HCV infection induces mitochondrial bioenergetic unbalance: causes and effects.

Cells infected by the hepatitis C virus (HCV) are characterized by endoplasmic reticulum stress, deregulation of the calcium homeostasis and unbalance of the oxido-reduction state. In this context, mitochondrial dysfunction proved to be involved and is thought to contribute to the outcome of the HCV-related disease. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This causes successive mitochondrial alterations comprising generation of reactive oxygen and nitrogen species and impairment of the oxidative phosphorylation. A progressive adaptive response results in an enhancement of the glycolytic metabolism sustained by up-regulation of the hypoxia inducible factor. Pathogenetic implications of the model are discussed.

Coexistence of mutations in PINK1 and mitochondrial DNA in early onset parkinsonism.

AIMS AND BACKGROUND: Various genes have been identified for monogenic disorders resembling Parkinson's disease. The products of some of these genes are associated with mitochondria and have been implicated in cellular protection against oxidative damage. In the present study we analysed fibroblasts from a patient carrying the homozygous mutation p.W437X in the PTEN-induced kinase 1 (PINK1), which manifested a very early onset parkinsonism. RESULTS: Patient's fibroblasts did not show variation in the mtDNA copy number or in the expression of the oxidative phosphorylation complexes. Sequence analysis of the patient's mtDNA presented two new missense mutations in the ND5 (m.12397A>G, p.T21A) and ND6 (m. 14319T>C, p.N119D) genes coding for two subunits of complex I. The two mutations were homoplasmic in both the patient and the patient's mother. Patient's fibroblasts resulted in enhanced constitutive production of the superoxide anion radical that was abrogated by inhibitor of the complex I. Moreover enzyme kinetic analysis of the NADH:ubiquinone oxidoreductase showed changes in the substrates affinity. CONCLUSION: To our knowledge, this is the first report showing co-segregation of a Parkinson's disease related nuclear gene mutation with mtDNA mutation(s). Our observation might shed light on the clinical heterogeneity of the hereditary cases of Parkinson's disease, highlighting the hitherto unappreciated impact of coexisting mtDNA mutations in determining the development and the clinical course of the disease.

Uncoupling protein-2 (UCP2) induces mitochondrial proton leak and increases susceptibility of non-alcoholic steatohepatitis (NASH) liver to ischaemia-reperfusion injury.

BACKGROUND: The mechanisms of progression from fatty liver to steatohepatitis and cirrhosis are not well elucidated. Mitochondrial dysfunction represents a key factor in the progression of non-alcoholic steatohepatitis (NASH) as mitochondria are the main cellular site of fatty acid oxidation, ATP synthesis and reactive oxygen species (ROS) production. AIMS: (1) To evaluate the role of the uncoupling protein 2 in controlling mitochondrial proton leak and ROS production in NASH rats and humans; and (2) to assess the acute liver damage induced by ischaemia-reperfusion in rats with NASH. METHODS: Mitochondria were extracted from the livers of NASH humans and rats fed a methionine and choline deficient diet. Proton leak, H(2)O(2) synthesis, reduced glutathione/oxidised glutathione, 4-hydroxy-2-nonenal (HNE)-protein adducts, uncoupling protein-2 (UCP2) expression and ATP homeostasis were evaluated before and after ischaemia-reperfusion injury. RESULTS: NASH mitochondria exhibited an increased rate of proton leak due to upregulation of UCP2. These results correlated with increased production of mitochondrial hydrogen peroxide and HNE-protein adducts, and decreased hepatic ATP content that was not dependent on mitochondrial ATPase dysfunction. The application of an ischaemia-reperfusion protocol to these livers strongly depleted hepatic ATP stores, significantly increased mitochondrial ROS production and impaired ATPase activity. Livers from patients with NASH exhibited UCP2 over-expression and mitochondrial oxidative stress. CONCLUSIONS: Upregulation of UCP2 in human and rat NASH liver induces mitochondrial uncoupling, lowers the redox pressure on the mitochondrial respiratory chain and acts as a protective mechanism against damage progression but compromises the liver capacity to respond to additional acute energy demands, such as ischaemia-reperfusion. These findings suggest that UCP2-dependent mitochondria uncoupling is an important factor underlying events leading to NASH and cirrhosis.

Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model.

BACKGROUND: Mitochondrial dysfunction is considered a key player in non-alcoholic steatohepatitis (NASH) but no data are available on the mitochondrial function and ATP homeostasis in the liver during NASH progression. In the present paper we evaluated the hepatic mitochondrial respiratory chain activity and ATP synthesis in a rodent model of NASH development. MATERIALS AND METHODS: Male Wistar rats fed a High Fat/Methionine-Choline Deficient (MCD) diet to induce NASH or a control diet (SHAM), and sacrificed after 3, 7 and 11 weeks. The oxidative phosphorylation, the F(0)F(1)ATPase (ATP synthase) and the ATP content were assessed in liver mitochondria. RESULTS: NASH mitochondria exhibited an increased rate of substrate oxidation at 3 weeks, which returned to below the normal level at 7 and 11 weeks, concomitantly with the coupling between the phosphorylation activity and the mitochondrial respiration (ADP/O). Uncoupling of NASH liver mitochondria did not allow the recovery of the maximal respiration rate at 7 and 11 weeks. The ATPase (ATP synthase) activity was similar in NASH and SHAM rats, but the mitochondrial ATP content was significantly lower in NASH livers. CONCLUSIONS: The loss of hepatic ATP stores is not dependent on the F(0)F(1)-ATPase but resides in the respiratory chain. Dysfunction of both Complex I and II of the mitochondrial respiratory chain during NASH development implies a mitochondrial adaptive mechanism occurring in the early stages of NASH.

Topological organization of NADPH-oxidase in haematopoietic stem cell membrane: preliminary study by fluorescence near-field optical microscopy.

The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O(2) sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane 'rafts'-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms.

Exposure to 1.8 GHz electromagnetic fields affects morphology, DNA-related Raman spectra and mitochondrial functions in human lympho-monocytes.

Blood is a fluid connective tissue of human body, where it plays vital functions for the nutrition, defense and well-being of the organism. When circulating in peripheral districts, it is exposed to some physical stresses coming from outside the human body, as electromagnetic fields (EMFs) which can cross the skin. Such fields may interact with biomolecules possibly inducing non thermal-mediated biological effects at the cellular level. In this study, the occurrence of biochemical/biological modifications in human peripheral blood lympho-monocytes exposed in a reverberation chamber for times ranging from 1 to 20 h to EMFs at 1.8 GHz frequency and 200 V/m electric field strength was investigated. Morphological analysis of adherent cells unveiled, in some of these, appearance of an enlarged and deformed shape after EMFs exposure. Raman spectra of the nuclear compartment of cells exposed to EMFs revealed the onset of biochemical modifications, mainly consisting in the reduction of the DNA backbone-linked vibrational modes. Respirometric measurements of mitochondrial activity in intact lympho-monocytes resulted in increase of the resting oxygen consumption rate after 20 h of exposure, which was coupled to a significant increase of the FoF1-ATP synthase-related oxygen consumption. Notably, at lower time-intervals of EMFs exposure (i.e. 5 and 12 h) a large increase of the proton leak-related respiration was observed which, however, recovered at control levels after 20 h exposure. Confocal microscopy analysis of the mitochondrial membrane potential supported the respiratory activities whereas no significant variations in the mitochondrial mass/morphology was observed in EMFs-exposed lympho-monocytes. Finally, altered redox homeostasis was shown in EMFs-exposed lympho-monocytes, which progressed differently in nucleated cellular subsets. This results suggest the occurrence of adaptive mechanisms put in action, likely via redox signaling, to compensate for early impairments of the oxidative phosphorylation system caused by exposure to EMFs. Overall the data presented warn for health safety of people involved in long-term exposure to electromagnetic fields, although further studies are required to pinpoint the leukocyte cellular subset(s) selectively targeted by the EMFs action and the mechanisms by which it is achieved.

Correction: Exposure to 1.8 GHz electromagnetic fields affects morphology, DNA-related Raman spectra and mitochondrial functions in human lympho-monocytes.

[This corrects the article DOI: 10.1371/journal.pone.0192894.].

Mitochondrial dysfunction in hepatitis C virus infection.

The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtDeltaPsi, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.

[Promoter effect induced by HgCl2 by studying the intercellular communication]. / Effetto promoter indoitto da HgCl2 attraverso lo studio della comunicazione intercellulare.

This work aims at assessing at molecular level the effect caused by the HgCl9 intercellular communication inhibition at non-cytotoxic doses. On the basis of our previous experiences, we exposed the human keratinocytes (HUKE) at 10 nM of HgCl2 for 24 hours Next, we estimated: a) the protein expression of connexines Cx43, Cx32 and Cx26 by western blotting; b) the amount of mRNA corresponding to the three connexines by semi-quantitative RT-PCR; and c) the production of reactive oxygen species in HgCl2 treated cells using a specific probe, i.e. DCF in confocal microscopy. Our study demonstrated a higher expression of the transcripts for Cx26, Cx32, Cx43, and a higher amount of proteins Cx43, Cx32 and Cx26, compared to the negative controls. Furthermore, we studied the effect of HgCl2 on the ROS production in keratinocytes, by the analysis in confocal microscopy carried out with the DCF, fit for marking the oxygen free radicals. In HgCl2 treated keratinocytes we obtained an increase of the ROS production compared to controls; and further the mitochondrions resulted the place of ROS production. The results of this study suggest that non-cytotoxic HgCl2 concentrations, might cause an unbalancing of the redox cellular state (ROS increased level), and we can assume that the activation of a redox signalling involves the inactivation of gap junctions.

Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells.

A study is presented of the effect of the cAMP cascade on oxygen metabolism in mammalian cell cultures. Serum-starvation of the cell cultures resulted in depression of the forward NADH-ubiquinone oxidoreductase activity of complex I, decreased content of glutathione, and enhancement of the cellular level of H2O2. Depressed transcription of cytosolic Cu/Zn-SOD 1, mitochondrial glutathione peroxidase and catalase was also observed. Activation of the cAMP cascade reversed the depression of the activity of complex I and the accumulation of H2O2. The effect of cAMP involved the cAMP-dependent protein kinase.

Proton-cation translocation in tumor cell mitochondria.

The capacity of mitochondria isolated from tumor cells to conserve the transmembrane electrochemical proton gradient set up by respiration has been studied. In a K+ medium, mitochondria from Ehrlich ascites tumor cells exhibit a capacity to conserve aerobic delta microH comparable to that displayed by normal rat liver mitochondria. Mitochondria from Morris hepatoma 3924A show a decreased capacity to store delta microH+, which is principally due to lowering of delta pH. In a Na+ medium, both species of tumor mitochondria show a significant decrease of aerobic delta pH, while delta psi is the same, with respect to rat liver mitochondria. Experiments on passive swelling show that mitochondria from ascites tumor cells have an enhanced permeability to chloride salts of monovalent cations and increased activity of the Na+ (K+)-H+ exchange system of the mitochondrial membrane with respect to normal mitochondria. The enhanced activity of this system in ascites cells is also shown by the characteristics of respiration-linked proton translocation in submitochondrial particles and subsequent anaerobic proton diffusion. It is concluded that the decreased capacity of mitochondria from tumor cells to conserve aerobic delta pH is due to enhanced cyclic flow of Na+ across the membrane.

Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement.

Using the high efficiency of homologous gene recombination in Bacillus subtilis, a strategy for mutational analysis of the proton pumping aa3-600 quinol oxidase of this organism has been developed. The qox operon with the qoxA, qoxB, qoxC and qoxD genes, coding for the four subunits of this oxidase, was deleted and then replaced with mutated copies in which qoxC (subunit III) or qoxD (subunit IV) genes were deleted. The complete deletion of the qox operon caused disappearance of heme aa3-600 and a slight depression of the overall respiratory activity, compensated by alternative oxidase with no proton pumping activity. Deletion of qoxC probably resulted in a defective assembly of the aa3-600 quinol oxidase. The strain with deletion of qoxD gene expressed normal content of heme aa3-600 but exhibited a reduced respiratory activity and a significantly depressed proton pumping activity. These results show that subunit IV is critical for the activity of the proton pumping aa3-600 quinol oxidase.

Redox-linked protolytic reactions in soluble cytochrome-c oxidase from beef-heart mitochondria: redox Bohr effects.

A study is presented of co-operative redox-linked protolytic reactions (redox Bohr effects) in soluble cytochrome-c oxidase purified from bovine-heart mitochondria. Bohr effects were analyzed by direct measurement, with accurate spectrophotometric and potentiometric methods, of H+ uptake and release by the oxidase associated with reduction and oxidation of hemes a and a3. CuA and CuB in the unliganded and in the CN- or CO-liganded enzyme. The results show that there are in the bovine oxidase four protolytic groups undergoing reversible pK shifts upon oxido-reduction of the electron transfer metals. Two groups with pKox and pKred values around 7 and > 12 respectively appear to be linked to redox transitions of heme a3. One group with pKox and pKred around 6 and 7 is apparently linked to CuB, a fourth one with pKox and pKred of 6 and 9 appears to be linked to heme a. The possible nature of the amino acids involved in the redox Bohr effects and their role in H+ translocation is discussed.

Ferrocyanide-peroxidase activity of cytochrome c oxidase

Redox interaction of mitochondrial cytochrome c oxidase (COX) with ferrocyanide/ferricyanide couple is greatly accelerated by polycations, such as poly-l-lysine [Musatov et al. (1991) Biological Membranes 8, 229-234]. This has allowed us to study ferrocyanide oxidation by COX at very high redox potentials of the ferrocyanide/ferricyanide couple either following spectrophotometrically ferricyanide accumulation or measuring proton uptake associated with water formation in the reaction. At low [ferrocyanide]/[ferricyanide] ratios (Eh values around 500 mV) and ambient oxygen concentration, the ferrocyanide-oxidase activity of COX becomes negligibly small as compared to the reaction rate observed with pure ferrocyanide. Oxidation of ferrocyanide under these conditions, is greatly stimulated by H2O2 or ethylhydroperoxide indicating peroxidatic reaction involved. The ferrocyanide-peroxidase activity of COX is strictly polylysine-dependent and is inhibited by heme a3 ligands such as KCN and NaN3. Apparently the reaction involves normal electron pathway, i.e. electron donation through CuA and oxidation via heme a3. The peroxidase reaction shows a pH-dependence similar to that of the cytochrome c oxidase activity of COX. When COX is preequilibrated with excess H2O2, addition of ferrocyanide shifts the initial steady-state concentrations of the Ferryl-Oxo and Peroxy compounds towards approximately 2:1 ratio of the two intermediates. It is suggested that in the peroxidase cycleferrocyanide donates electrons to both P and F intermediates with a comparable efficiency. Isolation of a partial redox activity of COX opens a possibility to study separately proton translocation coupled to the peroxidase half-reaction of the COX reaction cycle. Copyright 1998

The study of gap junctional intercellular communication in keratinocytes as screening of promoter effect induced by industrial and environmental toxic substances.

BACKGROUND: Disordered functioning of gap junctions between normal and initiated cells has been proposed as one possible mechanism of tumour promotion. Many putative carcinogens such as peroxisome proliferators, are known to activate various signal transduction mechanisms and modulate gap junctional intercellular communication (GJIC). They act as tumour promoters on pre-existing "initiated" cells, rather than as genotoxic initiators. OBJECTIVES: The aim of this article is to provide a screening-tool to evaluate the promoter carcinogen effect of environmental and occupational chemical contaminants, focusing on their ability to alter GJIC. METHODS: GJIC was investigated in serum-free cultured primary human keratinocytes, by directly evaluating the intercellular transfer of a microinjected fluorescent dye (Dye transfer). The expression of caspase 3, which is the ultimate target to be activated of both mitochondrial- and non-mitochondrial-linked pro-apoptotic pathways, was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Mercury chloride (10 nM), mono-methyl Mercury (250 nM) and Trichloroethylene (500 I1M) were shown to significantly inhibit GJIC. Conversely di-methyl mercury, lead acetate and epichloridine had no effect on GJIC. All Trans Retinoic Acid completely reversed the inhibitory effect on GJIC induced by HgCI2 but not that induced by mono-methyl mercury and trichloroethylene. The result of a RT-PCR assay on total RNA cell extract showed that treatment of keratinocytes with 10 nM HgCl2 resulted in a decrease of the pro-apoptotic caspase 3 expression. CONCLUSIONS: In this work a protocol is designed to study gap junction intercellular communication in primary cultures of human keratinocytes which could be used as a reliable screening tool to test the promoter carcinogen effect of various environmental and occupational contaminants.

A cooperative model for protonmotive heme-copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase.

Oxido-reductions of metal centers in cytochrome c oxidase are linked to pK shifts of acidic groups in the enzyme (redox Bohr effects). The linkage at heme a results in proton uptake from the inner space upon reduction and proton release in the external space upon oxidation of the metal. The relationship of this process to the features of the proton pump in cytochrome c oxidase and its atomic structure revealed by X-ray crystallography to 2.8-2.3 A resolution is examined. A mechanism for the proton pump of cytochrome c oxidase, based on cooperative coupling at heme a, is proposed.

Influence of surface charge on the incorporation and orientation of cytochrome c oxidase in liposomes.

Cytochrome c-oxidase is usually oriented 80-90% right-side-out when reconstituted with asolectin by the cholate dialysis method. Transformation of positively charged lysine groups at the matrix domain into negatively charged groups with succinic anhydride results in random orientation. A random orientation is also found after reconstitution in phosphatidylcholine, which can be changed into predominant right-side-out orientation by addition of cardiolipin. It is concluded that electrostatic interaction between positively charged groups of cytochrome c-oxidase with negative groups of phospholipids determines the asymmetric orientation of the enzyme in liposomes.

pH changes associated with cytochrome c oxidase reaction with H2O2. Protonation state of the peroxy and oxoferryl intermediates.

pH changes associated with the mitochondrial cytochrome oxidase reaction with H2O2 have been studied. In the presence of ferricyanide or Tris-phenanthroline complex of CoIII as electron acceptors, reaction of H2O2 with the oxidized cytochrome oxidase is accompanied by a steady proton release with a rate constant of ca. 3 M-1.s-1 at pH 6.8. The acidification is completely inhibited by superoxide dismutase and its pre-steady-state kinetics correlates with that of the oxoferryl compound (F) accumulation. Apparently, the proton release is linked to superoxide generation by cytochrome oxidase under these conditions. In the presence of superoxide dismutase and without the electron acceptors, the H2O2-induced transitions of cytochrome oxidase from the oxidized to the peroxy (P) and from the peroxy to the oxoferryl state are not associated with any significant proton release or uptake. The results point to the following mechanism of O2- generation and protonation states of the cytochrome oxidase compounds P and F: [formula: see text]

Proton pumping by cytochrome c oxidase is coupled to peroxidase half of its catalytic cycle.

The four-electron reaction cycle of cytochrome oxidase is comprised of an eu-oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a peroxidase phase in which the peroxy state formed in the eu-oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl-oxo state and oxidized form, respectively. Here we show that the ferrocyanide-peroxidase activity of cytochrome c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H+/e- ratio for the ferrocyanide-peroxidase partial reaction is twice higher than for the overall ferrocyanide-oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the eu-oxidase part (transfer of the first two electrons) may not be proton pumping.

Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase.

The vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase.