Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.

Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ER␣ and ER␤. ER␣ plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ER␤ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ER␤ localizes to the mitochondria. In immunocytochemistry studies, ER␤ was detected with two ER␤ antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ER␤ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ER␤ into the nucleus on 17␤-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ER␤, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS͞PAGE. The 50,000 -65,000 Mr band was digested with trypsin and subjected to matrix-assisted laser desorption͞ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ER␤. In summary, this study demonstrated that ER␤ is localized to mitochondria, suggesting a role for mitochondrial ER␤ in estrogen effects on this important organelle.nuclear receptor ͉ mitochondria E strogens play an important role in development, growth, and differentiation of both female and male secondary sex characteristics. Estrogen receptors (ERs) were the first identified nuclear receptor family member (1). The first ER, now called ER␣, was cloned in 1986 (2, 3). A second ER␤, was identified and cloned a decade later (4, 5). Like other members of the nuclear receptor superfamily, both ERs have a modular structure consisting of distinct functional domains (1). The DNA-binding domain (DBD) enables the receptor to bind its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AG-GTCA spaced by 3 bp, referred to as an estrogen response element (ERE). The ligand-binding domain enables estrogen binding to the receptors. ERs are highly conserved between ER␣ and ER␤, with Ͼ95% homology for the DBD and Ϸ50% homology for the ligand-binding domain. Less homology is observed for the transactivational domain between ER␣ and ER␤ (5, 6).Genomic actions of ER␣ are well described (7). On binding to ER␣, estrogens induce a conformational change in the ER␣ proteins, which is accompanied by the dissociation of the accessory protein, heat shock protein 90, thereby exposing the DBD. In the nucleus, the receptor-ligand complex binds to DNA and modulates gene transcription. This transcriptional͞translational activation is comparatively slow and sensitive to cyclohexi...

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The evidence is compelling that free radicals, plus increases in free cytosolic Ca2+ and Na+, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca2+ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (•OH) radicals, plus ascorbyl and other carbon‐centered radicals when exposed to 2.5 µM Ca2+, 14 mM Na+, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin‐induced neurodegeneration. In a feed‐forward cycle, exposure of isolated mitochondria to •OH significantly increases subsequent radical production five‐ to 16‐fold (average = 8.8 ± 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH‐CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after •OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.

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