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Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Succinate:quinone reductase (SQR) of Complex II occupies a unique central point in the mitochondrial respiratory system as a major source of electrons driving reactive oxygen species (ROS) production. It is an ideal pharmaceutical target for modulating ROS levels in normal cells to prevent oxidative stress-induced damage or alternatively,increase ROS in cancer cells, inducing cell death.The value of drugs like diazoxide to prevent ROS production,protecting normal cells, whereas vitamin E analogues promote ROS in cancer cells to kill them is highlighted. As pharmaceuticals these agents may prevent degenerative disease and their modes of action are presently being fully explored. The evidence that SDH/Complex II is tightly coupled to the NADH/NAD+ ratio in all cells,impacted by the available supplies of Krebs cycle intermediates as essential NAD-linked substrates, and the NAD+-dependent regulation of SDH/Complex II are reviewed, as are links to the NAD+-dependent dehydrogenases, Complex I and the E3 dihiydrolipoamide dehydrogenase to produce ROS. This review collates and discusses diverse sources of information relating to ROS production in different biological systems, focussing on evidence for SQR as the main source of ROS production in mitochondria, particularly its relevance to protection from oxidative stress and to the mitochondrial-targeted anti cancer drugs (mitocans) as novel cancer therapies [corrected].

Besides major NADH-, succinate-, and other substrate oxidase reactions resulting in four-electron reduction of oxygen to water, the mitochondrial respiratory chain catalyzes one-electron reduction of oxygen to superoxide radical O(2)(-.) followed by formation of hydrogen peroxide. In this paper the superoxide generation by Complex I in tightly coupled bovine heart submitochondrial particles is quantitatively characterized. The rate of superoxide formation during Deltamu(H(+))-controlled respiration with succinate depends linearly on oxygen concentration and contributes approximately 0.4% of the overall oxidase activity at saturating (0.25 mM) oxygen. The major part of one-electron oxygen reduction during succinate oxidation (approximately 80%) proceeds via Complex I at the expense of its Deltamu(H(+))-dependent reduction (reverse electron transfer). At saturating NADH the rate of O(2)(-.) formation is substantially smaller than that with succinate as the substrate. In contrast to NADH oxidase, the rate-substrate concentration dependence for the superoxide production shows a maximum at low (approximately 50 microM) concentrations of NADH. NAD+ and NADH inhibit the succinate-supported superoxide generation. Deactivation of Complex I results in almost complete loss of its NADH-ubiquinone reductase activity and in increase in NADH-dependent superoxide generation. A model is proposed according to which complex I has two redox active nucleotide binding sites. One site (F) serves as an entry for the NADH oxidation and the other one (R) serves as an exit during either the succinate-supported NAD+ reduction or superoxide generation or NADH-ferricyanide reductase reaction.

We have previously demonstrated regulatory roles for Rho subfamily of G-proteins in glucose- and calcium-induced insulin secretion. Herein, we examined regulation by these proteins of insulin secretion from betaTC3 cells elicited by mitochondrial fuels, such as the succinic acid methyl ester (SAME). Preincubation of these cells with Clostridium difficile toxin-B (200 ng/mL), which monoglucosylates and inactivates Cdc42 and Rac1, markedly decreased (> 70%) SAME-induced insulin secretion. Furthermore, exposure of betaTC3 cells to GGTI-2147 (20 microM), a selective inhibitor of the requisite prenylation of Rac1 and Cdc42, significantly reduced (> 80%) SAME-induced insulin release, suggesting that post-translational prenylation of these proteins is necessary for SAME-induced insulin release. Western blot analysis indicated localization of Cdc42, Rac1, and Ras in the beta cell mitochondrial fraction. Confocal microscopy revealed a modest, but inconsistent, increase in the association of either Rac1 or Cdc42 with Mitotracker, a mitochondrial marker, following exposure to SAME. These data suggest that activation of preexisting intramitochondrial Rac1 and Cdc42 may be sufficient to regulate SAME-induced insulin secretion. Together, our findings support a role for G-proteins in insulin secretion at a step dependent on mitochondrial metabolism. They also identify mevalonate-derived, isoprenoid modified Rho G-proteins as specific signaling molecules in recently proposed succinate mechanism of insulin release.

Fourteen antipsychotics of 5 different chemical classes were tested for their effects on mitochondrial succinate oxidation. Each of the compounds tested, with the exception of haloperidol, phenothiazine and prochlorperazine, inhibited succinate oxidation. The apparent inhibition constant for phenothiazine derivatives declined with increasing values of the partition coefficient.

Our study found the uptake of [14C]succinate into bovine adrenocortical cells to be sodium-dependent, inhibited by lithium, and to have an apparent K(m) of 146 mumol/l. Succinate uptake was inhibited by glutarate, fumarate, alpha-ketoglutarate, and maleate but not by 2,3-dimethylsuccinate or cis-aconitate, specific inhibitors of the basolateral Na(+)-dicarboxylate transporter of renal proximal tubule cells. Succinate uptake was highest at pH 6.0 and decreased with increasing pH. Transport of succinate was not significantly inhibited by citrate at pH 7.4 whereas at pH 6.0 inhibition of succinate uptake by citrate was small but significant. The affinity of the adrenal dicarboxylate transporter towards succinate ranges in between the low affinity of the renal luminal dicarboxylate transporter and the high affinity of the respective basolateral transporter. The pH dependency of succinate uptake and the missing inhibition by citrate at pH 7.4 differ from both the luminal and from the basolateral dicarboxylate transporters in kidney, liver, intestine, and placenta. These functional characteristics provide evidence for the existence of a Na(+)-dicarboxylate cotransporter in adrenocortical cells which may supply cholesterol metabolism with reducing substrates.