Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.

https://www.pnas.org/content/pnas/87/4/1620.full.pdf

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.

For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

/pdf/the-nox-family-of-ros-generating-nadph-oxidases-physiology-zjc88zvh59.pdf

The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

/pdf/nitric-oxide-and-peroxynitrite-in-health-and-disease-3uuuqgfdcz.pdf

Nitric Oxide and Peroxynitrite in Health and Disease

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.20957

Alcohol and oxidative liver injury

This review describes some of the biochemical and toxicological properties of CYP2E1, especially as it relates to alcohol metabolism and toxicity and the establishment of human hepatoma HepG2 cell lines that overexpress human CYP2E1. Ethanol, polyunsaturated fatty acids, and iron were found to be cytotoxic in HepG2 cells that overexpress CYP2E1. GSH appears to be essential in protecting HepG2 cells against the CYP2E1-dependent cytotoxicity, and GSH levels were elevated owing to a twofold increase in activity and expression of glutamate cysteine ligase. We suggest that this up-regulation of GSH synthesis was an adaptive response to attenuate CYP2E1-dependent oxidative stress and toxicity. Induction of a state of oxidative stress appears to play a central role in the CYP2E1-dependent cytotoxicity. Mitochondrial membrane potential decreased in the CYP2E1-expressing HepG2 cells, and this decrease shared similar characteristics with the developing toxicity. Alcohol-dependent liver injury is likely to be a multifactorial process involving several mechanisms. We believe that the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, and GSH homeostasis contribute to the toxic actions of ethanol on the liver.

Oxidative Stress, Toxicology, and Pharmacology of CYP2E1*

Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical-generating systems

Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in small amounts during the body's metabolic reactions and can react with and damage complex cellular molecules such as lipids, proteins, or DNA. This review describes pathways involved in ROS formation, why ROS are toxic to cells, and how the liver protects itself against ROS. Acute and chronic ethanol treatment increases the production of ROS, lowers cellular antioxidant levels, and enhances oxidative stress in many tissues, especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury.

Oxidative stress and alcoholic liver disease.

Fatty livers of obese fa/fa rats are vulnerable to injury when challenged by insults such as endotoxin, ischemia-reperfusion or acute ethanol treatment. The objective of this study was to evaluate whether a high-fat diet can act as a "second hit" and cause progression to liver injury in obese fa/fa rats compared with lean Fa/? rats. Accordingly, obese fa/fa rats and their lean littermates were fed a diet low in fat (12% of total calories) or a diet with 60% calories as lard for 8 weeks. Hyperglycemia and steatohepatitis occurred in the fa/fa rats fed the high-fat diet. This was accompanied by liver injury as assessed by alanine aminotransferase, hematoxilin and eosin staining, increased TNFalpha and stellate cell-derived TGFbeta, collagen deposition, and up-regulation of alpha-smooth muscle actin. Active MMP13 decreased in fa/fa rats independently of the diet, and TIMP1 expression increased with the high-fat diet, especially in fa/fa rats. Although UCP2 expression was higher in fa/fa rats regardless of the diet, minor changes in ATP levels were observed. Oxidative stress occurred in the fa/fa rats fed the high-fat diet as lipid peroxidation and protein carbonyls were elevated, while glutathione and antioxidant enzymes were very low. Expression and activity of cytochrome P450 2E1 and xanthine oxidase activity were down-regulated in fa/fa compared with Fa/? rats, and no effect was seen by the high-fat diet. However, NADPH oxidase activity increased 2.5-fold in fa/fa rats fed with the high-fat diet. In summary, a high-fat diet induces liver injury in fa/fa rats leading to periportal fibrosis. A role for oxidative stress is suggested via increased NADPH oxidase activity, lipid peroxidation, protein carbonyl formation, and low antioxidant defense.

Arthur I. Cederbaum

Papers

Alcohol and oxidative liver injury

Oxidative Stress, Toxicology, and Pharmacology of CYP2E1*

Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical-generating systems

Oxidative stress and alcoholic liver disease.

A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats