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Bilirubin, a byproduct of heme catabolism, has been shown to be an effective lipid-soluble antioxidant in vitro. Bilirubin is able to inhibit free radical chain reactions and protects against oxidant-induced damage in vitro and ex vivo. However, direct evidence for bilirubin's antioxidant effects in vivo remains limited. As bilirubin is formed from biliverdin by biliverdin reductase, we generated global biliverdin reductase-a gene knockout (Bvra -/- ) mice to assess the contribution of bilirubin as an endogenous antioxidant. Bvra -/- mice appear normal and are born at the expected Mendelian ratio from Bvra +/- x Bvra +/- matings. Compared with corresponding littermate Bvra +/+ and Bvra +/- animals, Bvra -/- mice have green gall bladders and their plasma concentrations of biliverdin and bilirubin are approximately 25-fold higher and 100-fold lower, respectively. Naïve Bvra -/- and Bvra +/+ mice have comparable plasma lipid profiles and low-molecular weight antioxidants, i.e., ascorbic acid, α-tocopherol and ubiquinol-9. Compared with wild-type littermates, however, plasma from Bvra -/- mice contains higher concentrations of cholesteryl ester hydroperoxides (CE-OOH), and their peroxiredoxin 2 (Prx2) in erythrocytes is more oxidized as assessed by the extent of Prx2 dimerization. These data show that Bvra -/- mice experience higher oxidative stress in blood, implying that plasma bilirubin attenuates endogenous oxidative stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.

Secondary degeneration contributes substantially to structural and functional deficits following traumatic injury to the CNS. While it has been proposed that oxidative stress is a feature of secondary degeneration, contributing reactive species and resultant oxidized products have not been clearly identified in vivo. The study is designed to identify contributors to, and consequences of, oxidative stress in a white matter tract vulnerable to secondary degeneration. Partial dorsal transection of the optic nerve (ON) was used to model secondary degeneration in ventral nerve unaffected by the primary injury. Reactive species were assessed using fluorescent labelling and liquid chromatography/tandem mass spectroscopy (LC/MS/MS). Antioxidant enzymes and oxidized products were semi-quantified immunohistochemically. Mitophagy was assessed by electron microscopy. Fluorescent indicators of reactive oxygen and/or nitrogen species increased at 1, 3 and 7days after injury, in ventral ON. LC/MS/MS confirmed increases in reactive species linked to infiltrating microglia/macrophages in dorsal ON. Similarly, immunoreactivity for glutathione peroxidase and haem oxygenase-1 increased in ventral ON at 3 and 7days after injury, respectively. Despite increased antioxidant immunoreactivity, DNA oxidation was evident from 1day, lipid oxidation at 3days, and protein nitration at 7days after injury. Nitrosative and oxidative damage was particularly evident in CC1-positive oligodendrocytes, at times after injury at which structural abnormalities of the Node of Ranvier/paranode complex have been reported. The incidence of mitochondrial autophagic profiles was also significantly increased from 3days. Despite modest increases in antioxidant enzymes, increased reactive species are accompanied by oxidative and nitrosative damage to DNA, lipid and protein, associated with increasing abnormal mitochondria, which together may contribute to the deficits of secondary degeneration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Abstract: NADPH oxidases (NOX) are superoxide anion radical (O2 −•)-generating enzymes. They form a family of seven members, each with a specific tissue distribution. They function as electron transport chains across membranes, using NADPH as electron donor to reduce molecular oxygen to O2 −•. NOX have multiple biological functions, ranging from host defense to inflammation and cellular signaling. Measuring NOX activity is crucial in understanding the roles of these enzymes in physiology and pathology. Many of the methods used to measure NOX activity are based on the detection of small molecules that react with NOX-generated O2 −• or its direct dismutation product hydrogen peroxide (H2O2) to form fluorescent, luminescent, or colored products. Initial techniques were developed to measure the activity of the phagocyte isoform NOX2 during the oxidative burst of stimulated polymorphonuclear leukocytes, which generate large quantities of O2 −•. However, other members of the NOX family generate much less O2 −• and hence H2O2, and their activity is difficult to distinguish from other sources of these reactive species. In addition, O2 −• and H2O2 are reactive molecules and most probes are prone to artifacts and therefore should be used with appropriate controls and the data carefully interpreted. This review gives an overview of current methods used to measure NOX activity and NOX-derived O2 −• and H2O2 in cells, tissues, isolated systems, and living organisms, describing the advantages and caveats of many established methods with emphasis on more recent technologies and future perspectives. [Copyright &y& Elsevier]

Significance: Oxidative stress is suggested to be a disease mechanism common to a wide range of disorders affecting human health. However, so far, the pharmacotherapeutic exploitation of this, for example, based on chemical scavenging of pro-oxidant molecules, has been unsuccessful. Recent Advances: An alternative emerging approach is to target the enzymatic sources of disease-relevant oxidative stress. Several such enzymes and isoforms have been identified and linked to different pathologies. For some targets, the respective pharmacology is quite advanced, that is, up to late-stage clinical development or even on the market; for others, drugs are already in clinical use, although not for indications based on oxidative stress, and repurposing seems to be a viable option. Critical Issues: For all other targets, reliable preclinical validation and drug ability are key factors for any translation into the clinic. In this study, specific pharmacological agents with optimal pharmacokinetic profiles are still lacking. Moreover, these enzymes also serve largely unknown physiological functions and their inhibition may lead to unwanted side effects. Future Directions: The current promising data based on new targets, drugs, and drug repurposing are mainly a result of academic efforts. With the availability of optimized compounds and coordinated efforts from academia and industry scientists, unambiguous validation and translation into proof-of-principle studies seem achievable in the very near future, possibly leading towards a new era of redox medicine. Antioxid. Redox Signal. 23, 1113-1129. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1 G93A ALS mouse model. A substantial (10–60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1 G93A mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1 G93A mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1 G93A mice in vivo . Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1 G93A mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Significance: Enhanced levels of reactive oxygen species (ROS) have been associated with different disease states. Most attempts to validate and exploit these associations by chronic antioxidant therapies have provided disappointing results. Hence, the clinical relevance of ROS is still largely unclear. Recent Advances: We are now beginning to understand the reasons for these failures, which reside in the many important physiological roles of ROS in cell signaling. To exploit ROS therapeutically, it would be essential to define and treat the disease-relevant ROS at the right moment and leave physiological ROS formation intact. This breakthrough seems now within reach. Critical Issues: Rather than antioxidants, a new generation of protein targets for classical pharmacological agents includes ROS-forming or toxifying enzymes or proteins that are oxidatively damaged and can be functionally repaired. Future Directions: Linking these target proteins in future to specific disease states and providing in each case proof of principle will be essential for translating the oxidative stress concept into the clinic. Antioxid. Redox Signal. 23, 1171-1185. [ABSTRACT FROM AUTHOR]