1,721,057 research outputs found
Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2.
No full textExposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A(2) [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539-1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain, In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A(2). This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A, inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A, inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A(2). In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA, isoform, probably localized in the mitochondria
Peroxynitrite damages U937 cell DNA via the intermediate formation of mitochondrial oxidants.
No full textEight years ago we published in this journal the first evidence that peroxynitrite does not directly produce DNA single-strand breakage in intact U937 cells (Guidarelli et al., IUBMB Life 50. 195-201). This event was rather attributed to the secondary reactive species produced at the mitochondrial level via a Ca2+-dependent reaction in which ubisemiquinone serves as an electron donor. Under these conditions, electrons are directly transferred to molecular oxygen and superoxide/H2O2, and the ensuing DNA damage can therefore be produced in a time dependent manner for at least 30 min. Formation of H2O2 and DNA single-strand breaks was therefore dependent on interference with electron transport at the complex Ill level as well as on mitochondrial Ca2+ accumulation. Further studies led to the demonstrations that peroxynitrite mobilizes Ca2+ from the ryanodine receptor. Finally. in U937 cells, a pro-monocytic cell line sharing with monocytes/macrophages the same signaling events to survive to peroxynitrite. mitochondrial H2O2 promotes inhibition of survival via tyrosine phosphatase activation, leading to ERK1/2 dephosphorylation and thus to upstream inhibition of the survival signaling
Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide.
Full text linkThe effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells. 2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1-30 microM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 microM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 microM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO). 3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH. 4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. 5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event
The inositol 1,4,5-trisphosphate-generating agonist ATP enhances DNA cleavage induced by tert-butylhydroperoxide.
No full textIn this paper we present experimental evidence indicating that DNA cleavage induced by tert-butylhydroperoxide in U937 cells can be enhanced via ATP-mediated activation of membrane receptors coupled with hydrolysis of phosphatidylinositol 4,5-bisphosphate. The mechanism whereby ATP exerts this effect involves release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive stores, further release of the cation from the ryanodine receptor, mitochondrial clearance of the fraction of Ca2+ derived from the ryanodine receptor, and Ca2+-dependent mitochondrial formation of DNA-damaging species. IP3-generating agonists must therefore be considered as potential modulators of the genotoxic effects of tert-butylhydroperoxide
Mitochondrial reactive oxygen species: the effects of mitochondrial ascorbic acid vs untargeted and mitochondria-targeted antioxidants
No full textPremise: Mitochondria represent critical sites for reactive oxygen species (ROS) production, which dependent on concentration is responsible for the regulation of both physiological and pathological processes.Purpose: Antioxidants in mitochondria regulate the redox balance, prevent mitochondrial damage and dysfunction and maintain a physiological ROS-dependent signaling. The aim of the present review is to provide critical elements for addressing this issue in the context of various pharmacological approaches using antioxidants targeted or non-targeted to mitochondria. Furthermore, this review focuses on the mitochondrial antioxidant effects of ascorbic acid (AA), providing clues on the complexities associated with the cellular uptake and subcellular distribution of the vitamin.Conclusions: Antioxidants that are not specifically targeted to mitochondria fail to accumulate in significant amounts in critical sites of mitochondrial ROS production and may eventually interfere with the ensuing physiological signaling. Mitochondria-targeted antioxidants are more effective, but are expected to interfere with the mitochondrial ROS-dependent physiologic signaling. AA promotes multiple beneficial effects in mitochondria. The complex regulation of vitamin C uptake in these organelles likely contributes to its versatile antioxidant response, thereby providing a central role to the vitamin for adequate control of mitochondrial dysfunction associated with increased mitochondrial ROS production
Alternative mechanism for hydroperoxide-induced DNA single strand breakage.
No full textThe results presented in this study point out the existence of similarities as well as differences in the DNA-damaging effects of organic vs. inorganic hydroper- oxides in human myeloid leukemia U937 cells. On the one hand, the formation of DNA single strand breaks (SSBs) induced by either hydrogen peroxide (H2O2) or tert-butylhydroperoxide (tBu-OOH) was prevented by iron chelators, was not affected by antioxidants or glucose omission before and during peroxide exposure and was enhanced by prior catalase depletion. Furthermore, H2O2- and tBu-OOH-induced DNA strand scission were also detected after treatment at 0 degrees C. On the other hand, H2O2, but not tBu-OOH or cumene hydroperoxide (cum-OOH), produced DNA strand scission in isolated nuclei and post-lysed DNA samples. In addition, lowering the basal intracellular calcium concentration with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) markedly reduced the DNA-damaging efficiency of tBu-OOH while promoting only a slight decline in the number of DNA SSBs induced by H2O2 Taken together, these results are consistent with the commonly held theory that DNA damage caused by H2O2 is mediated by the formation of hydroxyl radicals. tBu-OOH-induced DNA single strand breakage appears to involve both the formation of H2O2 and a rise in cytosolic calcium ions
Peroxynitrite-mediated release of arachidonic acid from PC12 cells
No full textA short term exposure of PC12 cells to a concentration of tert-butylhydroperoxide (tB-OOH) causing peroxynitrite-dependent DNA damage and cytotoxiticity promoted a release of arachidonic acid (AA) that was sensitive to phospholipase A(2) (PLA(2)) inhibitors and insensitive to phospholipase C or diacylglycerol lipase inhibitors. The extent of AA release was also mitigated by nitric oxide synthase (NOS) inhibitors and peroxpnitrite scavengers. Low levels (10 mu M) of authentic peroxynitrite restored the release of AA mediated by tB-OOH in NOS-inhibited cells whereas concentrations of peroxynitrite of 20 mu M, or higher, effectively stimulated a PLA(2) inhibitor-sensitive release of AA also in the absence of additional treatments. These results are consistent with the possibility that endogenous as well as exogenous peroxynitrite promotes activation of PLA(2)
Calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA in U937 cells exposed to tert-butylhydroperoxide: the role of arachidonate.
No full textTreatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801-806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca2+ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca2+ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca2+-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca2+-dependent activation of phospholipase A(2). Melittin, a potent phospholipase A(2) activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage
NON-TOXIC CONCENTRATIONS OF PEROXYNITRITE COMMIT U937 CELLS TO MITOCHONDRIAL PERMEABILITY TRANSITION-DEPENDENT NECROSIS THAT IS HOWEVER PREVENTED BY ENDOGENOUS ARACHIDONIC ACID
No full textThe present study was aimed at examining the mechanism whereby an otherwise non-toxic concentration of peroxynitrite promotes a rapid necrotic response in U937 cells in which cytosolic phospholipase A(2) is pharmacologically inhibited or genetically depleted. We found that loss of viable cells is appreciable 15 min after addition of peroxynitrite, does not further increase at 30 min and is mediated by mitochondrial permeability transition (MPT). Both MPT and toxicity were prevented by exogenous arachidonic acid (AA). Various experimental approaches produced results consistent with the notion that the AA-dependent protective mechanism takes place 10-15 min after addition of peroxynitrite. The observation that the extent of DNA strand scission induced by peroxynitrite did not vary under conditions of different AA availability suggests that this event is either upstream to mitochondrial dysfunction or irrelevant for cytotoxicity. Collectively, these data indicate that a non-toxic concentration of peroxynitrite commits U937 cells to MTP-dependent necrosis that is however prevented by endogenous AA. Thus, mitochondria are a likely target of the cytoprotective signalling triggered by AA
Low Concentrations of Arsenite Target the Intraluminal Inositol 1, 4, 5-Trisphosphate Receptor/Ryanodine Receptor Crosstalk to Significantly Elevate Intracellular Ca2
Full text linkArsenite is an established human carcinogen inducing cyto- and genotoxic effects through poorly defined mechanisms involving the formation of reactive oxygen species (ROS) and deregulated Ca2+ homeostasis. We used variants of the U937 cell line to address the central issue of the mechanism whereby arsenite affects Ca2+ homeostasis. We found that a 6 h exposure to the metalloid (2.5 μM), while not associated to an immediate or delayed toxicity, causes a significant increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism characterized by the following components: i) it was not affected by ROS produced under the same conditions; ii) a small amount of Ca2+ was mobilized from the inositol-1,4,5-trisphosphate receptor (IP3R). This response was not further augmented by greater concentrations of the metalloid; iii) large amounts of Ca2+ were instead dose-dependently mobilized from the ryanodine receptor (RyR) in response to IP3R stimulation; iv) the cells maintained an intact responsiveness to agonist-stimulated Ca2+ mobilization from both channels; v) arsenite, even at 5-10 μM, failed to directly mobilize Ca2+ from the RyR; vi) arsenite failed to enhance Ca2+ release from the RyR under conditions in which the [Ca2+]i was increased by either RyR agonists or ionophore-stimulated Ca2+ uptake. We therefore conclude that arsenite elevates the [Ca2+]i by directly targeting the IP3R and its intraluminal crosstalk with the RyR. This mechanism likely mediates mitochondrial superoxide formation, downstream damage on various biomolecules, including genomic DNA, and mitochondrial dysfunction/apoptosis eventually occurring after longer incubation to, or exposure to greater concentrations of, arsenite
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