Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinson's disease, including selective nigrostriatal dopaminergic degeneration and alpha-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 microm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a "replacement" for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by alpha-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinson's disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinson's disease.

IntroductionDespite advances in clinical therapy, metastasis is still the leading cause of death in breast cancer patients (1). A clearer understanding of molecular mechanisms that drive metastasis will help to develop more effective therapies (2). Our present study focused on metabolism as an essential driver of tumor growth and metastasis, potentially common to all breast cancer types. Normal cells primarily use mitochondrial oxidative phosphorylation (OXPHOS) for energy production, whereas cancer cells depend on aerobic glycolysis (the Warburg effect) to generate energy and glycolytic intermediates for enhanced growth (3, 4). Tumor cells also generate high levels of reduced forms of NAD + , NADH, and NADPH as important cofactors and redox components (4, 5). These altered metabolic activities can be linked to mitochondrial dysfunction that inhibits OXPHOS, increases ROS, promotes uncontrolled growth, and causes DNA damage that further supports a metastatic phenotype (6, 7). Mitochondrial dysfunctions can be caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes encoding mitochondrial proteins (6,8) that are essential for the respiratory chain/OXPHOS system. Due to the lack of protective histones and limited DNA repair (8), mtDNA mutations occur at high rates and were found in tumors including breast cancer (6,(9)(10)(11)(12)(13)(14), which suggests that defects in OXPHOS might contribute to tumorigenesis.By combining the nuclear genome of a recipient cell with the mitochondrial genome of a donor cell using cybrid technology, mitochondria from the triple-negative aggressive breast cancer cell lines MDA-MB-435 (15) and MDA-MB-231 facilitated tumor progression and metastasis in nonmetastatic tumor cells (7, 10). The donor cell lines harbor mtDNA mutations in tRNAs, in the

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Parkinson's disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for 3 H-dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR).Neuroblastoma cells stably expressing the ROT-insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants a-tocopherol and coenzyme Q 10 . PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.

The products of six unidentified reading frames of human mitochondrial DNA are precipitated from a mitochondrial lysate by antibodies against highly purified native beef heart NADH-ubiquinone oxidoreductase (complex I). These products are enriched greatly in a human submitochondrial fraction enriched in NADH-Q1 and NADH-K3Fe(CN)6 oxidoreductase activities. We conclude that the six reading frames encode components of the respiratory-chain NADH dehydrogenase.

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Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.aging | mitochondria | respiratory chain | free radicals M itochondria are key metabolic organelles whose oxidative phosphorylation (OXPHOS) system is considered to be one of the most efficient producers of bioenergy. When OX-PHOS function is compromized (e.g., by mutations or toxins), bioenergy supply and cellular homeostasis are seriously disrupted, which can be lethal.OXPHOS complex I plays a central role in the regulation of ATP production, intermediary metabolism, and apoptosis (1, 2), and mutations affecting it cause many human pathologies (3). It has also been proposed as a pacemaker of the aging process (4). Treatments inferred to decrease the production of reactive oxygen species (ROS) at the level of complex I can prolong lifespan in Drosophila (5). All these characteristics make it essential to understand better the role of complex I in vivo and its involvement in aging.Many organisms possess alternative enzymes that can bypass or replace the proton-translocating complexes of the mitochondrial respiratory chain. These include the alternative oxidases (AOX) and the NADH dehydrogenases of the Ndi and Nde families. Together these enzymes provide an alternative respiratory chain that potentially allows the maintenance of redox homeostasis and intermediary metabolism under conditions where flux through the "standard" respiratory chain is limited by high ATP levels, the action of toxins or other physiological restraints (6, 7). AOX acts as a bypass of complexes III and IV, whereas Nde or Ndi can bypass complex I.In previous studies these bypass enzymes were shown to be active when introduced into the mitochondria of higher metazoans such as mammals (8-12), arthropods (13), or nematodes (14), all of which lack endogenous alternative enzymes. Fu...