intermediates. Therefore, menadione toxicity may depend on the relative abundance of the flavoenzymes. In this study, the injection of PB, which is a well known inducer of cytochrome P-450 [32], resulted in an increase in menadione toxicity on the liver slices. This enhanced toxicity is presumably a result of more menadione being metabolized through the one electron reduction pathway. Utley and Mehendale [33] however, found no increase in menadione toxicity by PB treatment. This is in contrast to the result of this study and previous reports [11,32]. Pretreatment of rats with ZnS04 showed a significant induction of hepatic MT synthesis as reported previously [17]. Liver slices from rat injected with Zn showed a higher resistance to menadione toxicity as shown by the maintenance of cytosolic K and Ca concentrations and LDH leakage. However, high levels of MT alone did not lessen the decrease of GSH in liver slices as in the case of Cd toxicity reported in a previous study [17]. Induction of MT synthesis in GSH depleted rats provided only a partial recovery (GSH and K concentrations not recovered) of menadione toxicity unlike a total recovery (GSH in particular) in the case of Cd toxicity under similar experimental conditions [17]. These results suggests that GSH is a more sensitive target ligand to menadione toxicity compared with the effects of Cd and thus GSH may play a more important protective role in menadione toxicity. One of the major reasons for these differences in the protective effect of MT against Cd and menadione toxicities may be the specific binding of Cd and other metals to the induced MT, while such direct binding of menadione metabolites to metal bound MT may not occur. On the other hand, the reduced form of GSH can effectively protect the toxic effects of electrophilic reactive metabolites generated by menadione. However, we were unable to demonstrate any increased lipid peroxidation in menadione toxicity studies (data not shown). This can be explained by the inhibitory effect of menadione on the propagation reactions of lipid peroxidation [35]. Although a number of studies have reported that MT may play a role in protection against oxidative stress [13,14,36,37], little is known about the exact mechanism and its physiological significance [38]. Since menadione exerts its toxicity by arylation of intracellular thiols or nucleophiles as well as oxidative processes [6,39], MT may provide protection against menadione simply by its contribution to the intracellular thiol pool, by virtue of its high cysteine content. This hypothesis is supported by the evidence that intracellular MT concentrations were decreased with increasing concentrations of menadione in our experiment, particularly when the intracellular GSH concentrations were lowered by injection of BSO. Whether this loss of MT from tissue can be regenerated by increasing GSH levels is unclear from the present data. Our results, however, also show that MT can provide some protection in the enhanced toxicity caused by

122

PB injection. As mentioned above, PB induces cytochrome P-450 reductase which catalyses the one electron metabolic pathway and generates more free radicals. Therefore, this result suggests that MT can also suppress the oxidative damage caused by free radicals. Thornalley and Vasak [6] found that rabbit (Cd,Zn) MT is able to scavenge free hydroxyl and, to a lesser extent, superoxide radicals produced by the xanthine/xanthine oxidase reaction in a simple in vitro system. Matsubara [40] also found some evidences to suggest that MT may substitute GSH in the glutathione peroxidase system. Therefore, the protective mechanism of MT in the protection against menadione toxicity may involve both redox cycling and sulphydryl arylation. Their relative importance, however, has still to be investigated. In summary, we have shown that intracellular GSH is the principal defense mechanism against menadione toxicity. Intracellular MT may also play a similar role to GSH in protection of the liver slices against menadione toxicity. However, the protective effects of MT against menadione toxicity is not as efficient as that against cadmium toxicity. The mechanism of the protective effect of MT in menadione toxicity is not yet understood but may involve both redox cycling and sulphydryl arylation. ACKNOWLEDGEMENTS

We would like to acknowledge the technical help of Susanne Vesely. This project was supported by a research grant from the Medical Research Council of Canada. This work was initiated in freshly isolated rat liver cells in Dr. Donald J. Reeds' laboratory at the Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon where Dr. Cherian spent a sabbatical leave. Dr. Cherian wants to acknowledge NIEHS Center Grant ES00210. REFERENCES 1 M. Smith, Quinones as mutagens, carcinogens and anticancer agents, J. Toxicol. Environ. Health, 16 (1985) 665-672. 2 C. Lind, P. Hochstein and L. Ernester, DT-diaphorase as a quinone reductase: a cellular control devise against semiquinone and superoxide radical formation, Arch. Biochem. Biophys., 216 (1982) 178-185. 3 G. Rotilio, I. Mavelli, L. Rossi and Ciriolo, Biochemical mechanism of oxidative damage by redox cycling drugs, Environ. Health Perspect., 64 (1985) 259-264. 4 M. Comporti, Three models of free radical-induced cell injury, Chem.-Biol. Interact., 72 (1989) 1-56. 5 K.T. Finley, The addition and substitution chemistry of quinones, in: S. Patai (Ed.), The Chemistry of the Quinonoid Compounds, Part II, John Wiley, London, 1974, p. 878. 6 T.W. Gant, D.N.R. Rao, R.P. Mason and G.M. Cohen, Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes, Chem.-Biol. Interact., 65 {1988) 157-173. 7 P. Hochstein, Futile redox cycling: implications for oxygen radical toxicity, Fund. Appl. Toxicol., 3 (1983) 215-217. 8 T. Noll, H. de Groot and H. Sies, Oxygen dependence of menadione redox cycling: studies in a PO2-stat system, Arch. Pharmacol., 329 (1985) 81. 9 P. Starke and J. Farber, Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide, J. Biol. Chem., 260 (1985) 10099-10104.

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