NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.     

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.     

Thioredoxin reductase-2 is essential for keeping low levels of H(2)O(2) emission from isolated heart mitochondria

Respiring mitochondria produce H(2)O(2) continuously. When production exceeds scavenging, H(2)O(2) emission occurs, endangering cell functions. The mitochondrial peroxidase peroxiredoxin-3 reduces H(2)O(2) to water using reducing equivalents from NADPH supplied by thioredoxin-2 (Trx2) and, ultimately, thioredoxin reductase-2 (TrxR2). Here, the contribution of this mitochondrial thioredoxin system to the control of H(2)O(2) emission was studied in isolated mitochondria and cardiomyocytes from mouse or guinea pig heart. Energization of mitochondria by the addition of glutamate/malate resulted in a 10-fold decrease in the ratio of oxidized to reduced Trx2. This shift in redox state was accompanied by an increase in NAD(P)H and was dependent on TrxR2 activity. Inhibition of TrxR2 in isolated mitochondria by auranofin resulted in increased H(2)O(2) emission, an effect that was seen under both forward and reverse electron transport. This effect was independent of changes in NAD(P)H or membrane potential. The effects of auranofin were reproduced in cardiomyocytes; superoxide and H(2)O(2) levels increased, but similarly, there was no effect on NAD(P)H or membrane potential. These data show that energization of mitochondria increases the antioxidant potential of the TrxR2/Trx2 system and that inhibition of TrxR2 results in increased H(2)O(2) emission through a mechanism that is independent of changes in other redox couples.     

"Mitochondrial" photochemical drugs do not release toxic amounts of 1O(2) within the mitochondrial matrix space

Previously, we demonstrated that mitochondrial NAD(P)H is the primary target of singlet oxygen (1O(2)) generated by photoactivation of mitochondria-selective rhodamine derivatives. Hence, local NAD(P)H oxidation/fluorescence decrease may be used to reveal the site of intracellular 1O(2) generation. Therefore, in addition to the previously used tetramethylrhodamine methylester (TMRM), 2('),4('),5('),7(')-tetrabromorhodamine 123 bromide (TBRB) and rhodamine 123 (Rho 123), we tested here whether mitochondrial NAD(P)H of cultured hepatocytes is directly oxidized upon irradiation of different "mitochondrial" photosensitizers (Photofrin; protoporphyrin IX; Al(III) phthalocyanine chloride tetrasulfonic acid; meso-tetra(4-sulfonatophenyl)porphine dihydrochloride; Visudyne). In contrast to TMRM and Rho 123, which directly oxidized NAD(P)H upon irradiation, irradiation of intracellular TBRB and the photochemical drugs only indirectly affected mitochondrial NAD(P)H due to loss of mitochondrial integrity. In line with this result only TMRM and Rho 123 exclusively localized within the mitochondrial matrix. Due to these results it is doubtful whether real mitochondrial photosensitizers actually exist among the photochemical drugs applicable/used for photodynamic therapy.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Increased phospholipid methylation in the myocardium of alcoholic rats

NAD(P)H is known to be oxidized by singlet molecular oxygen, perhydroxyl radical, and hydroxyl radical. In marked contrast to these reactive oxygen species, NAD(P)H is stable in the presence of micromolar concentrations of H2O2. The experiments herein demonstrate that NADPH is rapidly oxidized by H2O2 in the presence of a heme-peptide. The oxidation product is enzymatically active NADP+. In the absence of NADPH, the heme-peptide undergoes rapid degradation via reaction with H2O2. In the presence of NADPH, the reduced nucleotide is oxidized to NADP and the heme-peptide is partially protected from oxidation. It is suggested that under certain conditions the reduced nucleotides may contribute to the protection of intracellular heme moieties from degradation engendered by endogenous or exogenous H2O2.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.     

Phorbol diester-induced H2O2 production by peritoneal macrophages. Different H2O2 production by macrophages from normal and BCG-infected mice despite comparable phorbol diester receptors

Mouse peritoneal macrophages respond to environmental stimuli in different ways depending on their state of differentiation. Macrophages from mice with bacillus Calmette--Guerin (BCG) infection produced large amounts of H2O2 in response to phorbol diesters (PDEs), while those from noninfected mice produced little or no H2O2. The effects of PDEs on cells are mediated by specific cellular receptors for these ligands. The purpose of this study was to determine if the varying responses of macrophages from different groups of mice were caused by differences in their receptors for the PDE ligands. By all parameters studied, the binding of [20-3H]phorbol 12,13-dibutyrate ( [3H]PDBu) was similar in all macrophages irrespective of their ability to produce H2O2 in response to PDEs. Binding of [3H]PDBu was rapid at 23 degrees C reaching a maximum at 10-20 min with a subsequent decline to 50-60% of maximum by 30-60 min. Binding was slower at 0 degrees C reaching a maximum at 90-120 min. The binding was reversible, with dissociation kinetics paralleling association kinetics. The binding was saturable; the Kd's (45 to 91 nM) and number of binding sites (about 7-14 X 10(5)/cell or 11-12 pmol/mg protein) were essentially the same for the different classes of macrophages. The binding was specific, and analogs of PDBu inhibited [3H]PDBu binding to macrophages with potencies comparable to their potencies in causing in vivo tumor promotion and elicitation of other cellular responses in vitro. The ligands [3H]PDBu and [3H]PMA were degraded to comparable degrees by macrophages from normal or BCG-infected mice. Macrophages from C3H/HeJ and C3H/HeN mice, although known to differ in their abilities to respond to stimuli such as lymphokines and LPS, did not differ in their ability to produce H2O2 in response to PDEs or in their receptors for PDEs. Results of this study suggest that in vivo "activation" of macrophages in mice infected with BCG is not associated with a change in the cells' receptors for PDEs, but may be associated with "postreceptor" changes such as linkage of the PDE receptor with NAD(P)H oxidase, a change in NAD(P)H oxidase, or induction of synthesis of NAD(P)H oxidase.     

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H(2)O(2) induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H(2)O(2), and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.     

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.     

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.     

Role of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, as an electron transfer mediator for NAD(P)(+) regeneration in Bifidobacterium longum.

2-Amino-3-carboxy-1,4-naphthoquinone (ACNQ) is a novel growth stimulator for bifidobacteria. The role of ACNQ as a mediator of the electron transfer from NAD(P)H to dioxygen (O(2)) and hydrogen peroxide (H(2)O(2)), proposed in our previous paper, was examined using the cell-free extract and whole cells of Bifidobacterium longum. Continuous monitoring of ACNQ, O(2) and H(2)O(2) by several amperometric techniques has revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase and that the reduced form of ACNQ is easily autoxidized and also acts as a better electron donor of NAD(P)H peroxidase than NAD(P)H. The generation of H(2)O(2) by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes. The accumulation of ACNQ in the cytosol has been also suggested. These characteristics of ACNQ seem to be responsible for the growth stimulation of bifidobacteria. Vitamin K(3), which has an extremely low growth-stimulating activity and was used as a reference compound, exhibits much lower activity as an electron transfer mediator. The difference in the activity is discussed in terms of the redox potential and partition property of the quinones.     

Interaction of myeloperoxidase with vascular NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular diseases

Vascular NAD(P)H oxidase-derived reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have emerged as important molecules in the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complications. Additionally, myeloperoxidase (MPO), a transcytosable heme protein that is derived from leukocytes, is also believed to play important roles in the above-mentioned inflammatory vascular diseases. Previous studies have shown that MPO-induced vascular injury responses are H2O2 dependent. It is well known that MPO can use leukocyte-derived H2O2; however, it is unknown whether the vascular-bound MPO can use vascular nonleukocyte oxidase-derived H2O2 to induce vascular injury. In the present study, ANG II was used to stimulate vascular NAD(P)H oxidases and increase their H2O2 production in the vascular wall, and vascular dysfunction was used as the vascular injury parameter. We demonstrated that vascular-bound MPO has sustained activity in the vasculature. MPO could use the vascular NAD(P)H oxidase-derived H2O2 to produce hypochlorus acid (HOCl) and its chlorinating species. More importantly, MPO derived HOCl and chlorinating species amplified the H2O2-induced vascular injury by additional impairment of endothelium-dependent relaxation. HOCl-modified low-density lipoprotein protein (LDL), a specific biomarker for the MPO-HOCl-chlorinating species pathway, was expressed in LDL and MPO-bound vessels with vascular NAD(P)H oxidase-derived H2O2. MPO-vascular NAD(P)H oxidase-HOCl-chlorinating species may represent a common pathogenic pathway in vascular diseases and a new mechanism involved in exacerbation of vascular diseases under inflammatory conditions.     

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.