Summary Here we report that Cyclooxygenase-2 (COX-2) mediates the formation of electrophilic fatty acid oxo-derivatives (EFOXs) from the omega-3 fatty acids (ω-3 FA) docosahexaenoic, docosapentaenoic and docosatetraenoic acid. EFOXs produced by activated macrophages were discovered by a mass spectrometry-based “fishing” method, using the nucleophile β-mercaptoethanol (BME) as bait. Intracellular EFOX concentrations ranged from 65 to 350 nM, with acetylsalicylic acid (ASA, aspirin) increasing both rate of production and intracellular concentrations. Due to an electrophilic nature, EFOXs adducted protein cysteine and histidine residues and the cysteine of glutathione (GSH) in activated macrophages. A role for EFOXs as signalling mediators was confirmed by the observations that 17-EFOX-D6 and 17-EFOX-D5 are PPARγ agonists and activate Nrf2-dependent antioxidant responses. They also inhibited cytokine production and inducible nitric oxide synthase (NOS-2) expression in activated macrophages within a biological concentration range. Thus, EFOXs are signalling mediators that can transduce the clinically beneficial effects of ω-3 FA, COX-2 and ASA.

This study reveals the de novo generation of fatty acid nitration products in vivo and reveals the anti-inflammatory and potential therapeutic actions of OA-NO(2) in myocardial I/R injury.

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Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid.

Aerobes require oxygen for metabolism and normal free radical formation. As a result, maintaining the redox homeostasis is essential for brain cell survival due to their high metabolic energy requirement to sustain electrochemical gradients, neurotransmitter release, and membrane lipid stability. Further, brain antioxidant levels are limited compared to other organs and less able to compensate for reactive oxygen and nitrogen species (ROS/RNS) generation which contribute oxidative/nitrative stress (OS/NS). Antioxidant treatments such as vitamin E, minocycline, and resveratrol mediate neuroprotection by prolonging the incidence of or reversing OS and NS conditions. Redox imbalance occurs when the antioxidant capacity is overwhelmed, consequently leading to activation of alternate pathways that remain quiescent under normal conditions. If OS/NS fails to lead to adaptation, tissue damage and injury ensue, resulting in cell death and/or disease. The progression of OS/NS-mediated neurodegeneration along with contributions from microglial activation, dopamine metabolism, and diabetes comprise a detailed interconnected pathway. This review proposes a significant role for OS/NS and more specifically, lipid peroxidation (LPO) and other lipid modifications, by triggering microglial activation to elicit a neuroinflammatory state potentiated by diabetes or abnormal dopamine metabolism. Subsequently, sustained stress in the neuroinflammatory state overwhelms cellular defenses and prompts neurotoxicity resulting in the onset or amplification of brain damage.

The peroxisome proliferator-activated receptor-␥ (PPAR␥) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPAR␥ include lipids and antihyperglycemic drugs such as thiazolidinediones (TZDsThe rapidly expanding global burden of type II diabetes mellitus (DM) 3 and the concomitant increased risk for cardiovascular disease (1, 2) have motivated better understanding of relevant cell signaling pathways and potential therapeutic strategies. One major characteristic of metabolic syndrome and DM is insulin resistance, leading to hyperglycemia and dyslipidemia. Following initial clinical use of TZDs as anti-hyperglycemic agents to treat DM in the late 1990s, the nuclear receptor PPAR␥ was discovered as their molecular target. This receptor is expressed primarily in adipose tissue, muscle, and macrophages, where it regulates glucose uptake, lipid metabolism/ storage, and cell proliferation/differentiation (3-5). Thus, PPAR␥ ligands and allied downstream signaling events play a pivotal role in both the development and treatment of DM (6, 7). This is underscored by the observation that mutations in the C-terminal helix 12 of the ligand-binding domain (LBD) of PPAR␥ (e.g. P467L or V290M) are linked with severe insulin resistance and the onset of juvenile DM (8).The oxidizing inflammatory milieu contributing to the pathogenesis of obesity, diabetes, and cardiovascular disease also promotes diverse biomolecule oxidation, nitrosation, and nitration reactions by O 2 and ⅐ NO-derived species. Although oxidized fatty acids typically propagate proinflammatory conditions, the recently detected class of NO 2 -FA act as anti-inflammatory mediators. Nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) have been detected in healthy human blood and murine cardiac tissue. The levels of free/unesterified OA-NO 2 are ϳ1-3 nM in human plasma (9, 10), with OA-NO 2 produced at increased rates and present at higher concentrations during inflammatory and metabolic stress (11-13). The signaling actions of NO 2 -FA are primarily ascribed to the electrophilic olefinic carbon situated ␤ to the electron-withdrawing NO 2 substituent, facilitating kinetically rapid and reversible Michael addition with nucleophilic amino acids (i.e. Cys and His) (14). NO 2 -FA adduction of proteins and GSH occurs in model systems and clinically, with this reaction

Nitrated derivatives of fatty acids (NO 2 -FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO 2 -FA signal transduction lacks insight into how or if NO 2 -FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO 2 ) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO 2 or metabolites were detected under basal conditions, whereas administered 18:1-NO 2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO 2 -FA are also metabolized via ␤-oxidation, with high performance liquid chromatographytandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO 2 as metabolites. Additionally, a significant proportion of 18:1-NO 2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO 2 hydration or nitric oxide ( ⅐ NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO 2 detected by exchange to added ␤-mercaptoethanol. This, coupled with the observation of 18:1-NO 2 release from glutathione-18:1-NO 2 adducts, supports that reversible and exchangeable NO 2 -FAthiol adducts occur under biological conditions. After administration of [ 3 H]18:1-NO 2 , 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and ␤-oxidation reactions that terminate at the site of acyl-chain nitration.

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury. Keywords: adriamycin-induced chemobrain, central nervous system toxicity, inducible nitric oxide synthase knockout mice, manganese superoxide dismutase, mitochondrial respiration, nitric oxide. Adriamycin (ADR), a prominent member of the anthracycline family, is an important therapeutic agent that has exhibited activity against a wide spectrum of human and experimental animal tumors. It is well established that ADR leads to generation of free radicals, which account for some of the normal tissue damage resulting from cancer treatment (Meredith and Reed 1983;Licinio 1997;Singal et al. 2000). This process may result from the redox cycling capability of anthracycline, its potential to bind nitric oxide (VasquezVivar et al. 1999;Weinstein et al. 2000;Kalivendi et al. 2001;Kalyanaraman et al. 2002) Abbreviations used: ADR, adriamycin; CuZnSOD, copper-zinc superoxide dismutase; DPTA NONOate, dipropylenetriamine NONOate; iNOS, inducible nitric oxide; iNOSKO, mice deficient in the inducible form of nitric oxide; L-NAME, NG-nitro-L-arginine-methyl ester; LPS, lipopolysaccharide; MnSOD, manganese superoxide dismutase; NO, nitric oxide; ONOO -, peroxynitrite; RCR, respiration control ratio; RNS, reactive nitrogen species; RONS, reactive oxygen and nitrogen species; ROS, reactive oxygen species; TNF-a, tumor necrosis factor alpha.