Respiratory Chain Inhibitors Research Articles

New Insights Into the Antibacterial Mechanism of Cryptotanshinone, a Representative Diterpenoid Quinone From Salvia miltiorrhiza Bunge.

The rapid rise of antibiotic resistance causes an urgent need for new antimicrobial agents with unique and different mechanisms of action. The respiratory chain is one such target involved in the redox balance and energy metabolism. As a natural quinone compound isolated from the root of Salvia miltiorrhiza Bunge, cryptotanshinone (CT) has been previously demonstrated against a wide range of Gram-positive bacteria including multidrug-resistant pathogens. Although superoxide radicals induced by CT are proposed to play an important role in the antibacterial effect of this agent, its mechanism of action is still unclear. In this study, we have shown that CT is a bacteriostatic agent rather than a bactericidal agent. Metabolome analysis suggested that CT might act as an antibacterial agent targeting the cell membrane. CT did not cause severe damage to the bacterial membrane but rapidly dissipated membrane potential, implying that this compound could be a respiratory chain inhibitor. Oxygen consumption analysis in staphylococcal membrane vesicles implied that CT acted as respiratory chain inhibitor probably by targeting type II NADH:quinone dehydrogenase (NDH-2). Molecular docking study suggested that the compound would competitively inhibit the binding of quinone to NDH-2. Consistent with the hypothesis, the antimicrobial activity of CT was blocked by menaquinone, and the combination of CT with thioridazine but not 2-n-heptyl-4-hydroxyquinoline-N-oxide exerted synergistic activity against Staphylococcus aureus. Additionally, combinations of CT with other inhibitors targeting different components of the bacterial respiratory chain exhibit potent synergistic activities against S. aureus, suggesting a promising role in combination therapies.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10–260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

SummaryIn addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

Computationally modeling mammalian succinate dehydrogenase kinetics identifies the origins and primary determinants of ROS production

Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of [Formula: see text] and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary [Formula: see text] source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, [Formula: see text] is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.

Study of mitophagy and ATP-related metabolomics based on β-amyloid levels in Alzheimer's disease

The aggregation of β-amyloid (Aβ) peptide in Alzheimer's disease (AD) is characterized by mitochondrial dysfunction and mitophagy impairment. Mitophagy is a homeostatic mechanism by which autophagy selectively eliminates damaged mitochondria. Valinomycin is a respiratory chain inhibitor that activates mitophagy via the PINK1/Parkin signaling pathway. However, the mechanism underlying the association between mitophagy and valinomycin in Aβ formation has not been explored. Here, we demonstrate that genetically modified (N2a/APP695swe) cells overexpressing a mutant amyloid precursor protein (APP) serve as an in vitro model of AD for studying mitophagy and ATP-related metabolomics. Our results prove that valinomycin induced a time-dependent increase in the mitophagy activation of N2a/APP695swe cells as indicated by increased levels of PINK1, Parkin, and LC3II as well as increased the colocalization of Parkin–Tom20 and fewer mitochondria (indicated by decreased Tom20 levels). Valinomycin significantly decreased Aβ1–42 and Aβ1–40 levels after 3 h of treatment. ATP levels and ATP-related metabolites were significantly increased at this time. Our findings suggest that the elimination of impaired mitochondria via valinomycin-induced mitophagy ameliorates AD by decreasing Aβ and improving ATP levels.

Total Synthesis of Ajudazol A by a Modular Oxazole Diversification Strategy.

The total synthesis of the potent respiratory chain inhibitor ajudazol A was accomplished by a concise strategy in 17 steps (longest linear sequence). The modular approach was based on a direct oxazole functionalization strategy involving a halogen dance reaction for selective halogenation in combination with a challenging combination of sp2-sp2 and sp2-sp3 Negishi cross coupling reactions. The applicability of this strategy for analogue synthesis was demonstrated by the synthesis of a simplified as well as stabilized ajudazol analogue.

A Fast, Simple, and Affordable Technique to Measure Oxygen Consumption in Living Zebrafish Embryos.

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.

Hypoxia/reoxygenation exacerbates drug-induced cytotoxicity by opening mitochondrial permeability transition pore: Possible application for toxicity screening

Recently, mitochondrial dysfunction is thought of as an important factor leading to a drug-induced liver injury. Our previous reports show that mitochondria-related toxicity, including respiratory chain inhibition (RCI) and reactive oxygen species (ROS) induction, can be detected by the modification of sugar resource substitution and high oxygen condition. However, this in vitro model does not detect mitochondrial permeability transition (MPT)-induced toxicity. Another study with a lipopolysaccharide-pre-administered rodent model showed that ischemia/reperfusion induced ROS, sensitized the susceptibility of MPT pore opening and, finally developed drug-induced liver toxicity. Based on this result, the present study investigated the effect of hypoxia/reoxygenation (H/R) treatment mimicking the ischemia/reperfusion on MPT-dependent toxicity, aiming to construct a system that can evaluate MPT by drugs in hepatocytes. Mitochondrial ROS were enhanced by H/R treatment only in the galactose culture condition. Amiodarone, benzbromarone, flutamide and troglitazone which induced MPT pore opening led to hepatocyte death only in combination with H/R and galactose. Moreover, this alteration was significantly suppressed in hepatocytes lacking cyclophilin D. In conclusion, MPT-induced cytotoxicity can be detected by activating mitochondrial function and H/R. This cell-based assay system could evaluate MPT induced-cytotoxicity by drugs, besides RCI and ROS induction.

Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex

The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential. The use of specific inhibitors of mitochondrial respiratory chain (complex I-IV) revealed no effects on the yeast growth, highlighting the metabolic shift to fermentative pathway in C. haemulonii strains. Also, C. haemulonii complex proved to be highly resistant to oxidative burst agents, which can be correlated with a high activity of antioxidant enzymes. Our data demonstrated primary evidence suggesting that ergosterol content, mitochondrial function, and fungal redox homeostasis are involved in AMB fungicidal effects and might explain the resistance presented in this multidrug-resistant, emergent, and opportunistic fungal complex.

The Functional Activity of Mitochondria in Deuterium Depleted Water

Abstract—The efficiency of phosphorylation in samples of isolated rat liver mitochondria in incubation media with variable isotopic composition at deuterium contents of 6, 60, 90, 120, 150 ppm, as well as 0.1, 1 and 2%, was studied. In media with a low deuterium content (up to 60 ppm), a monotonic decrease in the ADP/O ratio was observed, while at 6 ppm its value was the same as at 60 ppm. The generation of superoxide anion by complex I, II, and III of the mitochondrial respiratory chain was also investigated. For all studied modes of mitochondrial functioning using respiratory chain inhibitors, the generation of superoxide anion was independent of the deuterium concentration in the incubation medium.

Successful energy shift from glycolysis to mitochondrial oxidative phosphorylation in freshly isolated hepatocytes from humanized mice liver

Mitochondrial toxicity is a factor of drug-induced liver injury. Previously, we reported an in vitro rat hepatocyte assay where mitochondrial toxicity was more sensitively evaluated, using sugar resource substitution and increased oxygen supply. Although this method could be applicable to human cell-based assay, cryopreserved human hepatocyte (CHH) has some disadvantages/uncertainty, including unstable same donor supply and potential organelle damage due to cryopreservation. Herein, we compared the mitochondrial functions of freshly-isolated hepatocytes from humanized chimeric mice liver (PXB-cells) and three CHH lots to determine the better cell source for mitochondrial toxicity assay. Two CHH lots declined after replacing glucose with galactose. To confirm the shift in energy production from glycolysis to oxidative phosphorylation, lactate and oxygen consumption rate (indicators of glycolytic activity and mitochondrial oxidative phosphorylation, respectively) were measured. In PXB-cells, lactate amount decreased, while oxygen consumption in 100 min increased. These effects were less evident in CHH. The cytotoxicity of the select respiratory chain inhibitors was enhanced in PXB-cells upon sugar replacement, but no change occurred with negative control drugs (bicalutamide and metformin). Altogether, PXB-cells was less vulnerable to sugar resource substitution than CHH. The substitution activated mitochondrial function and enhanced cytotoxicity of respiratory chain inhibitors in PXB-cells.

Optimizing oxygen delivery in sepsis: A review

Multiple organ failure syndrome (MOFS) is a hallmark of sepsis. The continued dysfunction of microvascular perfusion has been implicated as the inciting factor for this. Multiple etiopathological factors are involved in producing this disequilibrium in the demand, supply, and extraction of oxygen as a result of derecruitment of microcirculation. This is further complicated by mitochondrial dysfunction in the form of the inhibition of mitochondrial respiratory chain of enzymes, leading to difficulty in extraction of the oxygen at the cellular level. Eventually, although hemodynamic stability of systemic/macrovascular circulation may have been achieved, the process of deficient and defective delivery of oxygen to the tissues goes on relentlessly. The indicators and monitoring of this process of impairment of oxygen delivery (DO2) have been discussed in this review. In addition, the review also encompasses various therapeutic modalities and their efficacy, based on the evidence. The latest guidelines regarding optimizing the DO2 in sepsis are also included here.

Protective effect of metformin against palmitate-induced hepatic cell death

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.

Mediated electron transfer efficiencies of Se(IV) bioreduction facilitated by meso-tetrakis (4-sulfonatophenyl) porphyrin

The redox-active sulfonated porphyrin meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) was novelly proposed to facilitate the mediated electron transfer (MET) of the otherwise time-consuming selenite (Se(IV)) bioreduction. TPPS (0.2 mM) increased the Se(IV) (39.5 ± 1.0 mg∙L−1) bioreduction rate by Shewanella oneidensis·MR-1 by 4- to 5-fold. UV–vis, redox potential, cyclic voltammetry, and electrochemical impedance spectroscopy showed that the underlying mechanism was the redox-active transformation of the porphyrin ring, which led to a 25% decrease of the activation energy. Furthermore, the Michaelis-Menten fitting and the experiments on specific respiratory chain inhibitors suggested that TPPS enhanced the MET efficiencies of NADH-CoQ, FAD-CoQ, and Cyt bL-Cyt bH with stronger affinity (Km−1TPPS = 50.0 mM−1 > Km−1control = 0.8 mM−1) between the Se(IV) and microorganisms. Interestingly, TPPS enhanced the MET efficiencies by increasing the contents of redox-active compounds in extracellular polymeric substances. This study offers a realistic application and further mechanistic understanding of porphyrins as redox mediators for the Se(IV) bioreduction.

Loss of ZBTB7A Enhances Glycolysis and Beta Oxidation in Myeloid Leukemia

ZBTB7A is a transcription factor with function in hematopoietic lineage fate decisions (reviewed in Lunardi et al., 2013, Blood). Moreover, ZBTB7A has been shown to also be involved in regulation of glycolysis in solid tumors (Liu et al., 2014, Genes Dev.). Recently, we found ZBTB7A frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. What is more, high expression of ZBTB7A correlated with better clinical outcome in cytogenetically normal AML (Hartmann et al., 2016, Nat Commun). The functional role of ZBTB7A and its alterations in myeloid malignancies, however, remains unclear, especially concerning the regulation of leukemia metabolism. To investigate the effect of ZBTB7A mutations in leukemia, we generated K562 ZBTB7A knockout (KO) cells using CRISPR/Cas9, followed by RNA-Seq in KO and control cells. Thereby, we confirmed de-repression of the previously reported ZBTB7A target gene SLC2A3 (glucose transporter 3) as well as upregulation of several other glycolysis related genes (PGM2, PGM3, SLC2A1 and ENO2). ZBTB7A binding to all of these candidate target genes was validated using publicly available ChIP-Seq data from K562 cells (ENCSR000BME). Interestingly, Gene Set Enrichment Analysis revealed deregulation of distinct pathways related to fatty acid metabolism in this model (Figure 1A). KO cells overexpress genes involved in the fatty acid beta oxidation pathway (ACAA2, ACOX1, ACSL1, ACADVL, CPT1A and CPT1B) as well as genes related to other fatty acid metabolism (EPHX2, FADS2 and others) (Figure 1B). We could further validate ACAA2, ACOX1, ACADVL and CPT1A as direct ZBTB7A targets using ChIP-Seq data. Of special interest are CPT1A and CPT1B, which are targetable through Etomoxir treatment. KO cells showed an increased sensitivity to this drug compared to control (IC50= 120.6 and 125.5 µM in KOs vs 228.4 µM in control, p&amp;lt;0.0001). Moreover, analysis of RNA-Seq data from patients with AML t(8;21) revealed a significantly higher expression of EPHX2 (p=0.049), FADS2 (p=0.003) and FASD1 (p=0.021) in patients harboring ZBTB7A mutations (Figure 1C) using a two-tailed unpaired Student's t-test. In order to evaluate our findings on a functional level, we performed metabolic flux assays in K562 ZBTB7A KO vs control cells. Using Seahorse technology (Agilent), we found that KO cells show a modest increase in extracellular acidification rate (ECAR), indicating a higher glycolysis. This effect becomes more obvious after mitochondrial respiratory chain inhibition: 41.20 and 51.66 mpH/min in KO clones vs 34.33 mpH/min in control (p=0.002 and p&amp;lt;0.001, respectively) (Figure 1D). This result suggests that loss of ZBTB7A may confer an advantage to cells in specific microenvironment with low oxygen availability, such as the bone marrow. Moreover, metabolic flux assays also revealed a nearly 50% increase in oxygen consumption rate (OCR) in KO cells after 1h glucose starvation: 140.53 and 148.53 pmol/min in KO clones vs 96.46 pmol/min in control (p&amp;lt;0.001 in both comparisons) (Figure 1E). Since the cells were deprived from glucose, the observed oxygen consumption may arise mainly from glutamate metabolism or fatty acid oxidation. Deprivation from glutamate reduced overall OCR but KO cells still showed increased oxygen consumption compared to control. These results therefore suggest that an increased beta oxidation of fatty acids leads to the higher OCR observed in KO cells. In summary, we have demonstrated that the previously described role of ZBTB7A as a regulator of glycolysis in solid tumors is also relevant in myeloid malignancies. In addition, we identified the beta oxidative pathway and fatty acid synthesis as novel mechanisms underlying the perturbed function of ZBTB7A in tumor metabolism. ZBTB7A downregulation or mutation may lead to an increased energy production providing an advantage to leukemia cells. These findings likely have therapeutic implications, as metabolic inhibitors such as 2-deoxy-d-glucose and Etomoxir may specifically target ZBTB7A deficient malignancies. Figure Disclosures Hiddemann: Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria.

The NADH-Oxidase Activity of Mitochondria under Hypotony with Respiratory Chain Inhibitors

We studied the contribution of NADH oxidase to mitochondrial respiration in a suspension under extreme conditions: in a hypotonic medium and with respiratory poisons rotenone, antimycin, and sodium azide. The experiment was carried out with rat liver mitochondria. It was shown that not only cytochrome oxidase but also NADH dehydrogenase of the respiratory chain notably consume oxygen to oxidize exogenous NADH. For this reason NADH-associated mitochondrial respiration is not completely inhibited by respiratory poisons. The insensitivity of respiration to poisoning is a result of a shunt of electron transfer from NADH to oxygen directly via NADH dehydrogenase. This residual respiration via NADH oxidase, which is insensitive to respiratory chain inhibitors, may account for one-half of the total oxygen consumption under extreme conditions.

Multiparametric real-time sensing of cytosolic physiology links hypoxia responses to mitochondrial electron transport.

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.

Induction of mitochondrial dysfunction in patients under cardiopulmonary by-pass: preliminary results.

Cardiac surgery is often performed by cardiopulmonary by-pass (CPB), generally associated with organ dysfunction. The aim of this work was to determine if and how this phenomenon is related to mitochondrial damage. To this purpose, the effect of the addition of serum from CPB patients to human fibroblasts cultures on mitochondrial respiratory chain and oxidative phosphorylation (OXPHOS) activities was investigated. Serum samples of five patients were obtained before (pre-CPB) and after 6 h CPB weaning (CPB). Mitochondrial OXPHOS activities were examined by polarographic and spectrophotometric assays, and reactive oxygen species (ROS) production was measured by a spectrofluorimeter. Addition of CPB serum to fibroblasts determined a decrease of mitochondrial oxygen consumption due to an inhibition of mitochondrial respiratory chain and some OXPHOS enzymes activities. This inhibition seems to be mainly related to a reduced activity of complex I. Our data represent the first translational research evidence showing that CPB determines mitochondrial dysfunction which leads to impairment of OXPHOX activities and to an increase in ROS production, compromising tissue bioenergetic efficiency.

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc–Gal–NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc–Gal–NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2–3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc–Gal–NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc–Gal–NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.

Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans.

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of kcat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.