Mitochondrial bioenergetics and redox state are unaltered in Trypanosoma cruzi isolates with compromised mitochondrial complex I subunit genes

In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims: In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods: Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings: cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondria! membrane. Significance: It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients. (C) 2010 Elsevier Inc. All rights reserved.

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

Efeito hiperpolarizante do isoproterenol na membrana da célula de sertoli de ratos imaturos : envolvimento dos receptores beta2-adrenérgicos e dos canais K+ATP

No presente estudo, foi investigado o mecanismo pelo qual, o isoproterenol hiperpolariza o potencial de membrana (PM) da célula de Sertoli em túbulos seminíferos de ratos, com quinze dias de idade (imaturos). Foram analisadas a modificação do potencial de membrana e a resistência, utilizando-se a técnica de registro intracelular. O isoproterenol (2x10-6M) induziu uma hiperpolarização imediata e significativa na membrana da célula de Sertoli. O antagonista β2-adrenérgico butoxamina (1x10-6M) anulou a ação do isoproterenol. O antagonista β1-adrenérgico metoprolol (1x10-6M) teve efeito de reduzir a ação do isoproterenol, porém sem ser significativo. A inibição dos canais K+ATP, com a sulfoniluréia glibenclamida, suprimiu a ação do isoproterenol. A testosterona, a qual atua despolarizando o potencial de membrana, através do fechamento de canais K+ATP, via PLC-PIP2, impediu a hiperpolarização produzida pelo agonista β-adrenérgico. Os policátions como: espermina e LaCl3 (cloreto de lantâno) reverteram o efeito hiperpolarizante do isoproterenol, despolarizando o potencial de membrana, provavelmente através de interações iônicas que neutralizam a ação do agonista β-adrenérgico nos canais K+ATP. O agonista da adenilato ciclase, forscolina (1x10-7M), rapidamente hiperpolariza o potencial de membrana da célula de Sertoli, mimetizando o efeito do isoproterenol; db-AMPc também hiperpolariza o potencial de membrana destas células. Estes efeitos indicam que o isoproterenol age nos canais K+ATP, provavelmente, envolvendo a cascata: receptor β-adrenérgico/Gs/AC/AMPc/PKA. Estes resultados sugerem que a hiperpolarização induzida por isoproterenol é mediada pela abertura de canais K+ATP, em células de Sertoli. Esta hiperpolarização β-adrenérgica, provavelmente, tem uma papel fisiológico, na modulação do potencial de membrana, opondo-se à despolarização produzida pela testosterona, através do fechamento dos canais K+ATP.

Effectiveness of high-throughput miniaturized sorbent- and solid phase microextraction techniques combined with gas chromatography–mass spectrometry analysis for a rapid screening of volatile and semi-volatile composition of wines: a comparative study

In this study the feasibility of different extraction procedures was evaluated in order to test their potential for the extraction of the volatile (VOCs) and semi-volatile constituents (SVOCs) from wines. In this sense, and before they could be analysed by gas chromatography–quadrupole first stage masss spectrometry (GC–qMS), three different high-throughput miniaturized (ad)sorptive extraction techniques, based on solid phase extraction (SPE), microextraction by packed sorbents (MEPS) and solid phase microextraction (SPME), were studied for the first time together, for the extraction step. To achieve the most complete volatile and semi-volatile signature, distinct SPE (LiChrolut EN, Poropak Q, Styrene-Divinylbenzene and Amberlite XAD-2) and MEPS (C2, C8, C18, Silica and M1 (mixed C8-SCX)) sorbent materials, and different SPME fibre coatings (PA, PDMS, PEG, DVB/CAR/PDMS, PDMS/DVB, and CAR/PDMS), were tested and compared. All the extraction techniques were followed by GC–qMS analysis, which allowed the identification of up to 103 VOCs and SVOCs, distributed by distinct chemical families: higher alcohols, esters, fatty acids, carbonyl compounds and furan compounds. Mass spectra, standard compounds and retention index were used for identification purposes. SPE technique, using LiChrolut EN as sorbent (SPELiChrolut EN), was the most efficient method allowing for the identification of 78 VOCs and SVOCs, 63 and 19 more than MEPS and SPME techniques, respectively. In MEPS technique the best results in terms of number of extractable/identified compounds and total peak areas of volatile and semi-volatile fraction, were obtained by using C8 resin whereas DVB/CAR/PDMS was revealed the most efficient SPME coating to extract VOCs and SVOCs from Bual wine. Diethyl malate (18.8 ± 3.2%) was the main component found in wine SPELiChrolut EN extracts followed by ethyl succinate (13.5 ± 5.3%), 3-methyl-1-butanol (13.2 ± 1.7%), and 2-phenylethanol (11.2 ± 9.9%), while in SPMEDVB/CAR/PDMS technique 3-methyl-1-butanol (43.3 ± 0.6%) followed by diethyl succinate (18.9 ± 1.6%), and 2-furfural (10.4 ± 0.4%), are the major compounds. The major VOCs and SVOCs isolated by MEPSC8 were 3-methyl-1-butanol (26.8 ± 0.6%, from wine total volatile fraction), diethyl succinate (24.9 ± 0.8%), and diethyl malate (16.3 ± 0.9%). Regardless of the extraction technique, the highest extraction efficiency corresponds to esters and higher alcohols and the lowest to fatty acids. Despite some drawbacks associated with the SPE procedure such as the use of organic solvents, the time-consuming and tedious sampling procedure, it was observed that SPELiChrolut EN, revealed to be the most effective technique allowing the extraction of a higher number of compounds (78) rather than the other extraction techniques studied.

Estudo do efeito dos metabólitos do benzeno e tolueno sobre as mitocôndris cerebrais e hepáticas de ratos

Aim: The aim of this work was to investigate the hypothesis that catechol and 3MC inhibit FADH2-linked basal respiration in mitochondria isolated from rat liver and brain homogenates. Moreover, catechol ability to induce DNA damage in rat brain cells through the comet assay (alkaline single-cell gel electrophoresis assay) was also observed. Methods: Two different catechols were evaluated: pirocatechol (derived from benzene) and 3-methylcatechol (derived from toluene); rat liver and brain homogenates were incubated with 1mM catechol at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate for a period of 12 minutes. In additional experiments, rat brain cells were treated with 1, 5 and 10mM pirocatechol for up to 20 minutes at 37º C, and submitted to electrophoresis. Results: Catechols (pirocatechol and 3methylcatechol) induced a time-dependent partial inhibition of FADH2-linked basal mitochondrial respiration. Indeed, pirocatechol was able to produce a dosedependent DNA oxidative damage in rat brain cells by 2 and 4 injury levels. These results suggest that reactive oxygen species generated by the oxidation of catechols, induced an impairment on mitochondrial respiration and a DNA damage, which might be related to their citotoxicity. Conclusion: Catechols produced an inhibition of basal respiration associated to FADH2 in isolated liver and brain mitochondria; 3-methylcatechol, at the same concentration, produced similar toxicity in the mitochondrial model. Indeed, pirocatechol induced a DNA damage in rat brain cells, mainly observed in comets formation and consequent DNA degradation

Chloramphenicol restores the polymorphonuclear accumulation in carrageenin-induced pleurisy in diabetic rats

Investigou-se o efeito do succinato de cloranfenicol (30 mg/kg, a cada 12 h, durante 4 dias, IP) sobre o acúmulo de leucócitos polimorfonucleares (PMN) na pleurisia induzida pela carragenina (150 mig) em ratos (Wistar, machos, 180-230 g, n = 12) diabéticos (40 mg/kg de aloxana, IV). O antibiótico produziu aumento de 36% no número de PMN (p<0,05) migrados para a cavidade pleural de animais normais. O estado diabético provocou redução de 45% dos PMN (p<0,05) acumulados no exsudato pleural de animais não tratados com o antibiótico. Por outro lado, animais diabéticos tratados com succinato de cloranfenicol apresentaram resposta de PMN que não diferiu estatisticamente do observado em animais controle, não tratados. A contagem total e diferencial dos leucócitos circulantes realizada antes e 4 h depois da aplicação da carragenina não diferiu estatisticamente entre os grupos.

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: Uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca2+ at different conditions, a mitochondrial permeability transition (MPT) linked to protein,thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 mu M on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 mu M Ca2+; inhibition of these processes was assessed in non-energized organelles in the presence of 300 mu M t-butyl hydroperoxide plus 500 mu M Ca2+. Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca2+. Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin. (c) 2006 Published by Elsevier B.V..

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

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

The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q(2). Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q(6), the coq10 mutant has near normal amounts of Q(6) in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the aromatic-rich protein family Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q(6)/mol of protein. We propose that Coq10p is a Q(6)-binding protein and that in the coq10 mutant Q(6) it is not able to act as an electron carrier, possibly because of improper localization.

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Glycerol: A promising and abundant carbon source for industrial microbiology

Petroleum is the main energy source utilized in the world, but its availability is limited and the search for new renewable energy sources is of major interest. Biofuels, such as ethanol and biodiesel, are among the most promising sources for the substitution of fossil fuels. Biodiesel can replace petroleum diesel, as it is produced from animal fats and vegetable oils, which generate about 10% (w/w) glycerol as the main by-product. The excess glycerol generated may become an environmental problem. since it cannot be disposed of in the environment. One of the possible applications is its use as carbon and energy source for microbial growth in industrial microbiology. Glycerol bioconversion in valuable chemicals, such as 1,3-propanediol, dihydroxyacetone, ethanol, succinate etc. is discussed in this review article. (C) 2008 Elsevier B.V. All rights reserved.

Enhanced pressor response to carotid occlusion in commNTS-lesioned rats: possible efferent mechanisms

Bilateral common carotid occlusion (BCO) over a period of 60 s in conscious rats produces a biphasic presser response, consisting of an early (peak) and late (plateau) phase. In this study we investigated 1) the effects of lesions of the commissural nucleus of the solitary tract (commNTS) on the cardiovascular responses produced by BCO in conscious rats and 2) the autonomic and humoral mechanisms activated to produce the presser response to BCO in sham- and commNTS-lesioned rats. Both the peak and plateau of the presser response produced by BCO increased in commNTS-lesioned rats despite the impairment of chemoreflex responses induced by intravenous potassium cyanide. In sham rats sympathetic blockade with intravenous prazosin and metoprolol, but not vasopressin receptor blockade with the Manning compound, reduced both components of BCO. In commNTS-lesioned rats the sympathetic blockade or vasopressin receptor blockade reduced both components of BCO. The results showed 1) the sympathetic nervous system, but not vasopressin, is important for the presser response to BCO during 60 s in conscious sham rats; 2) in commNTS-lesioned rats, despite chemoreflex impairment, BCO produces an increased presser response dependent on sympathetic activity associated with vasopressin release; and 3) the increment in the presser response to BCO in commNTS-lesioned rats seems to depend only on vasopressin secretion.

Dehydromonocrotaline inhibits mitochondrial complex 1. A potential mechanism accounting for hepatotoxicity of monocrotaline

Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/malate-supported state 3 respiration (respiratory chain complex 1), but did not affect succinate-supported respiration (complex II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH oxidase activity (IC50 = 62.06 mu M) through a non-competitive type of inhibition (K-I = 8.1 mu M). Therefore, dehydromonocrotaline is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-documented monocrotaline's hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I conformation resulting from modification of cysteine thiol groups by the metabolite. (c) 2007 Elsevier Ltd. All rights reserved.