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Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.

Diabetic hearts are susceptible to damage from inappropriate activation of the renin angiotensin system (RAS) and hyperglycemic events both of which contribute to increased oxidant production. Prolonged elevation of oxidants impairs mitochondrial enzyme function, further contributing to metabolic derangement. Nuclear factor erythriod-2-related factor 2 (Nrf2) induces antioxidant genes including those for glutathione (GSH) synthesis following translocation to the nucleus. We hypothesized that an acute elevation in glucose impairs Nrf2-related gene expression in diabetic hearts, while AT1 antagonism would aid in Nrf2-mediated antioxidant production and energy replenishment. We used four groups (n = 6-8/group) of 25-week-old rats: 1) LETO (lean strain-control), 2) type II diabetic OLETF, 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d × 8 wks), and 4) ARBM (4 weeks on ARB, 4 weeks off) to study the effects of acutely elevated glucose on cardiac mitochondrial function and Nrf2 signaling in the diabetic heart. Animals were gavaged with a glucose bolus (2 g/kg) and groups were dissected at T0, T180, and T360 minutes. Nrf2 mRNA was 32% lower in OLETF rats compared to LETO and remained suppressed in response to glucose. LETO Nrf2 mRNA increased 25% at T360 in response to glucose while no changes were observed in diabetic hearts. GCLC and GCLM mRNA decreased in diabetic hearts 33% and 44% respectively and remained suppressed in response to glucose while ARB treatment increased GCLM transcripts 90% at T180. These data illustrate that during T2DM and in response to glucose, cardiac Nrf2's adaptive response to environmental stressors such as glucose is impaired in diabetic hearts and that ARB treatment may aid Nrf2's impaired dynamic response.

The expression of the phospholipase A₂ activity (aiPLA₂) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA₂ assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA₂ activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA₂ activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H₂O₂ or the presence of oxidized phospholipid as substrate also significantly increased aiPLA₂ activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA₂ activity at pH 7 without effect on the activity of the enzyme at pH 4.

A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.

A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both the functionality of two hypoxia-response elements (HREs) within the promoter, GPx8 HRE1 and GPx8 HRE2, and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2'-bipyridyl (BP), induces GPx8 expression at both mRNA and protein level. Luciferase activity of pGL3(GPx8wt), containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8 HRE1 within pGL3(GPx8wt) resulted in a significantly higher inhibition of luciferase activity than mutating GPx8 HRE2. Electrophoretic mobility-shift assay showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8 HRE1. In DES-treated cells transfected with pGL3(GPx8wt) or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8 HREs, with GPx8 HRE1 playing the major role. Fibroblast growth factor (FGF) treatment increases GPx8 mRNA expression, and reporter gene experiments indicate that induction occurs via HIF. Comparing the effects of depleting GPx8 on the downstream effectors of FGF or insulin signaling revealed that absence of GPx8 results in a 16- or 12-fold increase in phosphorylated ERK1/2 by FGF or insulin treatment, respectively. Furthermore, in GPx8-depleted cells, phosphorylation of AKT by insulin treatment increases 2.5-fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al. provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of ‘mitoflashes’ as ‘bursts of superoxide’ radicals assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide. This interpretation has been criticised because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide-. We now provide direct evidence that purified cpYFP is completely unresponsive to superoxide. Therefore ‘mitoflashes’ do not reflect superoxide generation and are not evidence for a link between mitochondrial free radical dynamics and lifespan.

We present arguments for an evolution in our understanding of how antioxidants in fruits and vegetables exert their health-protective effects. There is much epidemiological evidence for disease prevention by dietary antioxidants and chemical evidence that such compounds react in one-electron reactions with free radicals in vitro. Nonetheless, kinetic constraints indicate that in vivo scavenging of radicals is ineffective in antioxidant defense. Instead, enzymatic removal of nonradical electrophiles, such as hydroperoxides, in two-electron redox reactions is the major antioxidant mechanism. Furthermore, we propose that a major mechanism of action for nutritional antioxidants is the paradoxical oxidative activation of the Nrf2 (NF-E2-related factor 2) signaling pathway, which maintains protective oxidoreductases and their nucleophilic substrates. This maintenance of "nucleophilic tone," by a mechanism that can be called "para-hormesis," provides a means for regulating physiological nontoxic concentrations of the nonradical oxidant electrophiles that boost antioxidant enzymes, and damage removal and repair systems (for proteins, lipids, and DNA), at the optimal levels consistent with good health.

Abstract There is a large published literature presenting information regarding the toxicology of CO in humans. There is information showing that CO-Hb levels below 15% are not associated with clinical adverse signs or symptoms in healthy individuals and smokers have CO-Hb levels around 6%, but may be above 10% on occasion. It has also been found that CO at low levels may have positive effects in SCD. It has been reported that non-toxic levels of CO extend the red cell life span in SCD patients (Beutler E, 1975.). Further, CO might play a role in preventing sickle cell formation by preventing the polymerization of hemoglobin (Sirs JA, 1963), and it has been reported that CO might have a preventative effect on the occurrence of clinical symptoms of SCD (Yallop D et al, 2007). Inhaled CO administered in low concentrations to transgenic sickle mice for one hour per day, 3 days per week for 10 weeks, was non-toxic and substantially decreased white cell counts and cellular inflammatory signals (Beckman J et al, 2009). This research program hypothesizes that CO, when administered orally at non-toxic levels, results in CO-Hb levels that will result in benefit to patients with SCD through two mechanisms: reducing the sickling phenomenon of red cells and by reducing the inflammatory response in these patients. It also hypothesizes that the safest and most efficient mode of administration of CO is by the oral route via a solution with a known dose of CO. Methods The carbon monoxide (CO) is obtained from a commercial source. The liquid formulation (LF) is composed of a mixture of the following components: water, proteins, lipids and carbohydrates ( PLC Mixture). The CO is instilled into this LF and tested for CO concentration. The principle of the method used for CO measurement is to allow the CO and added Hb to interact and to measure the CO-Hb formed by using the dithionite reduction method employing a spectrophotometer. (A.H. Chalmers 1991).The container is weighed before and after CO instillation to determine the occurrence of CO gas leakage. The container for the CO-LF is of low gas permeability. Once CO instillation in the LF is complete, the containers of CO-LF are sealed and labeled. The prepared containers are stored at 2-8°C for no longer than 72 hours. The CO content in blood is expressed as ‘%’ of total hemoglobin (CO-Hb) and mg/L in the CO-LF. CO-LF was administered via esophageal-placed gavage using Sprague-Dawley rats with jugular venous catheters. Results In the first study, there were 5 groups of 4 non-anemic rats studied, with each group comprising of a different LF made with the following percent of PLC Mixture combined with water: 50% PLC Mixture, 20% PLC Mixture, 10% PLC Mixture, 5% PLC Mixture, and 0% PLC Mixture (water only). Two 3 mL doses of CO-LF were administered to each rat one hour apart. Data generated showed levels of CO-Hb at baseline varying between 0 and 1.1 %. After the administration of the test article, the highest CO-Hb levels were 6.1% in one animal at 1 hour after the first dose, and 9.6% one hour after the second dose in one animal. The levels of CO-Hb fell at different rates depending upon the dose and formulation. The PK data for the first study is shown in Table 1. Differences among the test articles were not statistically different. The PK parameters evaluated over 24 hours were not impacted by the administered PLC Mixture, but animals receiving CO-50% PLC Mixture showed an initial fall to baseline levels, then a second rise in CO-Hb levels peaking in one animal at 3.1% at 48 hours. Such late increments were not seen with other CO-LF’s. The second PK study was comprised of 2 groups of 4 anemic rates, and employed CO-0% and CO-10% PLC Mixture. This study was carried out on rats made anemic by blood draw to an estimated 50% blood volume. The PK characteristics show data similar to that seen in non-anemic rats. Conclusion This research has documented the feasibility in a proof-of-concept rat model that orally administered CO via a CO liquid formulation can produce CO-Hb levels that would be expected to impact the pathophysiology of the Sickle Cell Disease-associated micro-vasculature. The terminal half-life of orally administered CO in the rat is similar to that reported via inhaled CO. Disclosures: Gomperts: Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties. Forman:Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties.

BACKGROUND: Rodent models for urban air pollution show consistent induction of inflammatory responses in major brain regions. However, the initial impact of air pollution particulate material on olfactory gateways has not been reported. OBJECTIVE: We evaluated the olfactory neuroepithelium (OE) and brain regional responses to a nanosized subfraction of urban traffic ultrafine particulate matter (nPM, < 200 nm) in vivo, ex vivo, and in vitro. METHODS: Adult mice were exposed to reaerosolized nPM for 5, 20, and 45 cumulative hours over 3 weeks. The OE, the olfactory bulb (OB), the cerebral cortex, and the cerebellum were analyzed for oxidative stress and inflammatory responses. Acute responses of the OE to liquid nPM suspensions were studied with ex vivo and primary OE cultures. RESULTS: After exposure to nPM, the OE and OB had rapid increases of 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosine (3-NT) protein adducts, whereas the cerebral cortex and cerebellum did not respond at any time. All brain regions showed increased levels of tumor necrosis factor-α (TNFα) protein by 45 hr, with earlier induction of TNFα mRNA in OE and OB. These responses corresponded to in vitro OE and mixed glial responses, with rapid induction of nitrite and inducible nitric oxide synthase (iNOS), followed by induction of TNFα. CONCLUSIONS: These findings show the differential time course of oxidative stress and inflammatory responses to nPM between the OE and the brain. Slow cumulative transport of inhaled nPM into the brain may contribute to delayed responses of proximal and distal brain regions, with potential input from systemic factors. http://dx.doi.org/10.1289/EHP134, Introduction The brain has emerged as a target of air pollution. Population-based studies have shown that cognitive impairments increase in proportion to levels of [PM.sub.2.5] (Ailshire and Crimmins 2014; Gatto [...]