In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning
ABSTRACT: Graphical abstract Highlights • Cyanide is a cytochrome c oxidase inhibitor that results in bioenergetic failure.• There is a cell-permeable succinate prodrug, NV118, that releases succinate.• Our mitochondrial-directed therapy (NV118) but not B12a improved cellular function.• There was attenuation of ROS with NV118 but not with B12a treatment. The objective of this study was to compare the use of hydroxocobalamin (B12a) and a succinate prodrug to evaluate for improvement in mitochondrial function in an in vitro model of cyanide poisoning. Peripheral blood mononuclear cells (PBMC) and human aortic smooth muscle cells (HASMC) incubated with 50?mM of sodium cyanide (CN) for five minutes serving as the CN group compared to controls. We investigated the following: (1) Mitochondrial respiration; (2) Superoxide and mitochondrial membrane potential with microscopy; (3) Citrate synthase protein expression. All experiments were performed with a cell concentration of 2?3?×?106 cells/ml for both PBMC and HASMC. There were four conditions: (1) Control (no exposure); (2) Cyanide (exposure only); (3) B12a (cyanide exposure followed by B12a treatment); (4) NV118 (cyanide followed by NV118 treatment). In this study the key findings include: (1) Improvement in key mitochondrial respiratory states with the succinate prodrug (NV118) but not B12a; (2) Attenuation of superoxide production with treatment of NV118 but not with B12a treatment; (3) The changes in respiration were not secondary to increased mitochondrial content as measured by citrate synthase; (4) The use of easily accessible human blood cells showed similar mitochondrial response to both cyanide and treatment to HASMC. The use of a succinate prodrug to circumvent partial CIV inhibition by cyanide with clear reversal of cellular respiration and superoxide production that was not attributed to changes in mitochondrial content not seen by the use of B12a.
Project description:Metformin is the most common pharmacological treatment for type 2 diabetes. It is considered safe but has been associated with the development of lactic acidosis under circumstances where plasma concentrations exceed therapeutic levels. Metformin-induced lactic acidosis has been linked to the drug's toxic effect on mitochondrial function. Current treatment strategies aim to remove the drug and correct for the acidosis. With a mortality of 20%, complementary treatment strategies are needed. In this study, it was investigated whether targeting mitochondria with pharmacological agents that bypass metformin-induced mitochondrial dysfunction can counteract the energetic deficit linked to toxic doses of metformin.The redox agent methylene blue and the cell-permeable succinate prodrug NV118 were evaluated by measuring mitochondrial respiration and lactate production of human platelets exposed to metformin and co-treated with either of the two pharmacological bypass agents.The cell-permeable succinate prodrug NV118 increased mitochondrial respiration which was linked to phosphorylation by the ATP-synthase and alleviated the increase in lactate production induced by toxic doses of metformin. The redox agent methylene blue, in contrast, failed to mitigate the metformin-induced changes in mitochondrial respiration and lactate generation.The cell-permeable succinate prodrug NV118 bypassed the mitochondrial dysfunction and counteracted the energy deficit associated with toxic doses of metformin. If similar effects of NV118 prove translatable to an in vivo effect, this pharmacological strategy presents as a promising complementary treatment for patients with metformin-induced lactic acidosis.
Project description:Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.
Project description:1. Chemiluminescence of Acanthomoeba castellanii in the presence of O2 was of similar intensity in organisms harvested early or late during exponential growth [when cyanide (1 mM) stimulates or inhibits respiration respectively]. 2. Cyanide (up to 1.5 mM) stimulated photoemission in both types of organism by 250--300 photons/s per 10(7) cells above the value observed under aerobic conditions. 3. 'Dibromothymoquinone' (2,5-dibromo-6-isopropyl-3-methyl-p-benzoquinone) (up to 80 microM) further increased chemiluminescence. 4. Similar responses were also demonstrated in whole homogenates and in subcellular fractions; 36% of the chemiluminescence was provided by a fraction sedimenting at 100000g-min, and 20% in that fraction that was non-sedimentable at 200000g-min. 5. Mitochondrial substrates (succinate, 2-oxoglutarate, NADH) in the presence or absence of ADP and Pi or peroxisomal substrates (glycollate, urate or ethanol) gave no increases in light emission by whole homogenates or in any of the fractions. 6. It is suggested that reactions responsible for production of chemiluminescence are those primarily producing superoxide anions and leading to lipid peroxidation and singlet-oxygen formation. Photoemission enhancement and superoxide dismutase inhibition showed similar cyanide concentration-dependencies.
Project description:Acetaminophen is one of the most common over-the-counter pain medications used worldwide and is considered safe at therapeutic dose. However, intentional and unintentional overdose accounts for up to 70% of acute liver failure cases in the western world. Extensive research has demonstrated that the induction of oxidative stress and mitochondrial dysfunction are central to the development of acetaminophen-induced liver injury. Despite the insight gained on the mechanism of acetaminophen toxicity, there still is only one clinically approved pharmacological treatment option, N-acetylcysteine. N-acetylcysteine increases the cell's antioxidant defense and protects liver cells from further acetaminophen-induced oxidative damage. Because it primarily protects healthy liver cells rather than rescuing the already injured cells alternative treatment strategies that target the latter cell population are warranted. In this study, we investigated mitochondria as therapeutic target for the development of novel treatment strategies for acetaminophen-induced liver injury. Characterization of the mitochondrial toxicity due to acute acetaminophen overdose in vitro in human cells using detailed respirometric analysis revealed that complex I-linked (NADH-dependent) but not complex II-linked (succinate-dependent) mitochondrial respiration is inhibited by acetaminophen. Treatment with a novel cell-permeable succinate prodrug rescues acetaminophen-induced impaired mitochondrial respiration. This suggests cell-permeable succinate prodrugs as a potential alternative treatment strategy to counteract acetaminophen-induced liver injury.
Project description:1. The interference mechanism of carbonyl cyanide m-chlorophenylhydrazone with the respiratory process and with phosphorylation coupled to respiration has been investigated in resting cells of Escherichia coli. 2. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone in the absence of substrate caused strong inhibition of succinate oxidation. The inactivation of the respiratory system proved to be time-dependent and temperature-dependent and could be arrested by adding the substrate. Inhibition of incorporation of (32)P into acid-soluble organic phosphate esters exceeded the inhibition of oxygen uptake. 3. In contrast with succinate, the rate of oxidation of glucose was increased by carbonyl cyanide m-chlorophenylhydrazone. The sensitivity of other substrates to the inhibitor was less than that of succinate. 4. Various observations are described in support of the view that respiratory inhibition induced by carbonyl cyanide m-chlorophenylhydrazone is a result of its interference with ATP synthesis. The capacity of a given substrate to increase intracellular ATP concentration appeared to be directly related to its resistance to inhibition. In cell-free extracts carbonyl cyanide m-chlorophenylhydrazone still suppressed (32)P incorporation but had no effect on respiration. 5. Carbonyl cyanide m-chlorophenylhydrazone-induced stimulation of glucose oxidation and the acceleration of succinate oxidation by ADP or AMP in cells rendered permeable to nucleotides are tentatively interpreted as an indication that a certain part of respiration in E. coli is under phosphate-acceptor-mediated control.
Project description:Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.
Project description:Superoxide reductases (SORs) are nonheme iron-containing enzymes that reduce HO(2) to H(2)O(2). Exogenous substrates such as N(3)(-) and CN(-) have been shown to bind to the catalytic iron site of SOR, and cyanide acts as an inhibitor. To understand how these exogenous ligands alter the physical and reactivity properties of the SOR iron site, acetate-, azide-, and cyanide-ligated synthetic models of SOR have been prepared. The x-ray crystal structures of azide-ligated [Fe(III)(S(Me2)N(4)(tren))(N(3))](+) (3), dimeric cyanide-bridged ([Fe(III)(S(Me2)N(4)(tren))](2)-mu-CN)(3+) (5), and acetate-ligated [Fe(III)(S(Me2)N(4)(tren))(OAc)](+) (6) are described, in addition to x-ray absorption spectrum-derived and preliminary crystallographic structures of cyanide-ligated [Fe(III)(S(Me2)N(4)(tren))(CN)](+) (4). Cyanide coordination to our model (4) causes the redox potential to shift anodically by 470 mV relative to acetate-ligated 6 and 395 mV relative to azide-ligated 3. If cyanide coordination were to cause a similar shift in redox potential with SOR, then the reduction potential of the catalytically active Fe(3+) center would fall well below that of its biological reductants. These results suggest therefore that cyanide inhibits SOR activity by making the Fe(2+) state inaccessible and thus preventing the enzyme from turning over. Cyanide inhibits activity in the metalloenzyme superoxide dismutase via a similar mechanism. The reduced five-coordinate precursor to 3, 4, and 6 [Fe(II)(S(Me2)N(4)(tren))](+) (1) was previously shown by us to react with superoxide to afford H(2)O(2) via an [Fe(III)(S(Me2)N(4)(tren))(OOH)](+) intermediate. Cyanide and azide do not bind to 1 and do not prevent 1 from reducing superoxide.
Project description:Major depressive disorder (MDD) has been linked to mitochondrial defects, which could manifest in mitochondrial DNA (mtDNA) polymorphisms or mutations. Additionally, copy number of mtDNA (mtDNA-cn) can be quantified in peripheral blood mononuclear cells (PBMC)s, indirectly reflecting cellular energetics, or in the circulating cell-free mtDNA (ccf-mtDNA) levels, which may reflect a fraction of the mitochondrial genome released during cellular stress. Few studies have examined ccf-mtDNA in MDD, and no studies have tested its relationship with intracellular mtDNA-cn or with antidepressant treatment response. Here, mtDNA levels were quantified in parallel from: (i) PBMCs and (ii) cell-free plasma of 50 unmedicated MDD subjects and 55 controls, in parallel with PBMC telomere length (TL) and antioxidant enzyme glutathione peroxidase (GpX) activity. MtDNA measures were repeated in 19 MDD subjects after 8 weeks of open-label SSRI treatment. In analyses adjusted for age, sex, BMI, and smoking, MDD subjects had significantly elevated levels of ccf-mtDNA (F?=?20.6, p?=?0.00002). PBMC mtDNA-cn did not differ between groups (p?>?0.4). In preliminary analyses, we found that changes in ccf-mtDNA with SSRI treatment differed between SSRI responders and non-responders (F?=?6.47, p?=?0.02), with the non-responders showing an increase in ccf-mtDNA and responders not changing. Baseline ccf-mtDNA was positively correlated with GpX (r?=?0.32, p?=?0.001), and PBMC mtDNA correlated positively with PBMC TL (r?=?0.38, p?=?0.0001). These data suggest that plasma ccf-mtDNA and PBMC mtDNA-cn reflect different cellular processes and that the former may be more reflective of certain aspects of MDD pathophysiology and of the response to SSRI antidepressants.
Project description:We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant ("clamped") at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed.
Project description:1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.