Efeitos da abamectina na bioenergética de mitocôndrias isoladas de fígado de rato: Juliana Carla Castanha Zanoli. -

Pós-graduação em Ciência Animal - FMVA

Mecanismos da infertilidade causada por Gossipol em ratos e efeito protetor da vitamina E

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Análise funcional das proteínas desacopladas mitocondriais de plantas utilizando RNA-seq e mutantes de inserção

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fipronil na bioenergética de mitocôndrias isoladas de figado de rato

Pós-graduação em Ciência Animal - FMVA

Effect of fipronil on energy metabolism in the perfused rat liver

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative effects of fipronil and its metabolites sulfone and desulfinyl on the isolated rat liver mitochondria

Fipronil is an insecticide extensively used to control pests in crops and animals. There are relates of poisoning due to exposure of fipronil in mammals and the liver has been suggested as potential target. In this study, we evaluated the effects of fipronil and its metabolites sulfone and desulfinyl on the bioenergetics, reactive oxygen species (ROS) production and calcium efflux from mitochondria isolated from rat liver. Fipronil (5-25 μM) inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I of the respiratory chain and decreased the mitochondrial membrane potential resulting in inhibition of ATP synthesis. Fipronil also caused uncoupling in succinate-energized mitochondria and calcium efflux. The metabolites sulfone and desulfinyl also acted as mitochondrial inhibitors and uncouplers and caused calcium efflux, but with different potencies, being the sulfone the more potent one. These effects of fipronil and its metabolites on mitochondrial bioenergetics and calcium homeostasis may be related to toxic effects of the insecticide in the liver.

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

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Identification of epsilon PKC Targets During Cardiac Ischemic Injury

Background: Epsilon-protein kinase C (epsilon PKC) protects the heart from ischemic injury. However, the mechanism(s) of epsilon PKC cardioprotection is still unclear. Identification of the epsilon PKC targets may aid in elucidating the epsilon PKC-mediated cardioprotective mechanisms. Previous studies, using epsilon PKC transgenic mice and difference in gel electrophoresis, identified proteins involved in glucose metabolism, the expression of which was modified by epsilon PKC. Those studies were accompanied by metabolomic analysis, suggesting that increased glucose oxidation may be responsible for the cardioprotective effect of epsilon PKC. Whether these epsilon PKC-mediated alterations were because of differences in protein expression or phosphorylation was not determined. Methods and Results: In the present study, we used an epsilon PKC -specific activator peptide, psi epsilon RACK, combined with phosphoproteomics, to find epsilon PKC targets, and identified that the proteins whose phosphorylation was altered by selective activation of epsilon PKC were mostly mitochondrial proteins. Analysis of the mitochondrial phosphoproteome led to the identification of 55 spots, corresponding to 37 individual proteins, exclusively phosphorylated, in the presence of psi epsilon RACK. The majority of the proteins identified were involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins. Conclusions: The protective effect of epsilon PKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose and lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by epsilon PKC phosphorylation may lead to epsilon PKC-mediated cardioprotection induced by psi epsilon RACK. (Circ J 2012; 76: 1476-1485)

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Composti organostannici nell'ambiente marino e membrane biologiche: risposte molecolari e biochimiche nei molluschi bivalvi

Organotin compounds are worldwide diffused environmental contaminants, mainly as consequence of their extensive past use as biocides in antifouling paints. In spite of law restrictions, due to unwanted effects, organotin still persist in waters, being poorly degraded, easily resuspended from sediments and bioaccumulated in exposed organisms. The widespread toxicity and the possible threat to humans, likely to be organotin-exposed through contaminated seafood, make organotin interactions with biomolecules an intriguing biochemical topic, apart from a matter of ecotoxicological concern. Among organotins, tributyltin (TBT) is long known as the most dangerous and abundant chemical species in the Mediterranean Sea. Due to its amphiphilic nature, provided by three lipophilic arms and an electrophilic tin core, TBT can be easily incorporated in biomembranes and affect their functionality. Accordingly, it is known as a membrane-active toxicant and a mitochondrial poison. Up to now the molecular action modes of TBT are still partially unclear and poorly explored in bivalve mollusks, even if the latter play a not neglectable role in the marine trophic chain and efficiently accumulate organotins. The bivalve mollusk Mytilus galloprovincialis, selected for all experiments, is widely cultivated in the Mediterranean and currently used in ecotoxicological studies. Most work of this thesis was devoted to TBT effects on mussel mitochondria, but other possible targets of TBT were also considered. A great deal of literature points out TBT as endocrine disrupter and the masculinization of female marine gastropods, the so-called imposex, currently signals environmental organotin contamination. The hormonal status of TBT-exposed mussels and the possible interaction between hormones and contaminants in modulating microsomal hydroxilases, involved in steroid hormone and organotin detoxification, were the research topics in the period spent in Barcelona (Marco Polo fellowship). The variegated experimental approach, which consisted of two exposure experiments and in vitro tests, and the choice of selected tissues of M. galloprovincialis, the midgut gland for mitochondrial and microsomal preparations for subsequent laboratory assays and the gonads for the endocrine evaluations, aimed at drawing a clarifying pattern on the molecular mechanisms involved in organotin toxicity. TBT was promptly incorporated in midgut gland mitochondria of adult mussels exposed to 0.5 and 1.0 μg/L TBT, and partially degraded to DBT. TBT incorporation was accompanied by a decrease in the mitochondrial oligomycin-sensitive Mg-ATPase activity, while the coexistent oligomycin-insensitive fraction was unaffected. Mitochondrial fatty acids showed a clear rise in n-3 polyunsaturated fatty acids after 120 hr of TBT exposure, mainly referable to an increase in 22:6 level. TBT was also shown to inhibit the ATP hydrolytic activity of the mitochondrial F1FO complex in vitro and to promote an apparent loss of oligomycin sensitivity at higher than 1.0 μM concentration. The complex dose-dependent profile of the inhibition curve lead to the hypothesis of multiple TBT binding sites. At lower than 1.0 μM TBT concentrations the non competitive enzyme inhibition by TBT was ascribed to the non covalent binding of TBT to FO subunit. On the other hand the observed drop in oligomycin sensitivity at higher than 1.0 μM TBT could be related to the onset of covalent bonds involving thiolic groups on the enzyme structure, apparently reached only at high TBT levels. The mitochondrial respiratory complexes were in vitro affected by TBT, apart from the cytocrome c oxidase which was apparently refractory to the contaminant. The most striking inhibitory effect was shown on complex I, and ascribed to possible covalent bonds of TBT with –SH groups on the enzyme complexes. This mechanism, shouldered by the progressive decrease of free cystein residues in the presence of increasing TBT concentrations, suggests that the onset of covalent tin-sulphur bonds in distinct protein structures may constitute the molecular basis of widespread TBT effects on mitochondrial complexes. Energy production disturbances, in turn affecting energy consuming mechanisms, could be involved in other cellular changes. Mussels exposed to a wide range of TBT concentrations (20 - 200 and 2000 ng/L respectively) did not show any change in testosterone and estrogen levels in mature gonads. Most hormones were in the non-biologically active esterified form both in control and in TBT-treated mussels. Probably the endocrine status of sexually mature mussels could be refractory even to high TBT doses. In mussel digestive gland the high biological variability of microsomal 7-benzyloxy-4-trifluoromethylcoumarin-O-Debenzyloxylase (BFCOD) activity, taken as a measure of CYP3A-like efficiency, probably concealed any enzyme response to TBT exposure. On the other hand the TBT-driven enhancement of BFCOD activity in vitro was once again ascribed to covalent binding to thiol groups which, in this case, would stimulate the enzyme activity. In mussels from Barcelona harbour, a highly contaminated site, the enzyme showed a decreased affinity for the 7-benzyloxy-4-trifluoromethylcoumarin (BCF) substrate with respect to mussel sampled from Ebro Delta, a non-polluted marine site. Contaminant exposure may thus alter the kinetic features of enzymes involved in detoxification mechanisms. Contaminants and steroid hormones were clearly shown to mutually interact in the modulation of detoxification mechanisms. The xenoestrogen 17α-ethylenyl estradiol (EE2) displayed a non-competitive mixed inhibition of CYP3A-like activity by a preferential bond to the free enzyme both in Barcelona harbour and Ebro Delta mussels. The possible interaction with co-present contaminants in Barcelona harbour mussels apparently lessened the formation of the ternary complex enzyme-EE2-BCF. The whole of data confirms TBT as membrane toxicant in mussels as in other species and stresses TBT covalent binding to protein thiols as a widespread mechanism of membrane-bound-enzyme activity modulation by the contaminant.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Relevanz der Methioninoxidation aus evolutionärer Sicht und im akuten oxidativen Stressmodell der Alzheimerschen Krankheit

Im Laufe der Evolution müssen Sauerstoff-metabolisierende Organismen eine Reihe von Anpassungen entwickelt haben, um in der zytotoxischen oxidativen Umgebung der sauerstoff-haltigen Erdatmosphäre überleben zu können. Die im Rahmen dieser Arbeit durchgeführten vergleichenden Analysen mitochondrial kodierter und kern-kodierter Proteome mehrerer hundert Spezies haben ergeben, dass die Evolution eines alternativen genetischen Codes in Mitochondrien eine moderne Adaptation in diesem Sinne war. Viele aerobe Tiere und Pilze dekodieren in Abweichung vom genetischen Standard-Code das Codon AUA als Methionin. In der vorliegenden Arbeit wird gezeigt, dass diese Spezies dadurch eine massive Akkumulation der sehr leicht oxidierbaren Aminosäure Methionin in ihren Atmungskettenkomplexen erreichen, die generell ein bevorzugtes Ziel reaktiver Sauerstoffspezies sind. Der gewonnene Befund lässt sich widerspruchsfrei nur unter Annahme einer antioxidativen Wirkung dieser Aminosäure erklären, wie sie erstmals 1996 von R. Levine anhand von Oxidationsmessungen in Modellproteinen postuliert worden war. In der vorliegenden Arbeit wird diese Hypothese nun direkt mittels neuartiger Modellsubstanzen in lebenden Zellen bestätigt. Die durchgeführten bioinformatischen Analysen und zellbiologischen Experimente belegen, dass kollektive Proteinveränderungen die Triebkraft für die Evolution abweichender genetischer Codes sein können.rnDie Bedeutung von oxidativem Stress wurde darüber hinaus auch im Referenzrahmen einer akuten oxidativen Schädigung im Einzelorganismus untersucht. Da oxidativer Stress in der Pathogenese altersassoziierter neurodegenerativer Erkrankungen wie der Alzheimerschen Krankheit prominent involviert zu sein scheint, wurden die Auswirkungungen von Umwelt-induziertem oxidativem Stress auf den histopathologischen Verlauf in einem transgenen Modell der Alzheimerschen Krankheit in vivo untersucht. Dabei wurden transgene Mäuse des Modells APP23 im Rahmen von Fütterungsversuchen einer lebenslangen Defizienz der Antioxidantien Selen oder Vitamin E ausgesetzt. Während die Selenoproteinexpression durch die selendefiziente Diät gewebespezifisch reduziert wurde, ergaben sich keine Anzeichen eines beschleunigten Auftretens pathologischer Marker wie amyloider Plaques oder Neurodegeneration. Es war vielmehr ein unerwarteter Trend hinsichtlich einer geringeren Plaquebelastung in Vitamin E-defizienten Alzheimermäusen zu erkennen. Auch wenn diese Daten aufgrund einer geringen Versuchstiergruppengröße nur mit Vorsicht interpretiert werden dürfen, so scheint doch ein Mangel an essentiellen antioxidativen Nährstoffen die Progression in einem anerkannten Alzheimermodell nicht negativ zu beeinflussen.rn

Evolutionäre Veränderungen in der Nutzung redox-aktiver Aminosäuren und ihre Ursachen

Gewebe, Zellen und speziell Zellkompartimente unterscheiden sich in ihrer Sauerstoffkonzentration, Stoffwechselrate und in der Konzentration an gebildeten reaktiven Sauerstoffspezies. Um eine mögliche Änderung in der Aminosäurennutzung durch den Einfluss von Sauerstoff und seinen reaktiven Spezies untersuchen zu können wurden, Bereiche bzw. Kompartimente der menschlichen Zelle definiert, die einen Referenzrahmen bildeten und bekannt dafür sind, einen relativ hohen Grad an reaktiven Sauerstoffspezies aufzuweisen. Aus dem Vergleich wurde deutlich, dass vor allem die beiden redox-aktiven und schwefeltragenden Aminosäuren Cystein und Methionin durch eine besondere Verteilung und Nutzung charakterisiert sind. Cystein ist hierbei diejenige Aminosäure mit den deutlichsten Änderungen in den fünf untersuchten Modellen der oxidativen Belastung. In all diesen Modellen war die Nutzung von Cystein deutlich reduziert, wohingegen Methionin in Proteinen des Mitochondriums und der Elektronentransportkette angereichert war. Dieser auf den ersten Blick paradoxe Unterschied zwischen Cystein und Methionin wurde näher untersucht, indem die differenzierte Methioninnutzung in verschiedenen Zellkompartimenten von Homo sapiens charakterisiert wurde.rnDie sehr leicht zu oxidierende Aminosäure Methionin zeigt ein ungewöhnliches Verteilungsmuster in ihrer Nutzungshäufigkeit. Entgegen mancher Erwartung wird Methionin in zellulären Bereichen hoher oxidativer Belastung und starker Radikalproduktion intensiv verwendet. Dieses Verteilungsmuster findet man sowohl im intrazellulären Vergleich, als auch im Vergleich verschiedener Spezies untereinander, was daraufhin deutet, dass es einen lokalen Bedarf an redox-aktiven Aminosäuren gibt, der einen sehr starken Effekt auf die Nutzungshäufigkeit von Methionin ausübt. Eine hohe Stoffwechselrate, die im Allgemeinen mit einer erhöhten Produktion von Oxidantien assoziiert wird, scheint ein maßgeblicher Faktor der Akkumulation von Methionin in Proteinen der Atmungskette zu sein. Die Notwendigkeit, oxidiertes Antioxidans wieder zu reduzieren, findet auch bei Methionin Anwendung, denn zu Methioninsulfoxid oxidiertes Methionin wird durch die Methioninsulfoxidreduktase wieder zu Methionin reduziert. Daher kann die spezifische Akkumulation von Methionin in Proteinen, die verstärkt reaktiven Sauerstoffspezies ausgesetzt sind, als eine systematische Strategie angesehen werden, um andere labile Strukturen vor ungewollter Oxidation zu schützen. rnDa Cystein in allen untersuchten Modellen der oxidativen Belastung und im Besonderen in Membranproteinen der inneren Mitochondrienmembran lebensspannenabhängig depletiert war, wurde dieses Merkmal näher untersucht. Deshalb wurde die Hypothese getestet, ob ein besonderer Redox-Mechanismus der Thiolfunktion für diese selektive Depletion einer im Allgemeinen als harmlos oder antioxidativ geltenden Aminosäure verantwortlich ist. Um den Effekt von Cysteinresten in Membranen nachzustellen, wurden primäre humane Lungenfibroblasten (IMR90) mit diversen Modellsubstanzen behandelt. Geringe Konzentrationen der lipophilen Substanz Dodecanthiol verursachten eine signifikante Toxizität in IMR90-Zellen, die von einer schnellen Zunahme an polyubiquitinierten Proteinen und anderen Indikatoren des proteotoxischen Stresses, wie Sequestosom 1 (P62), HSP70 und HSP90 begleitet wurde. Dieser Effekt konnte spezifisch der Chemie der Thiolfunktion in Membranen zugeordnet werden, da Dodecanol (DOH), Dodecylmethylsulfid (DMS), Butanthiol oder wasserlösliche Thiole weder eine cytotoxische Wirkung noch eine Polyubiquitinierung von Proteinen verursachten. Die Ergebnisse stimmen mit der Hypothese überein, dass Thiole innerhalb von biologischen Membranen als radikalische Kettentransferagentien wirken. Diese Eigenschaft wird in der Polymerchemie durch Nutzung von lipophilen Thiolen in hydrophoben Milieus technisch für die Produktion von Polymeren benutzt. Da die Thiylradikal-spezifische Reaktion von cis-Fettsäuren zu trans-Fettsäuren in 12SH behandelten Zellen verstärkt ablief, kann gefolgert werden, dass 12SH zellulär radikalisiert wurde. In lebenden Organismen kann demnach die Oxidation von Cystein die Schädigung von Membranen beschleunigen und damit Einfallstore für die laterale Radikalisierung von integralen Membranproteinen schaffen, welche möglicherweise der Langlebigkeit abträglich ist, zumindest, wenn sie in der inneren Mitochondrienmembran auftritt.

Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes

During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.