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Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging. Nuclear DNA damage downstream of mitochondrial ROS is often cited to contribute to cancer initiation and aging. However, here the authors show that although H2O2 induces DNA mutations when produced near DNA, it does not when released by mitochondria. [ABSTRACT FROM AUTHOR]

Reactive Oxygen Species (ROS) in the form of H 2 O 2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H 2 O 2 were often studied making use of exogenously added H 2 O 2 , generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H 2 O 2 production by for instance mitochondrial respiration. The enzyme d -Amino Acid Oxidase (DAAO) catalyzes H 2 O 2 formation using d -amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H 2 O 2. However, a method to directly quantify the amount of H 2 O 2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H 2 O 2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H 2 O 2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H 2 O 2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d -Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H 2 O 2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H 2 O 2 production to be able to discriminate the effects of H 2 O 2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward. [Display omitted] • Oxygen consumption rate (OCR) can serve to quantify chemogenetic H 2 O 2 production. • Cellular d -amino acid uptake is a rate-limiting step in DAAO-dependent H 2 O 2 production. • The lethal rate of H 2 O 2 production depends on the subcellular localization of DAAO. • Media Glucose concentration is a key determinant of HyPer7 reduction rates. [ABSTRACT FROM AUTHOR]

Reactive oxygen species are required for cell proliferation but can also induce apoptosis[1]. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis[2]. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans[3-6], and we conclude that this model also extends to mammalian systems. [ABSTRACT FROM AUTHOR]

Stabilization and activation of the p53 tumor suppressor are triggered in response to various cellular stresses, including DNA damaging agents and elevated Reactive Oxygen Species (ROS) like H 2 O 2. When cells are exposed to exogenously added H 2 O 2 , ATR/CHK1 and ATM/CHK2 dependent DNA damage signaling is switched on, suggesting that H 2 O 2 induces both single and double strand breaks. These collective observations have resulted in the widely accepted model that oxidizing conditions lead to DNA damage that subsequently mediates a p53-dependent response like cell cycle arrest and apoptosis. However, H 2 O 2 also induces signaling through stress-activated kinases (SAPK, e.g., JNK and p38 MAPK) that can activate p53. Here we dissect to what extent these pathways contribute to functional activation of p53 in response to oxidizing conditions. Collectively, our data suggest that p53 can be activated both by SAPK signaling and the DDR independently of each other, and which of these pathways is activated depends on the type of oxidant used. This implies that it could in principle be possible to modulate oxidative signaling to stimulate p53 without inducing collateral DNA damage, thereby limiting mutation accumulation in both healthy and tumor tissues. [Display omitted] • Oxidizing conditions can stabilize and activate p53 independent of DNA damage signaling. • Oxidant signaling stablizes p53 through inhibition of protein ubiquitinylation and degradation. • p38 MAPK is required for oxidant signaling-mediated p53 activation. • Both DNA damage signaling and oxidant signaling induces p53-dependent transcription and apoptosis. [ABSTRACT FROM AUTHOR]