Mitochondrial oxygen consumption inhibition importance for TMT-dependent cell death in undifferentiated PC12 cells Francesco Misiti a, * , Federica Orsini b , M. Elisabetta Clementi c , Wanda Lattanzi d , Bruno Giardina b,c , Fabrizio Michetti d a Department of Health and Motor Sciences, University of Cassino, Viale Bonomi, 03043 Cassino (FR), Italy b Institute of Biochemistry and Clinical Biochemistry, Catholic University, Largo F. Vito 1, 00168 Rome, Italy c CNR, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Largo F. Vito 1, 00168 Rome, Italy d Institute of Anatomy and Cell Biology, Catholic University, Largo F. Vito 1, 00168 Rome, Italy Received 17 July 2007; received in revised form 8 November 2007; accepted 19 November 2007 Available online 4 December 2007 Abstract The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24 h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (DC) in PC12 cells was also determined. After 24 h in culture, a strong loss of mitochondrial membrane potential (DC) was observed in TMT- treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin. # 2007 Elsevier Ltd. All rights reserved. Keywords: Pheochromocytoma 12; Trimethyltin; Mitochondria; Mitochondrial membrane potential DC; Apoptosis 1. Introduction Trimethyltin is an organotin compound that induces a distinct pattern of selective neuronal degeneration in the central nervous system. Brain damage is evident in limbic structures, especially in the hippocampus, with the pyramidal cells in CA1 and CA3 subfields being predominantly affected (Balaban et al., 1988; Whittington et al., 1989; Geloso et al., 1997, 2002). TMT-induced limbic degeneration is accompanied by beha- vioural alterations including cognitive deficits with a delayed onset (Brown et al., 1979; Dyer et al., 1982; Boyer, 1989) so that, owing to its characteristics, TMT intoxication is regarded as a suitable model of chronic neurodegeneration with cognitive impairment, also useful in study of Alzheimer’s disease (Kasseda et al., 2003). The neuropathological, neurochemical and neurobehavioural alterations exerted in vivo have been analyzed in rodents as well as in human cases of accidental poisoning (Fortemps et al., 1978; Brown et al., 1979; Chang et al., 1982; Noland et al., 1982; Stanton et al., 1991; Feldman et al., 1993; Geloso et al., 1997, 2002; Gui-bin et al., 2000; Harry and Lefebvre d’Hellencourt, 2003). As a result of information actually available, the molecular and cellular pathogenesis of TMT-induced neurodegeneration is believed to be a composite picture involving several steps, as TMT is able to affect multiple processes in sensitive cells. In particular, the role of oxidative stress in TMT-mediated toxicity have been previously demonstrated by different authors, suggesting a direct involvement of the mitochondrial function in the response to the neurotoxicant (Aldridge et al., 1977). Interestingly the gene expression profile induced by sub-lethal TMT exposure in cell cultures suggests the possible implication of oxygen-related processes along with membrane transport impairment, delineating basic stress adaptation mechanisms possibly shared by cells responding to different death-inducing noxae (Lattanzi et al., 2007). In this respect, since the role of mitochondria as a site where multiple cell death signalling www.elsevier.com/locate/neuint Available online at www.sciencedirect.com Neurochemistry International 52 (2008) 1092–1099 * Corresponding author. Tel.: +39 7762994423; fax: +39 7762993902. E-mail address: f.misiti@unicas.it (F. Misiti). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.11.008