Mecanismos de toxicidade dos metabólitos do fipronil, dessulfinil e sulfona, em mitocôndrias isoladas de fígado de rato

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

UNLABELLED Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but thatbokwas not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found thatbok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injuryin vitroand seizure-induced neuronal injuryin vivo Deletion ofbokalso increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death inbax-deficient neurons. Single-cell imaging demonstrated thatbok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bokdeficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death inbok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injuryin vitroandin vivo SIGNIFICANCE STATEMENT Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activitiesin vitroandin vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins.

Inhibition of mitochondrial oxidative phosphorylation by 2-methyl-4-dimethylaminoazobenzene

The azodye 2-methyl-4-dimethylaminoazobenzene inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. Phosphorylation was more sensitive to the inhibitory action of the azodye than was the oxidation of succinate or ascorbate. The oxidation of NAD+-linked substrate was severely inhibited by the compound. In submitochondrial particles, only NADH oxidation was sensitive. The site of inhibition has been identified to lie between the dehydrogenase flavoprotein and ubiquinone.

Influence of cerebral ischemia and post-ischemic reperfusion on mitochondrial oxidative phosphorylation

Unilateral ischemia in the right cerebral hemisphere of the rat was induced by ligation of the right common carotid artery coupled with controlled hemorrhage to produce hypotension (25±8 mm/Hg). Where indicated after 30 min of ischemia, the withdrawn blood was reinfused to restore arterial pressure to normal. Mitochondria isolated from the ipsilateral hemisphere after 30 min of ischemia showed significantly lower respiratory rates than the organelles isolated from the contralateral side. Oxidation of NAD+-linked substrates was more sensitive to inhibition in ischemia (30%) than was of ferrocytochromec (12%), succinate oxidation being intermediate. The activities of membrane-bound dehydrogenases (both NADH and succinate-linked) were also significantly lowered. Ischemia did not affect the cytochrome content of mitochondria. Respiratory activity (NAD+-linked) of mitochondria isolated from the ipsilateral hemisphere was twice as sensitive to inhibition by fatty acid as was of preparations from the contralateral side. Mitochondria isolated from cerebral cortex after 90 min of post-ischemic reperfusion showed no significant improvement in the rate of substrate oxidation. Adenine nucleotide translocase activity and energy-dependent Ca2+ uptake, both of which decreased significantly in mitochondria isolated from the ischemic brain, showed little recovery, on reperfusion. These observations suggested the strong possibility that the deleterious effects of ischemia on mitochondrial respiratory function might be mediated by free fatty acids that are known to accumulate in large amounts in ischemic tissues. The pattern of inhibition of ATPase activity was consistent with this view.

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(->+)/2e(-) indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(->+)/2e(-). This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme. (c) 2006 Elsevier B.V. All rights reserved.

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III + IV (S) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

Characterisation of the active/de-active transition of mitochondrial complex I

Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39 kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Effect of monovalent cations on the kinetics of hypoxic conformational change of mitochondrial complex i

Mitochondrial complex I is a large, membrane-bound enzyme central to energy metabolism, and its dysfunction is implicated in cardiovascular and neurodegenerative diseases. An interesting feature of mammalian complex I is the so-called A/D transition, when the idle enzyme spontaneously converts from the active (A) to the de-active, dormant (D) form. The A/D transition plays an important role in tissue response to ischemia and rate of the conversion can be a crucial factor determining outcome of ischemia/reperfusion. Here, we describe the effects of alkali cations on the rate of the D-to-A transition to define whether A/D conversion may be regulated by sodium.At neutral pH (7–7.5) sodium resulted in a clear increase of rates of activation (D-to-A conversion) while other cations had minor effects. The stimulating effect of sodium in this pH range was not caused by an increase in ionic strength. EIPA, an inhibitor of Na+/H+antiporters, decreased the rate of D-to-A conversion and sodium partially eliminated this effect of EIPA. At higher pH (> 8.0), acceleration of the D-to-A conversion by sodium was abolished, and all tested cations decreased the rate of activation, probably due to the effect of ionic strength.The implications of this finding for the mechanism of complex I energy transduction and possible physiological importance of sodium stimulation of the D-to-A conversion at pathophysiological conditions in vivo are discussed.

Ischemic A/D transition of mitochondrial complex I and its role in ROS generation

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in cellular energy metabolism and provides approximately 40% of the proton-motive force that is utilized during mitochondrial ATP production. The dysregulation of complex I function – either genetically, pharmacologically, or metabolically induced – has severe pathophysiological consequences that often involve an imbalance in the production of reactive oxygen species (ROS). Slow transition of the active (A) enzyme to the deactive, dormant (D) form takes place during ischemia in metabolically active organs such as the heart and brain. The reactivation of complex I occurs upon reoxygenation of ischemic tissue, a process that is usually accompanied by an increase in cellular ROS production. Complex I in the D-form serves as a protective mechanism preventing the oxidative burst upon reperfusion. Conversely, however, the D-form is more vulnerable to oxidative/nitrosative damage. Understanding the so-called active/deactive (A/D) transition may contribute to the development of new therapeutic interventions for conditions like stroke, cardiac infarction, and other ischemia-associated pathologies. In this review, we summarize current knowledge on the mechanism of A/D transition of mitochondrial complex I considering recently available structural data and site-specific labeling experiments. In addition, this review discusses in detail the impact of the A/D transition on ROS production by complex I and the S-nitrosation of a critical cysteine residue of subunit ND3 as a strategy to prevent oxidative damage and tissue damage during ischemia–reperfusion injury.

Mitochondrial Transfer via Tunneling Nanotubes (TNT) is an Important Mechanism by which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the in vitro and in vivo Models of ARDS

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in pre-clinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the anti-microbial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC anti-microbial effect in the in vivo model of E.coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct co-culture of MSC with monocyte-derived macrophages (MDMs) enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through TNT-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the anti-microbial effect of MSC in vivo.

Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the anti-microbial effect of MSC in ARDS.

trans, trans-2,4-decadienal induces mitochondrial dysfunction and oxidative stress

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2, 4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 mu M) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 mu M) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved.