Abstract
Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH–bimane-conjugate (GS–B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS–B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.
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Hroboňová K, Machyňáková A, Čižmárik J (2018) Determination of dicoumarol in Melilotus officinalis L. by using molecularly imprinted polymer solid-phase extraction coupled with high performance liquid chromatography. J Chromatogr 1539:93–102
Last JA (2002) The missing link: the story of Karl Paul Link. Toxicol Sci 66:4–6
Poulton JE, McRee DE, Conn EE (1980) Intracellular localization of two enzymes involved in coumarin biosynthesis in Melilotus alba. Plant Physiol 65:171–175
Timson D (2017) Dicoumarol: a drug which hits at least two very different targets in vitamin K metabolism. Curr Drug Targets 18:500–510
Stafford DW (2005) The vitamin K cycle. J Thromb Haemost 3:1873–1878
Aras D, Cinar O, Cakar Z, Ozkavukcu S, Can A (2016) Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study. Mol Hum Reprod 22:57–67
Hong J, Zhang P, Yoon IN, Kim H (2016) Inhibition of NAD(P)H:quinone oxidoreductase 1 by dicumarol reduces tight junction in human colonic epithelial cells. J Life Sci 26:531–536
Deponte M (2017) The incomplete glutathione puzzle: just guessing at numbers and figures? Antioxid Redox Signal 27:1130–1161
Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 62:649–671
Dringen R, Brandmann M, Hohnholt MC, Blumrich E-M (2015) Glutathione-dependent detoxification processes in astrocytes. Neurochem Res 40:2570–2582
Hirrlinger J, Dringen R (2010) The cytosolic redox state of astrocytes: maintenance, regulation and functional implications for metabolite trafficking. Brain Res Rev 63:177–188
Schmidt M, Dringen R (2012) Glutathione (GSH) synthesis and metabolism. In: Choi I-Y, Gruetter R (eds) Neural metabolism in vivo. Springer, New York, pp 1029–1050
Flohé L (2016) The impact of thiol peroxidases on redox regulation. Free Radic Res 50:126–142
Jiao Y, Wang Y, Guo S, Wang G (2017) Glutathione peroxidases as oncotargets. Oncotarget 8:80093
Couto N, Wood J, Barber J (2016) The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med 95:27–42
Mazzetti AP, Fiorile MC, Primavera A, Bello ML (2015) Glutathione transferases and neurodegenerative diseases. Neurochem Int 82:10–18
Tew KD, Townsend DM (2012) Glutathione-S-transferases as determinants of cell survival and death. Antioxid Redox Signal 17:1728–1737
Deeley RG, Westlake C, Cole SP (2006) Transmembrane transport of endo-and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 86:849–899
Keppler D (2011) Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy. In: Fromm MF, Kim RB (eds) Drug transporters. Springer, New York, pp 299–323
Cole S (2014) Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future. Annu Rev Pharmacol Toxicol 54:95–117
Cole SP (2014) Multidrug resistance protein 1 (MRP1, ABCC1): A ‘multitasking’ ABC transporter. J Biol Chem 289:30880–30888
Allen NJ, Barres BA (2009) Neuroscience: glia more than just brain glue. Nature 457:675–677
Argente-Arizon P, Guerra-Cantera S, Garcia-Segura LM, Argente J, Chowen JA (2017) Glial cells and energy balance. J Mol Endocrinol 58:R59–R71
Parpura V, Heneka MT, Montana V, Oliet SHR, Schousboe A, Haydon PG, Stout RF, Spray DC, Reichenbach A, Pannicke T, Pekny M, Pekna M, Zorec R, Verkhratsky A (2012) Glial cells in (patho)physiology. J Neurochem 121:4–27
Schmidt MM, Dringen R (2012) Glutathione (GSH) synthesis and metabolism. In: Choi I-Y, Gruetter R (eds) Neural metabolism in vivo. Springer, Boston, pp 1029–1050
McBean GJ (2017) Cysteine, glutathione, and thiol redox balance in astrocytes. Antioxidants 6:62
Hirrlinger J, König J, Dringen R (2002) Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones. J Neurochem 82:716–719
Ballerini P, Di Iorio P, Ciccarelli R, Nargi E, D’alimonte I, Traversa U, Rathbone MP, Caciagli F (2002) Glial cells express multiple ATP binding cassette proteins which are involved in ATP release. Neuroreport 13:1789–1792
Dallas S, Miller DS, Bendayan R (2006) Multidrug resistance-associated proteins: expression and function in the central nervous system. Pharmacol Rev 58:140–161
Hirrlinger J, Moeller H, Kirchhoff F, Dringen R (2005) Expression of multidrug resistance proteins (Mrps) in astrocytes of the mouse brain: a single cell RT-PCR study. Neurochem Res 30:1237–1244
Nies A, Jedlitschky G, König J, Herold-Mende C, Steiner H, Schmitt H-P, Keppler D (2004) Expression and immunolocalization of the multidrug resistance proteins, MRP1–MRP6 (ABCC1–ABCC6), in human brain. Neuroscience 129:349–360
Hirrlinger J, Schulz JB, Dringen R (2002) Glutathione release from cultured brain cells: multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells. J Neurosci Res 69:318–326
Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R (2006) The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes. J Neurochem 97:373–384
Dringen R, Pfeiffer B, Hamprecht B (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19:562–569
Hirrlinger J, König J, Keppler D, Lindenau J, Schulz JB, Dringen R (2001) The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress. J Neurochem 76:627–636
Akerboom TP, Sies H (1989) Transport of glutathione, glutathione disulfide, and glutathione conjugates across the hepatocyte plasma membrane. Methods Enzymol 173:523–534
Waak J, Dringen R (2006) Formation and rapid export of the monochlorobimane–glutathione conjugate in cultured rat astrocytes. Neurochem Res 31:1409–1416
Jedlitschky G, Keppler D (2002) Transport of leukotriene C4 and structurally related conjugates. Vitam Horm 64:153–184
Leier I, Jedlitschky G, Buchholz U, Center M, Susan P, Deeley RG, Keppler D (1996) ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump. Biochem J 314:433–437
Beyer RE, Segura-Aguilar J, Di Bernardo S, Cavazzoni M, Fato R, Fiorentini D, Galli MC, Setti M, Landi L, Lenaz G (1996) The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc Natl Acad Sci USA 93:2528–2532
Cullen JJ, Hinkhouse MM, Grady M, Gaut AW, Liu J, Zhang YP, Weydert CJ, Domann FE, Oberley LW (2003) Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism. Cancer Res 63:5513–5520
Huenchuguala S, Muñoz P, Graumann R, Paris I, Segura-Aguilar J (2016) DT-diaphorase protects astrocytes from aminochrome-induced toxicity. Neurotoxicology 55:10–12
Karczewski JM, Peters JG, Noordhoek J (1999) Quinone toxicity in DT-diaphorase-efficient and-deficient colon carcinoma cell lines. Biochem Pharmacol 57:27–37
Ruiz-Larrea MB, Garrido MJ, Lacort M (1993) Estradiol-induced effects on glutathione metabolism in rat hepatocytes. J Biochem 113:563–567
Drukarch B, Jongenelen CA, van Muiswinkel FL (2001) NAD(P)H:quinone oxidoreductase (NQO1) protects astroglial cells against L-Dopa toxicity. In: Dansette PM, Snyder R, Delaforge M, Gibson GG, Greim H, Jollow DJ, Monks TJ, Sipes IG (eds) Biological reactive intermediates VI. Springer, Boston, pp 237–240
Kapinya KJ, Harms U, Harms C, Blei K, Katchanov J, Dirnagl U, Hörtnagl H (2003) Role of NAD(P)H:quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischaemia in vivo. J Neurochem 84:1028–1039
Tulpule K, Hohnholt M, Hirrlinger J, Dringen R (2014) Primary cultures of astrocytes and neurons as model systems to study the metabolism and metabolite export from brain cells. In: Hirrlinger J, Waagepetersen HS (eds) Neuromethods: brain energy metabolism. Springer, New York, pp 45–72
Dringen R, Kranich O, Hamprecht B (1997) The γ-glutamyl transpeptidase inhibitor acivicin preserves glutathione released by astroglial cells in culture. Neurochem Res 22:727–733
Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Protoc 2:223–228
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Luther EM, Koehler Y, Diendorf J, Epple M, Dringen R (2011) Accumulation of silver nanoparticles by cultured primary brain astrocytes. Nanotechnology 22:375101
Abe K, Saito H (1996) Menadione toxicity in cultured rat cortical astrocytes. Jpn J Pharmacol 72:299–306
Klotz L-O, Hou X, Jacob C (2014) 1, 4-Naphthoquinones: from oxidative damage to cellular and inter-cellular signaling. Molecules 19:14902–14918
Ross D, Thor H, Orrenius S, Moldeus P (1985) Interaction of menadione (2-methyl-1, 4-naphthoquinone) with glutathione. Chem-Biol Interact 55:177–184
Vogel R, Wiesinger H, Hamprecht B, Dringen R (1999) The regeneration of reduced glutathione in rat forebrain mitochondria identifies metabolic pathways providing the NADPH required. Neurosci Lett 275:97–100
Dinkova-Kostova AT, Talalay P (2010) NAD (P) H: quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector. Arch Biochem Biophys 501:116–123
Li J, Lin JC, Wang H, Peterson JW, Furie BC, Furie B, Booth SL, Volpe JJ, Rosenberg PA (2003) Novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons. J Neurosci 23:5816–5826
Dringen R, Hamprecht B (1997) Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells. Brain Res 759:67–75
Ehrke E, Arend C, Dringen R (2015) 3-Bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes. J Neurosci Res 93:1138–1146
Schmidt MM, Dringen R (2010) Fumaric acid diesters deprive cultured primary astrocytes rapidly of glutathione. Neurochem Int 57:460–467
Tulpule K, Dringen R (2011) Formaldehyde stimulates Mrp1-mediated glutathione deprivation of cultured astrocytes. J Neurochem 116:626–635
Meyer N, Koehler Y, Tulpule K, Dringen R (2013) Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures. Neurochem Int 62:1012–1019
Tadepalle N, Koehler Y, Brandmann M, Meyer N, Dringen R (2014) Arsenite stimulates glutathione export and glycolytic flux in viable primary rat brain astrocytes. Neurochem Int 76:1–11
Arend C, Brandmann M, Dringen R (2013) The antiretroviral protease inhibitor ritonavir accelerates glutathione export from cultured primary astrocytes. Neurochem Res 38:732–741
Brandmann M, Tulpule K, Schmidt MM, Dringen R (2012) The antiretroviral protease inhibitors indinavir and nelfinavir stimulate Mrp1-mediated GSH export from cultured brain astrocytes. J Neurochem 120:78–92
Tulpule K, Schmidt MM, Boecker K, Goldbaum O, Richter-Landsberg C, Dringen R (2012) Formaldehyde induces rapid glutathione export from viable oligodendroglial OLN-93 cells. Neurochem Int 61:1302–1313
Hirrlinger J, Dringen R (2005) Multidrug resistance protein 1-mediated export of glutathione and glutathione disulfide from brain astrocytes. Methods Enzymol 400:395–409
Hirrlinger J, Schulz JB, Dringen R (2002) Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson’s disease. J Neurochem 82:458–467
Homma M, Suzuki H, Kusuhara H, Naito M, Tsuruo T, Sugiyama Y (1999) High-affinity efflux transport system for glutathione conjugates on the luminal membrane of a mouse brain capillary endothelial cell line (MBEC4). J Pharmacol Exp Ther 288:198–203
Kishi T, Takahashi T, Mizobuchi S, Mori K, Okamoto T (2002) Effect of dicumarol, a NAD (P) H: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes. Free Radic Res 36:413–419
Nemeikait A, Šarlauskas J, Anusevičius Ž, Nivinskas H, Narimantas Č (2003) Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD (P) H: quinone oxidoreductase (NQO1) and oxidative stress. Arch Biochem Biophys 416:110–118
Renes J, de Vries EE, Hooiveld GJ, Krikken I, Jansen PL, Müller M (2000) Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal. Biochem J 350:555–561
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Christian Arend and Ralf Dringen would like to acknowledge the substantial financial support of the Tönjes-Vagt-Foundation.
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Raabe, J., Arend, C., Steinmeier, J. et al. Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes. Neurochem Res 44, 333–346 (2019). https://doi.org/10.1007/s11064-018-2680-y
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DOI: https://doi.org/10.1007/s11064-018-2680-y