The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O ) and hydrogen peroxide (H₂O₂). The electron transfer chain of mitochondria is a well documented source of H₂O₂; however, the release of O from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O , elucidating the mechanisms underlying the release of O from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O . Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochromec proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O release from mitochondria were estimated at ∼0.04 nmol/min/mg protein, a value increased ∼8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-basedp-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O production from mitochondria by ∼55%, thus suggesting that a large portion of O exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro‐apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O‐methyl‐serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 − but not cathepsins B or D − causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.

Hydrogen peroxide (H₂O₂) has been implicated in the regulation of the transcription factor NF-κB, a key regulator of the inflammatory process and adaptive immunity. However, no consensus exists regarding the regulatory role played by H₂O₂. We discuss how the experimental methodologies used to expose cells to H₂O₂ produce inconsistent results that are difficult to compare, and how the steady-state titration with H₂O₂ emerges as an adequate tool to overcome these problems. The redox targets of H₂O₂ in the NF-κB pathway—from the membrane to the post-translational modifications in both NF-κB and histones in the nucleus—are described. We also review how H₂O₂ acts as a specific regulator at the level of the single gene, and briefly discuss the implications of this regulation for human health in the context of κB polymorphisms. In conclusion, after near 30 years of research, H₂O₂ emerges not as an inducer of NF-κB, but as an agent able to modulate the activation of the NF-κB pathway by other agents. This modulation is generic at the level of the whole pathway but specific at the level of the single gene. Therefore, H₂O₂ is a fine-tuning regulator of NF-κB-dependent processes, as exemplified by its dual regulation of inflammation. Antioxid. Redox Signal. 11, 2223–2243.