Kinetics Of Reactive Oxygen Species Production Research Articles

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

Computational Modeling Analysis of Generation of Reactive Oxygen Species by Mitochondrial Assembled and Disintegrated Complex II.

Reactive oxygen species (ROS) function as critical mediators in a broad range of cellular signaling processes. The mitochondrial electron transport chain is one of the major contributors to ROS formation in most cells. Increasing evidence indicates that the respiratory Complex II (CII) can be the predominant ROS generator under certain conditions. A computational, mechanistic model of electron transfer and ROS formation in CII was developed in the present study to facilitate quantitative analysis of mitochondrial ROS production. The model was calibrated by fitting the computer simulated results to experimental data obtained on submitochondrial particles (SMP) prepared from bovine and rat heart mitochondria upon inhibition of the ubiquinone (Q)-binding site by atpenin A5 (AA5) and Complex III by myxothiazol, respectively. The model predicts that only reduced flavin adenine dinucleotide (FADH2) in the unoccupied dicarboxylate state and flavin semiquinone radical (FADH•) feature the experimentally observed bell-shaped dependence of the rate of ROS production on the succinate concentration upon inhibition of respiratory Complex III (CIII) or Q-binding site of CII, i.e., suppression of succinate-Q reductase (SQR) activity. The other redox centers of CII such as Fe-S clusters and Q-binding site have a hyperbolic dependence of ROS formation on the succinate concentration with very small maximal rate under any condition and cannot be considered as substantial ROS generators in CII. Computer simulation results show that CII disintegration (which results in dissociation of the hydrophilic SDHA/SDHB subunits from the inner membrane to the mitochondrial matrix) causes crucial changes in the kinetics of ROS production by CII that are qualitatively and quantitatively close to changes in the kinetics of ROS production by assembled CII upon inhibition of CIII or Q-binding site of CII. Thus, the main conclusions from the present computational modeling study are the following: (i) the impairment of the SQR activity of CII resulting from inhibition of CIII or Q-binding site of CII and (ii) CII disintegration causes a transition in the succinate-dependence of ROS production from a small-amplitude sigmoid (hyperbolic) shape, determined by Q-binding site or [3Fe-4S] cluster to a high-amplitude bell-shaped kinetics with a shift to small subsaturated concentrations of succinate, determined by the flavin site.

Kinetics of ROS generation induced by polycyclic aromatic hydrocarbons and organic extracts from ambient air particulate matter in model human lung cell lines

Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min–24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods.

Study of the Photodynamic Effect on the A549 Cell Line by Atomic Force Microscopy and the Influence of Green Tea Extract on the Production of Reactive Oxygen Species

We studied the morphology of the A549 cell line (human lung carcinoma cells) before and after photodynamic therapy (PDT) by atomic force microscopy. PDT was induced by an efficient light-emitting diode source with total light dose of 15 J cm(-2) in the presence of the sensitizer zinc-5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrine. In the presence of molecular oxygen, light activation of the photosensitizer, which accumulates in cancer cells, leads to the local production of reactive oxygen species (ROS). This is one of several reasons leading to cell death, and in some cases we could observe signs of apoptosis. We detected the kinetics of ROS production to be dependent on the presence of green tea extract.

Transcriptional activation of the gp91phoxNADPH oxidase subunit by TPA in HL-60 cells

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

Neuregulin induces sustained reactive oxygen species generation to mediate neuronal differentiation.

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate NRG-induced differentiation through the MAPK signaling pathway. Here we demonstrate that NRG induces an increase in the intracellular concentration of reactive oxygen species (ROS). N-acetylcysteine, a ROS scavenger, inhibited NRG-induced activation of Ras and Erk and PC12-ErbB-4 cell differentiation. These results suggest that ROS production is involved in NRG-mediated neuronal differentiation and that ROS can regulate activation of Ras and Erk. Constitutively active Ras enhanced ROS production and dominant negative Ras inhibited NRG-induced ROS production, suggesting, a positive regulatory loop between Ras and ROS. The mitogen, EGF, induced short-term ROS production whereas NRG and NGF, which induce cell differentiation, induced prolonged ROS production. These results strongly suggest that the kinetics of ROS production may determine whether the cells will differentiate or proliferate.