Protective action of tamoxifen on carboxyatractyloside-induced mitochondrial permeability transition Luz Hernández-Esquivel, Natalia-Pavón, Cecilia Zazueta, Noemí García, Francisco Correa, Edmundo Chávez ⁎ Departamento de Bioquímica, Instituto Nacional de Cardiología, Ignacio Chávez, Mexico D.F. 014080, Mexico abstract article info Article history: Received 17 June 2010 Accepted 27 January 2011 Keywords: Mitochondria Mitochondrial calcium Tamoxifen Permeability transition Adenine nucleotide translocase Aims: Mitochondrial permeability transition is established after massive Ca 2+ accumulation inside the matrix, in addition to an inducer. The closure of the pore can be accomplished by adenosine diphosphate and the immunosuppressant cyclosporin A. Recently, the estrogen antagonist, tamoxifen, has been introduced as an inhibitor of the opening of the permeability transition pore. However, the mechanism by which this drug inhibits pore opening is still under discussion. This work was performed with the purpose of establishing the membrane system involved in tamoxifen-induced pore closure. For this purpose, permeability transition was induced after the addition of carboxyatractyloside, which is a specific reagent that interacts with the adenine nucleotide translocase. Main methods: Permeability transition was assessed by analyzing matrix Ca 2+ release, transmembrane electric gradient, and mitochondrial swelling in aged, as well as in freshly prepared mitochondria. Also, cytochrome c content was analyzed in membrane mitochondria as well as in the supernatant. Key findings: In freshly prepared mitochondria, tamoxifen, at the concentration of 10 μM, totally inhibited nonspecific membrane permeability induced by 1 μM carboxyatractyloside. In addition, tamoxifen inhibited non-specific permeability in aged mitochondria and diminished membrane fluidity. Significance: Plausibly, the inhibitory effect of tamoxifen on nonspecific membrane permeability, as induced by carboxyatractyloside, should be ascribed to a diminution, of membrane fluidity by this drug. © 2011 Elsevier Inc. All rights reserved. Introduction Mitochondrial Ca 2+ overload induces the opening of a transmem- brane non-specific pore with a diameter size of around 2 to 3 nm, through which DNA can even be released (García and Chávez, 2007). Such a process, named membrane permeability transition (MPT), allows the release of matrix metabolites up to a molecular weight of 1.5 kDa. Opening of the non-specific pore underlies also the process of cytochrome c release from the intermembrane space; thus, MPT is involved in the mitochondrial pathway of apoptosis (Bernardi et al., 1994; Kroemer et al., 1995; Zoratti and Szabó, 1995; Correa et al., 2007). Besides Ca 2+ , opening of the transmembrane pore requires an inducer such as carboxyatractyloside (García et al., 2009a), among several other inducers. The closure of such a pore is accomplished by adenosine diphosphate (ADP) (Haworth and Hunter, 2000), as well as by the immunosuppressant cyclosporin A, in addition to a wide array of reagents, such as antioxidants (Broekemeier et al., 1989; Zoratti and Szabó, 1995). Modulation of the increased permeability has been associated with the orientation of the ADP/ATP translocase (ANT) across the inner membrane. In this context it is well established that atractyloside and carboxyatractyloside by stabilizing ANT in the cytosol side of the inner membrane promote pore opening (Asimakis and Sordahl, 1977; Le Quoc and Le Quoc, 1988; Nogorodov et al., 1990). Recently, the drug tamoxifen has been introduced as an inhibitor of membrane permeability transition (Custodio et al., 1998; Cardoso et al., 2004). This drug is a selective estrogen receptor antagonist used in chemotherapy of breast cancer (Wang et al., 2009). However, there are reports indicating that, in mitochondria, tamoxifen promotes oxidative injury, causing lipid membrane peroxidation (Parvez et al., 2008). In addition to the above, there is also information about the cytotoxic role of tamoxifen as an inducer of apoptosis in cancer cell lines (Nazarewicz et al., 2007; Parihar et al., 2008). Nevertheless, several reports point out that tamoxifen acts as an antioxidant preventing brain and heart damages induced by oxidative stress (Wakade et al., 2008; Ek et al., 2008). The aim in this work was addressed to further contribute to the understanding of the action of tamoxifen, as an inhibitor of pore opening. Carboxyatractyloside (CAT), which is well known, does not act as a generator of reactive oxygen derived species (ROS). The results obtained show that tamoxifen, at the concentration of 10 μM, inhibited Life Sciences 88 (2011) 681–687 ⁎ Corresponding author. Fax: + 52 155 5573 0927. E-mail address: echavez@salud.gob.mx (E. Chávez). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.02.006 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie