What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4–State 3 transition) decreases the succinate- and glutamate plus malate-supported H₂O₂ production 8- and 1.3-times, respectively. NH₄⁺ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H₂O₂ production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H₂O₂ diffusion by alamethicin treatment are only partially (～ 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (～ 6-fold) stimulated by ammonium chloride. NAD⁺ inhibits both Complex I-mediated and ammonium-stimulated H₂O₂ production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

The anticancer agent doxorubicin disrupts mitochondrial energy metabolism and redox balance in skeletal muscle

The combined loss of muscle strength and constant fatigue are disabling symptoms for cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and premature fatigue along with an increase in reactive oxygen species (ROS). As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin could negatively affect mitochondria by inhibiting respiratory capacity, leading to an increase in H₂O₂-emitting potential. Here we demonstrate a biphasic response of skeletal muscle mitochondria to a single doxorubicin injection (20 mg/kg). Initially at 2 h doxorubicin inhibits both complex I- and II-supported respiration and increases H₂O₂ emission, both of which are partially restored after 24 h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24 h, indicating elevated reducing pressure within the electron transport system (ETS). Respiratory capacity is further decreased at a later time point (72 h) along with H₂O₂-emitting potential and an increased sensitivity to mitochondrial permeability transition pore (mPTP) opening. These novel findings suggest a role for skeletal muscle mitochondria as a potential underlying cause of doxorubicin-induced muscle dysfunction.     

Glutamate induces H2O2 synthesis in nonsynaptic brain mitochondria

Mitochondrial reactive oxygen species regulate many important biological processes. We studied H₂O₂ formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H₂O₂ generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca²⁺ (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5 mM). We have shown that the H₂O₂ formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H₂O₂ formation through mitochondrial respiratory chain complex II. The H₂O₂ formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria.     

Oxidative stress caused by pyocyanin impairs CFTR Cl− transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H₂O₂ threefold above the endogenous H₂O₂ production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 μM) oxidized the cytosol from a resting value of − 318 ± 5 mV by 48.0 ± 4.6 mV within 2 h; a comparable oxidation was induced by 100 μM H₂O₂. Whereas resting Cl⁻ secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl⁻ secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for ΔF508 CFTR failed to secrete Cl⁻ in response to pyocyanin or H₂O₂, indicating that these oxidants specifically target the CFTR and not other Cl⁻ conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H₂O₂, depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Effects of temperature on complexes I and II mediated respiration,ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata

Effects of fluctuations in habitat temperature (18–30°) on mitochondrial respiratory behavior and oxidative metabolic responses in the euryhaline ectotherm Scylla serrata are not fully understood. In the present study, effects of different temperatures ranging from 12 to 40 °C on glutamate and succinate mediated mitochondrial respiration, respiratory control ratio (RCR), ATP generation rate, ratio for the utilization of phosphate molecules per atomic oxygen consumption (P/O), levels of lipid peroxidation and H₂O₂ in isolated gill mitochondria of S. serrata are reported. The pattern of variation in the studied parameters was similar for the two substrates at different temperatures. The values recorded for RCR (≥3) and P/O ratio (1.4–2.7) at the temperature range of 15–25 °C were within the normal range reported for other animals (3–10 for RCR and 1.5–3 for P/O). Values for P/O ratio, ATP generation rate and RCR were highest at 18 °C when compared to the other assay temperatures. However, at low and high extreme temperatures, i.e. at 12 and 40 °C, states III and IV respiration rates were not clearly distinguishable from each other indicating that mitochondria were completely uncoupled. Positive correlations were noticed between temperature and the levels of both lipid peroxidation and H₂O₂. It is inferred that fluctuations on either side of ambient habitat temperature may adversely influence mitochondrial respiration and oxidative metabolism in S. serrata. The results provide baseline data to understand the impacts of acute changes in temperature on ectotherms inhabiting estuarine or marine environments.     

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b₅₆₂ by 71%, in an [O₂] independent manner. Hyperbolic increases in O₂^•- (up to 1.3±0.1 nmol/min. mg protein) and H₂O₂ (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O₂^•-/H₂O₂ ratio was 1.98 in accordance with the stoichiometry of the O₂^•- disproportionation. Moreover, H₂O₂ production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N₂ atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH^•]_ss which, in turn, leads to an increase in O₂^•- and H₂O₂ mitochondrial production rates.     

Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity

Effects of exogenous nitric oxide (NO) on starch degradation, oxidation in mitochondria and K⁺/Na⁺ accumulation during seed germination of wheat were investigated under a high salinity level. Seeds of winter wheat (Triticum aestivum L., cv. Huaimai 17) were pre-soaked with 0 mM or 0.1 mM of sodium nitroprusside (SNP, as nitric oxide donor) for 20 h just before germination under 300 mM NaCl. At 300 mM NaCl, exogenous NO increased germination rate and weights of coleoptile and radicle, but decreased seed weight. Exogenous NO also enhanced seed respiration rate and ATP synthesis. In addition, seed starch content decreased while soluble sugar content increased by exogenous NO pre-treatment, which was in accordance with the improved amylase activities in the germinating seeds. Exogenous NO increased the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6); whereas decreased the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and superoxide anions (O₂^??) release rate in the mitochondria. Exogenous NO also decreased Na⁺ concentration while increased K⁺ concentration in the seeds thereby maintained a balance between K⁺ and Na⁺ during germination under salt stress. It is concluded that exogenous NO treatment on wheat seeds may be a good option to improve seed germination and crop establishment under saline conditions.     

α7 nicotinic acetylcholine receptors control cytochrome c release from isolated mitochondria through kinase-mediated pathways

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of both excitable and non-excitable cells. Previously we reported that nicotinic receptors containing α7 subunits were present in the outer membranes of mitochondria to regulate the early apoptotic events like cytochrome c release. Here we show that signaling of mitochondrial α7 nicotinic receptors affects intramitochondrial protein kinases. Agonist of α7 nicotinic receptors PNU 282987 (30 nM) prevented the effect of phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in isolated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either 90 μM Ca²⁺ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90 μM Ca²⁺) or of Src kinase(s) (upon 0.5 mM H₂O₂) and of protein kinase C. Cytochrome c release from mitochondria could be also attenuated by α7 nicotinic receptor antagonist methyllicaconitine or α7-specific antibodies. Allosteric modulator PNU 120526 (1 μM) did not improve the effect of agonist PNU 282987. Acetylcholine (1 μM) and methyllicaconitine (10 nM) inhibited superoxide release from mitochondria measured according to alkalization of Ca²⁺-containing medium. It is concluded that α7 nicotinic receptors regulate mitochondrial permeability transition pore formation through ion-independent mechanism involving activation of intramitochondrial PI₃K/Akt pathway and inhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2 and paraquat

This paper aims to investigate the effect of H₂O₂ and paraquat on the activities of superoxide dismutase (SOD) and catalase (CAT), and membrane lipid peroxidation (LPO) levels in newly isolated Streptomyces sp. M3004. SOD activities of Streptomyces sp. M3004, grown in 10 mM and 30 mM H₂O₂, were significantly lower than the control cultures. On the other hand, as an antioxidant enzyme, CAT activity in both H₂O₂ treatment conditions increased significantly compared with the control. These activity values in 10 mM and 30 mM H₂O₂ treatment on the 48th hour of incubation were 3.8- and 6.6-fold higher than the control, respectively. SOD activity decreased significantly with respect to paraquat concentration, which was added at the start of the incubation. CAT activities increased significantly in 1.0 mM and 3.0 mM paraquat treatments compared to control. As an indicative marker of membrane damage, LPO levels of the novel isolate Streptomyces sp. M3004 treated with H₂O₂, and paraquat stress conditions were significantly higher than the control. Nevertheless, compared with the 30 mM H₂O₂ in both treatment conditions, LPO levels in 10 mM H₂O₂ were significantly higher. The decreases in SOD activities in paraquat and H₂O₂ treatment conditions resulted in the increases in the LPO levels although it increases in CAT activities.     

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H₂O₂). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O₂⁻/H₂O₂ formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O₂⁻/H₂O₂ formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H₂O₂. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O₂⁻/H₂O₂ production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O₂⁻/H₂O₂ formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O₂⁻/H₂O₂ production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H₂O₂.     

Diabetes impairs heart mitochondrial function without changes in resting cardiac performance

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28 days of Streptozotocin injection (STZ, 60 mg kg⁻¹, ip.). Heart O₂ consumption was declined, mainly due to the impairment of mitochondrial O₂ uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22–26%). An enhancement in mitochondrial H₂O₂ (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a β‐adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O₂ consumption and oxidative phosphorylation efficiency are decreased, and H₂O₂ and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H₂O₂, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.     

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

H₂O₂ production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H₂O₂ production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H₂O₂ production was dominated by complex II (44% from site II_F in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site I_F). With added carnitine, H₂O₂ production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site III_Qo) and 25% from site I_F. Thus complex II (site II_F in the forward reaction) is a major source of H₂O₂ production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H₂O₂ production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site II_F in the reverse reaction). About half the rest was from site I_F, but a significant portion, ∼40 pmol H₂O₂·min⁻¹·mg protein⁻¹, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200 pmol H₂O₂·min⁻¹·mg protein⁻¹ under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H₂O₂ productionduring fatty acid oxidation under appropriate conditions.     

Comparative diel oxygen cycles preceding and during a Karenia bloom in Sarasota Bay,Florida, USA

The diel change in dissolved oxygen concentrations were recorded with an automated incubator containing a pulsed oxygen sensor in Sarasota Bay, Florida. The deployments occurred during a ‘pre-bloom’ period in May to June 2006, and during a harmful algal bloom dominated by Karenia brevis in September 2006. The diurnal (daylight) increase in dissolved oxygen concentrations varied from 16 to 104 μmol O₂ l⁻¹ with the corresponding nocturnal decrease in oxygen varying from 16 to 77 μmol O₂ l⁻¹. Nocturnal respiration consumed 42–113% of the diurnal net oxygen production with the minimum and maximum during the pre-bloom period. Hourly production rates closely followed fluctuations in irradiance with maximum rates in the late morning. Hourly oxygen utilization rates (community respiration) at night were highest during the first few hours after sunset.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.