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E Cadenas

Publications and source records attributed to E Cadenas.

At least 19 recordsLinked to original sources
PubMed2001

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.

Apoptosis↗
PubMed2001

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium.

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

1-Butanol↗
PubMed2001

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state.

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

2,2'-Dipyridyl↗
PubMed2001

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.

Animals↗
PubMed2000

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage.

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

Albumins↗
PubMed2000

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.

Catalase↗
PubMed2000

p21WAF1 regulates anchorage-independent growth of HCT116 colon carcinoma cells via E-cadherin expression.

The cyclin-dependent kinase inhibitor p21WAF1 has been characterized as an important effector of the tumor suppressor p53 and has been linked to various growth-regulatory processes. To identify a potential role of p21 in anchorage-dependent growth control, we analyzed a pair of HCT116 human colon carcinoma cell lines that differed only in their p21 status. We found that during suspension culture, HCT116 cells (which contain wildtype p53 and p21) continued to proliferate and formed compact multicellular spheroids (MCSs). In contrast, HCT116 cells engineered to lack functional p21 (HCTp21-/-) were unable to form MCSs in suspension culture, ceased proliferation, and eventually died through apoptosis. The parental HCT116 cells underwent the same fate when treated with hyaluronidase, indicating that cell-cell contact might be required for survival in suspension culture. We established that E-cadherin was induced in HCT116 but not in HCTp21-/- cells and accounted for the formation of MCSs. Forced expression of E-cadherin or p21 in HCTp21-/- cells restored the ability to form MCSs and to grow independently of anchorage. Moreover, HCTp21-/- cells exhibited a severely reduced transformed phenotype and demonstrated greatly enhanced chemosensitivity in suspension culture. Thus, our results link an important regulator of the cell cycle machinery to the expression of a cell-cell adhesion molecule involved in tumor formation. Because our results indicate that loss of p21 severely impairs the ability of HCT cells to grow independently of anchorage, it may not be coincidental that inactivating mutations of this gene are very rarely found in tumor cells.

Animals↗
PubMed2000

Mitochondrial free radical generation, oxidative stress, and aging.

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.

Animals↗
PubMed2000

Analysis of the pathways of nitric oxide utilization in mitochondria.

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen- and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.

Animals↗
PubMed2000

Regulation of mitochondrial respiration by oxygen and nitric oxide.

Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2-) and peroxynitrite (ONOO-). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. No exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

Electron Transport↗
PubMed1999

The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol.

The reversible inhibitory effects of nitric oxide (.NO) on mitochondrial cytochrome oxidase and O(2) uptake are dependent on intramitochondrial.NO utilization. This study was aimed at establishing the mitochondrial pathways for.NO utilization that regulate O-(2) generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO(-)) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO(-)-supplemented mitochondrial membranes were used. The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for.NO utilization in mitochondria, which effectively compete with the binding of.NO to cytochrome oxidase, thereby releasing this inhibition and restoring O(2) uptake. The pathways for.NO utilization are discussed in terms of the steady-state levels of.NO and O-(2) and estimated as a function of O(2) tension. These calculations indicate that mitochondrial.NO decays primarily by pathways involving ONOO(-) formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.

Animals↗
PubMed1999

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance.

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.

Aerobiosis↗
PubMed1999

Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation.

This study addresses the dynamic interactions among alpha-tocopherol, caffeic acid, and ascorbate in terms of a sequence of redox cycles aimed at accomplishing optimal synergistic antioxidant protection. Several experimental models were designed to examine these interactions: UV irradiation of alpha-tocopherol-containing sodium dodecyl sulfate micelles, one-electron oxidations catalyzed by the hypervalent state of myoglobin, ferrylmyoglobin, and autoxidation at appropriate pHs. These models were assessed by ultraviolet (UV) and electron paramagnetic resonance (EPR), entailing direct- and continuous-flow experiments, spectroscopy and by separation and identification of products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by caffeic acid and ascorbate; in the former case, no other EPR signal was observed at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical EPR signal was replaced by a doublet EPR spectrum corresponding to the ascorbyl radical (A*-). The potential interactions between caffeic acid and ascorbate were further analyzed by assessing, on the one hand, the ability of ascorbate to reduce the caffeic acid o-semiquinone (generated by oxidation of caffeic acid by ferrylmyoglobin) and, on the other hand, the ability of caffeic acid to reduce ascorbyl radical (generated by autoxidation or oxidation of ascorbate by ferrylmyoglobin). The data presented indicate that the reductive decay of ascorbyl radical (A*-) and caffeic acid o-semiquinone (Caf-O*) can be accomplished by caffeic acid (Caf-OH) and ascorbate (AH-), respectively, thus pointing to the reversibility of the reaction Caf-O* + AH- <--> Caf-OH + A*-. Continuous-flow EPR measurements of mixtures containing ferrylmyoglobin, alpha-tocopherol-containing micelles, caffeic acid, and ascorbate revealed that ascorbate is the ultimate electron donor in the sequence encompassing transfer of the radical character from the micellar phase to the phase. In independent experiments, the effects of caffeic acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and results indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synergistically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of three domains: its localization and that of the antioxidant-derived radical, its reduction potential, and the predominant decay pathways for the antioxidant-derived radical, that exert kinetic control on the process.

Ascorbic Acid↗
PubMed1998

The role of mitochondrial glutathione in DNA base oxidation.

The objective of this study was to elucidate the role of mitochondrial GSH in the reactions leading to mitochondrial DNA oxidative damage in terms of 8-hydroxy-desoxyguanosine (8-HOdG) accumulation. With this purpose, tightly coupled mitochondria depleted of matrix GSH were used and the effects of H2O2 (generated during the oxidation of substrates) on 8-HOdG levels were investigated. Mitochondrial integrity, assessed by O2 uptake, respiratory control and P/O ratios, was conserved upon depletion of GSH up to 95%. The rates of H2O2 production linked to the oxidation of endogenous substrates by control and GSH-depleted mitochondria were similar. Succinate (in the absence or presence of antimycin A) enhanced the rate H2O2 production to a similar extent in both control and GSH-depleted mitochondria. These rates of H2O2 production accounted for 1.5-2.5% of the rate of O2 uptake. The levels of 8-HOdG in GSH-depleted mitochondria were 35-50% lower than those in control mitochondria, when measured at different H2O2 production rates. Conversely, in experiments carried out with calf thymus DNA with different Cu/Fe content, GSH increased 1.4-2.4-fold the accumulation of 8-HOdG. These values were further enhanced (44-50%) by superoxide dismutase and decreased by catalase. The lower levels of 8-HOdG in GSH-depleted mitochondria and the higher levels in GSH-supplemented calf thymus DNA suggest a role for the non-protein thiol in the reactions leading to mtDNA oxidative damage. These findings are interpreted in terms of the redox transitions involving O2, GSH, and metal catalysts bound to DNA. A mechanism is proposed by which GSH plays a critical role in the reduction of DNA-Cu complexes and decays by free radical pathways kinetically regulated by superoxide dismutase.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Role of p53 in aziridinylbenzoquinone-induced p21waf1 expression.

Quinones are the second largest family of anticancer drugs clinically used in the United States. However, their exact mode of action at the cellular and molecular level is not completely understood. We have shown earlier that the quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) leads to the increased expression of p21waf1/cip1/sdi1 protein, an inhibitor of cyclin-dependent kinases. Because p21 has been established as an important negative regulator of the cell cycle, we further investigated the molecular basis of p21 induction by DZQ. Here we report that the induction of p21 by DZQ is regulated at the transcriptional level, and requires the activation of p53, a tumor suppressor protein. In cells that lack functional p53 protein, DZQ-mediated p21 induction is greatly diminished. However, the introduction of a wild type p53 gene into p53-negative cells restores the strong DZQ-inducibility of p21. Restoration of wild type p53 status in HL60 myeloid leukemia cells significantly increases the cells' sensitivity to the cytotoxic effects of DZQ. Thus, our results indicate that the p53-p21 pathway may play a central role in mediating the gene-regulatory and cytotoxic effects of aziridinylbenzoquinones.

Animals↗