There is substantial evidence to support the theory that oxidative stress plays an important role in the pathophysiology of CVD (1). Accordingly, several naturally-occurring antioxidant compounds have been utilized to counteract oxidative cardiac injury (2,3). Another promising strategy for protecting cardiac cells against oxidative stress may be through the induction of endogenous antioxidants and phase II enzymes. Sulforaphane (SF) is a naturally-occurring isothiocyanate that is highly concentrated in Cruciferous vegetables. Many studies have shown a strong chemopreventive effect of SF through its ability to induce phase II detoxifying enzymes by activating antioxidant-response element (ARE) through the induction of Nrf2 (4), but no data are available to support a similar role for SF in cardioprotection. Using cultured rat cardiomyocytes, the time-dependent induction of cellular antioxidants and phase II enzymes by SF and its ability to protect cardiac cells against oxidative stress have been characterised, and the translocation of nrf2 to the nucleus after SF supplementation has been investigated. Cultured rat cardiomyocytes were prepared and grown as described previously (5). Cells were supplemented with 5 mM-SF for different time periods (6, 12, 24 and 48 h). The activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and NAD(P)H-quinone reductase 1 (NQO1) were determined spectrophotometrically, and the content of intracellular reduced glutathione (GSH) was estimated using the fluorescent indicator monochlorobimane. Western-blot analyses of GR, GST, GPx and NQO1 were performed using specific antibodies and following the manufacturer’s recommended protocols. ROS formation was determined by a spectrofluorimetric method using 2',7'-dichlorofluorescein diacetate. Cell viability was evaluated by MTT assay and Nrf2 translocation to the nucleus by laser confocal microscopy using specific antibodies. Incubation of cardiomyocytes with SF resulted in a significant elevation of cellular GSH content for all exposure times. SF supplementation also led to a time-dependent increase in GR, GST and NQO1 activities. Accordingly, a significant increase in GR, GST and NQO1 expression was observed. In contrast, incubation of cardiomyocytes with SF for 6–48 h did not result in any significant increase in cellular GPx activity and expression, in agreement with published data (6). Laser confocal microscopy revealed the translocation to the nucleus of Nrf2 after SF supplementation. SF pretreatment led to a decreased intracellular accumulation of ROS and marked cytoprotection after exposure to 100 μM-H2O2. The results demonstrate, for the first time, that a number of endogenous antioxidants and phase II enzymes can be induced in cultured cardiomyocytes by low micromolar concentrations of SF, and that this nutritionally-mediated up-regulation of cellular defences is accompanied by a markedly increased resistance to cardiac cell injury elicited by peroxide. 1. Ceconi C, Boraso A, Cargnoni A & Ferrari R (2003) Arch Biochem Biophys 420, 217 -21. 2. Angeloni C, Spencer JP, Leoncini E, Biagi PL & Hrelia S (2007) Biochimie 89, 73-82. 3. Bandyopadhyay D, Chattopadhyay A, Ghosh G & Datta AG (2004) Curr Med Chem 11, 369-387. 4. Fimognari C & Hrelia P (2007) Mutat Res 635, 90-104. 5. Hrelia S, Fiorentini D, Maraldi T, Angeloni C, Bordoni A, Biagi PL & Hakim G (2002) Biochim Biophys Acta 1567, 150–156. 6. Cao Z & Li Y (2004) Eur J Pharmacol 489, 39–48.

Induction of phase II enzymes by sulforaphane leads to cardioprotection

LEONCINI, EMANUELA;CALONGHI, NATALIA;PAGNOTTA, ELEONORA;BIAGI, PIERLUIGI;HRELIA, SILVANA
2007

Abstract

There is substantial evidence to support the theory that oxidative stress plays an important role in the pathophysiology of CVD (1). Accordingly, several naturally-occurring antioxidant compounds have been utilized to counteract oxidative cardiac injury (2,3). Another promising strategy for protecting cardiac cells against oxidative stress may be through the induction of endogenous antioxidants and phase II enzymes. Sulforaphane (SF) is a naturally-occurring isothiocyanate that is highly concentrated in Cruciferous vegetables. Many studies have shown a strong chemopreventive effect of SF through its ability to induce phase II detoxifying enzymes by activating antioxidant-response element (ARE) through the induction of Nrf2 (4), but no data are available to support a similar role for SF in cardioprotection. Using cultured rat cardiomyocytes, the time-dependent induction of cellular antioxidants and phase II enzymes by SF and its ability to protect cardiac cells against oxidative stress have been characterised, and the translocation of nrf2 to the nucleus after SF supplementation has been investigated. Cultured rat cardiomyocytes were prepared and grown as described previously (5). Cells were supplemented with 5 mM-SF for different time periods (6, 12, 24 and 48 h). The activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and NAD(P)H-quinone reductase 1 (NQO1) were determined spectrophotometrically, and the content of intracellular reduced glutathione (GSH) was estimated using the fluorescent indicator monochlorobimane. Western-blot analyses of GR, GST, GPx and NQO1 were performed using specific antibodies and following the manufacturer’s recommended protocols. ROS formation was determined by a spectrofluorimetric method using 2',7'-dichlorofluorescein diacetate. Cell viability was evaluated by MTT assay and Nrf2 translocation to the nucleus by laser confocal microscopy using specific antibodies. Incubation of cardiomyocytes with SF resulted in a significant elevation of cellular GSH content for all exposure times. SF supplementation also led to a time-dependent increase in GR, GST and NQO1 activities. Accordingly, a significant increase in GR, GST and NQO1 expression was observed. In contrast, incubation of cardiomyocytes with SF for 6–48 h did not result in any significant increase in cellular GPx activity and expression, in agreement with published data (6). Laser confocal microscopy revealed the translocation to the nucleus of Nrf2 after SF supplementation. SF pretreatment led to a decreased intracellular accumulation of ROS and marked cytoprotection after exposure to 100 μM-H2O2. The results demonstrate, for the first time, that a number of endogenous antioxidants and phase II enzymes can be induced in cultured cardiomyocytes by low micromolar concentrations of SF, and that this nutritionally-mediated up-regulation of cellular defences is accompanied by a markedly increased resistance to cardiac cell injury elicited by peroxide. 1. Ceconi C, Boraso A, Cargnoni A & Ferrari R (2003) Arch Biochem Biophys 420, 217 -21. 2. Angeloni C, Spencer JP, Leoncini E, Biagi PL & Hrelia S (2007) Biochimie 89, 73-82. 3. Bandyopadhyay D, Chattopadhyay A, Ghosh G & Datta AG (2004) Curr Med Chem 11, 369-387. 4. Fimognari C & Hrelia P (2007) Mutat Res 635, 90-104. 5. Hrelia S, Fiorentini D, Maraldi T, Angeloni C, Bordoni A, Biagi PL & Hakim G (2002) Biochim Biophys Acta 1567, 150–156. 6. Cao Z & Li Y (2004) Eur J Pharmacol 489, 39–48.
Diet and Chronic Disease
241
241
E. Leoncini; N. Calonghi; E. Pagnotta; P.L. Biagi; S. Hrelia
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/46554
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