Enrique Cadenas

Metabolic shift in lung alveolar cell mitochondria following acrolein exposure

Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

A novel biologically active seleno-organic compound--I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen).

a synthetic seleno-organic compound, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (PZ 51), exhibits GSH peroxidase-like activity in vitro, in contrast to its sulfur analog, PZ 25. In addition, PZ 51 behaves as an antioxidant shown by a temporary protection of rat liver microsomes against ascorbate/ADP-Fe-induced lipid peroxidation, an effect also elicited by PZ 25 but to a smaller extent. This protection against lipid peroxidation is independent of GSH and of P-450 monooxygenase activity.

Handbook of Antioxidants

General Topics Food-Derived Antioxidants: How to Evaluate Their Importance in Food and In Vivo Barry Halliwell Measurement of Total Antioxidant Capacity in Nutritional and Clinical Studies Guohua Cao and Ronald L. Prior Quantification of Isoprostanes as Indicators of Oxidant Stress In Vivo Jason D. Morrow, William E. Zackert, Daniel S. Van der Ende, Erin E. Reich, Erin S. Terry, Brian Cox, Stephanie C. Sanchez, Thomas J. Montine, and L. Jackson Roberts Vitamin E Efficacy of Vitamin E in Human Health and Disease Sharon V. Landvick, Anthony T. Diplock, and Lester Packer Vitamin E Bioavailability, Biokinetics, and Metabolism Maret G. Traber Biological Activity of Tocotrienols Stefan U. Weber and Gerald Rimbach Vitamin C Vitamin C: From Molecular Mechanisms to Optimum Uptake Sebastian J. Padayatty, Rushad Daruwala, Yaohui Wang, Peter Eck, Jian Song, Woo S. Koh, and Mark Levine Vitamin C and Cardiovascular Diseases Anitra C. Carr and Balz Frei Epidemiological and Clinical Aspects of Ascorbate and Cancer James E. Enstrom Carotenoids Carotenoids: Linking Chemistry, Absorption, and Metabolism to Potential Roles in Human Health and Disease Denise M. Deming, Thomas W. M. Boileau, Kasey H. Heintz, Christine A. Atkinson, and John W. Erdman, Jr. Antioxidant Effects of Carotenoids: Implication in Photoprotection in Humans Wilhelm Stahl and Helmut Sies Oxidative Breakdown of Carotenoids and Biological Effects of Their Metabolites Werner G. Siems, Olaf Sommerburg, and Frederik J. G. M. van Kuijk Carotenoids in the Nutrition of Infants Olaf Sommerburg, Werner G. Siems, Kristina Meissner, and Michael Leichsenring Human Studies on Bioavailability and Serum Response of Carotenoids Elizabeth J. Johnson Polyphenols and Flavonoids Caffeic Acid and Related Antioxidant Compounds: Biochemical and Cellular Effects Joao Laranjinha Polyphenols and Flavonoids Protect LDL Against Atherogenic Modifications Bianca Fuhrman and Michael Aviram Phytoestrogen Content in Foods and Their Role in Cancer Anna H. Wu and Malcolm C. Pike Peroxynitrite Scavenging by Mitochondrial Reductants and Plant Polyphenols Alberto Boveris, Silvia Alvarez, Silvia Lores Arnaiz, and Laura B. Valdez Antioxidants in Beverages and Herbal Products Antioxidant and Other Properties of Green and Black Tea Philip J. Rijken, Douglas A. Balentine, C. A. J. van Mierlo, I. Paetau-Robinson, F. van de Put, Paul T. Quinlan, Ute M. Weisgerber, and Sheila A. Wiseman The Phenolic Wine Antioxidants Andrew L. Waterhouse French Maritime Pine Bark: Pycnogenol Gerald Rimbach, Fabio Virgili, and Lester Packer Spices as Potent Antioxidants with Therapeutic Potential Bharat B. Aggarwal, Nihal Ahmad, and Hasan Mukhtar Lipoic Acid and Glutathione Lipoic Acid: Cellular Metabolism, Antioxidant Activity, and Clinical Relevance Oren Tirosh, Sashwati Roy, and Lester Packer Cellular Effects of Lipoic Acid and Its Role in Aging Regis Moreau , Wei-Jian Zhang, and Tory M. Hagen Vascular Complications in Diabetes: Mechanisms and the Influence of Antioxidants Peter Rosen, Hans-Jurgen Tritschler, and Lester Packer Therapeutic Effects of Lipoic Acid on Hyperglycemia and Insulin Resistance Erik J. Henriksen Bioavailability of Glutathione Dean P. Jones Melatonin Antioxidant Capacity of Melatonin Russel J. Reiter, Dun-xian Tan, Lucien C. Manchester, and Juan R. Calvo Radical and Reactive Intermediate-Scavenging Properties of Melatonin in Pure Chemical Systems Maria A. Livrea, Luisa Tesoriere, Dun-xian Tan, and Russel J. Reiter Selenium Selenium: An Antioxidant? Regina Brigelius-Flohe, Matilde Maiorino, Fulvio Ursini, and Leopold Flohe Selenium Status and Prevention of Chronic Diseases Paul Kneckt Nitric Oxide Antioxidant Properties of Nitric Oxide Homero Rubbo and Rafael Radi

Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space.

It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the intermembrane space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the intermembrane space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium trioxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the intermembrane space. The generation of superoxide anion towards the intermembrane space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.

Estimation of H2O2 gradients across biomembranes.

When cells are exposed to an external source of H2O2, the rapid enzymatic consumption of H2O2 inside the cell provides the driving force for the formation of the gradient across the plasma and other subcellular membranes. By using the concepts of enzyme latency, the following gradients – formed after a few seconds following the exposure to H2O2 – were estimated in Jurkat T‐cells: [H2O2]cytosol/[H2O2]peroxisomes=3; [H2O2]extracellular/[H2O2]cytosol=7. The procedure presented in this work can easily be applied to other cell lines and provides a quantitative framework to interpret the data obtained when cells are exposed to an external source of H2O2.

Redox and addition chemistry of quinoid compounds and its biological implications

The overall biological activity of quinones is a function of the physico-chemical properties of these compounds, which manifest themselves in a critical bimolecular reaction with bioconstituents. Attempts have been made to characterize this bimolecular reaction as a function of the redox properties of quinones in relation to hydrophobic or hydrophilic environments. The inborn physico-chemical properties of quinones are discussed on the basis of their reduction potential and dissociation constants, as well as the effect of environmental factors on these properties. Emphasis is given on the effect of methyl-, methoxy-, hydroxy-, and glutathionyl substituents on the reduction potential of quinones and the subsequent electron transfer processes. The redox chemistry of quinoid compounds is surveyed in terms of a) reactions involving only electron transfer, as those accomplished during the enzymic reduction of quinones and the non-enzymic interaction with redox couples generating semiquinones, and b) nucleophilic addition reactions. The addition of nucleophiles, entailing either oxidation or reduction of the quinone, are exemplified in reactions with oxygen- or sulfur nucleophiles, respectively. The former yields quinone epoxides, whereas the latter yields thioether-hydroquinone adducts as primary molecular products. The subsequent chemistry of these products is examined in terms of enzymic reduction, autoxidation, cross-oxidation, disproportionation, and free radical interactions. The detailed chemical mechanisms by which quinoid compounds exert cytotoxic, mutagenic and carcinogenic effects are considered individually in relation to redox cycling, alterations of thiol balance and Ca++ homeostasis, and covalent binding.

MAPK signaling in neurodegeneration: influences of flavonoids and of nitric oxide

Oxidative and nitrosative stress is increasingly associated with the pathology of neurodegeneration and aging. The molecular mechanisms underlying oxidative/nitrosative stress-induced neuronal damage are emerging and appear to involve a mode of death in which mitogen-activated protein kinase (MAPK) signaling pathways are strongly implicated. Thus, attention is turning towards the modulation of intracellular signaling as a therapeutic approach against neurodegeneration. Both endogenous and dietary agents have been suggested as potent modulators of intracellular signal transduction, e.g. nitric oxide and flavonoids, respectively. This review addresses recent findings on the biological effects of flavonoids and nitric oxide in neurodegeneration and aims to elucidate the rationale for their prospective use as modulators of cellular signal transduction.

[30] DT-diaphorase: Purification, properties, and function

Publisher Summary DT-diaphorase is a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzes the oxidation of nicotine adenine dinucleotide, reduced nicotine adenine dinucleotide hydrogenase (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH) at equal rates. This chapter discusses the purification, properties, and function of DT-diaphorase. Most of the present knowledge about DT-diaphorase has been obtained in studies of the cytosolic enzyme, which was first purified from both rat and beef liver 44 by employing conventional methods. The introduction of affinity chromatography for the purification of DTdiaphorase reduced the number of purification steps considerably. In the method presented in the chapter for the purification of DT-diaphorase, the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, which is a potent competitive inhibitor of the enzyme with respect to NAD(P)H. A gel with both a high affinity and a high binding capacity for DT-diaphorase is obtained by coupling dicoumarol through an azo linkage to divinyl sulfonate (DVS)-activated Sepharose 6B. In the assay, DT-diaphorase activity is measured routinely with NADH or NADPH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) or 2-methyl-l,4-naphthoquinone (menadione) as the electron acceptor. The standard assay system contains 50 m M Tris-HC1, pH 7.5, 0.08% Triton X-100, 0.5 m M NADH or NADPH, and 40 μM DCPIP or 10 μM menadione.

Nitric oxide inhibits mitochondrial NADH : ubiquinone reductase activity through peroxynitrite formation

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture.

We have re-examined the lysosomal hypothesis of oxidative-stress-induced apoptosis using a new technique for exposing cells in culture to a low steady-state concentration of H(2)O(2). This steady-state technique mimics the situation in vivo better than the bolus-administration method. A key aspect of H(2)O(2)-induced apoptosis is that the apoptosis is evident only after several hours, although cells may become committed within a few minutes of exposure to this particular reactive oxygen species. In the present work, we were able to show, for the first time, several correlative links between the triggering effect of H(2)O(2) and the later onset of apoptosis: (i) a short (15 min) exposure to H(2)O(2) caused almost immediate, albeit limited, lysosomal rupture; (ii) early lysosomal damage, and later apoptosis, showed a similar dose-related response to H(2)O(2); (iii) both events were inhibited by pre-treatment with iron chelators, including desferrioxamine. This compound is known to be taken up by endocytosis only and thus to become localized in the lysosomal compartment. After exposure to oxidative stress, when cells were again in standard culture conditions, a time-dependent continuous increase in lysosomal rupture was observed, resulting in a considerably lowered number of intact lysosomes in apoptotic cells, whereas non-apoptotic cells from the same batch of oxidative-stress-exposed cells showed mainly intact lysosomes. Taken together, our results reinforce earlier findings and strongly suggest that lysosomal rupture is an early upstream initiating event, and a consequence of intralysosomal iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.