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.

A comparison of the actions of ethanol and acetaldehyde on glucose production from various precursors by isolated rate liver cells was made to evaluate the role of acetaldehyde in the effects of ethanol. Both ethanol and acetaldehyde stimulated glucose production from pyruvate, inhibited glucose production from glycerol, xylitol, and sorbitol, and both had no effect with fructose. Ethanol, but not acetaldehyde, inhibited glucose production from lactate, dihydroxyacetone and alanine. The inhibitory effects of ethanol and acetaldehyde were prevented by methylene blue, an aritificial electron acceptor. The similarities in the actions of ethanol and acetaldehyde on glucose production from some substrates, e.g. pyruvate, glycerol, xylitol and sorbitol suggest that the metabolism of acetaldehyde may contribute to the effects of ethanol on gluconeogenesis from these substrates. Differences in the actions of ethanol and acetaldehyde on glucose production from other substrates reflect the different compartments, cytosol for ethanol, mitochondrial for acetaldehyde, in which these compounds are metabolized in the liver.

A comparison of the effects of ethanol and acetaldehyde on glucose production from various precursors by isolated rat liver cells.

There is current interest in the toxicity associated with formaldehyde and its role in biological systems in view of its increasing use. The oxidation of formaldehyde to formate proceeds mainly via two pathways, one involving the low K , mitochondria1 aldehyde dehydrogenase and the other a glutathione-dependent cytosolic aldehyde dehydrogenase, specific for formaldehyde.' Previous experimen t~~ ' showed that oxidation of exogenous formaldehyde by the glutathionedependent formaldehyde dehydrogenase was not affected by cyanamide, a potent inhibitor of aldehyde dehydrogenase. By contrast, cyanamide inhibited the oxidation of formaldehyde by rat liver mitochondria by 75 to 90%. In isolated hepatocytes, cyanamide inhibited the uptake of formaldehyde as well as the production of C02 and formate by approximately 50%. These results indicated that formaldehyde can be oxidized by cyanamide-sensitive, presumably low K, aldehyde dehydrogenase, and cyanamide-insensitive, formaldehyde dehydrogenase reactions. The experiments described in this report were carried out to investigate the metabolism of formaldehyde generated by the oxidative demethylation of aminopyrine by the cytochrome P-450-dependent monooxygenase system. Measurements of ''TO2 production after administration of labeled aminopyrine to humans or animals is used as a liver function test4 and applied for the diagnosis of liver diseases.s

Pathways of Metabolism of Formaldehyde Produced from the Oxidation of Aminopyrine by Intact Rat Hepatocytes

Pyrazole, and particularly pyrazole derivatives with a substitution on the four position, for example, 4-methylpyrazole (4-MP), are powerful inhibitors of alcohol dehydrogenase and of ethanol metabolism and are widely used in the alcohol field.’ However, 4-methylpyrazole blocks cytochrome P-45Mependent oxidation of drugs, for example, aminopyrine and ethanol,2 although the mechanism for this is not known. Results to be reported below indicate that 4-MP interacts with cytochrome P-450, induces cytochrome P-450, and can alter the metabolism of alcohols and aminopyrine by microsomes. Male Sprague-Dawley rats, weighing about 135 to 150 g, were injected i.p. either with saline or varying doses of 4-MP for one, two or three days. Rats were fasted overnight and microsomes prepared 24 hours after the last injection. The various assays were performed by previously described methods.’.‘ The content of cytochrome P-450 was 0.79 nmol per mg microsomal protein for saline controls and 1 .O, 1.5 1, and 1.54 nmol per mg protein after treating rats with 200 mg per kg body wt of 4-MP for one, two or three days, respectively. However, 4-MP-treatment did not change the activity of NADPH-cytochrome c reductase. Associated with this increase in content of P-450 was an increase in the microsomal oxidation of alcohols; for example, the oxidation of ethanol was increased by 73, 74, and 139%. while the oxidation of 2-butanol was increased 86,136, and 188% after one, two or three days of treatment, respectively, with 4-MP. The oxidation of aminopyrine was only slightly increased (5, 17, and 52%) after one, two or three days of 4-MP treatment. TABLE 1 shows a dose-response curve for the effect of three days of treatment with 4-MP on content of P-450 and microsomal oxidation of substrates. Some effects could be noticed even at a dose of 50 mg 4-MP per kg body wt, and these effects increased as the dose increased. However, the increase in oxidation of alcohols and aminopyrine appeared to reflect the increase in total content of P-450 induced by 4-MP. When the results were expressed as product formation per nmole P-450 (rather than per mg microsomal protein), there was little or no increase in oxidation of alcohols or aminopyrine (TABLE 1, Exp. B). Dimethylsulfoxide (DMSO) and 2-butanol were previously shown to produce type 11 binding spectra with microsomes from ethanolor pyrazole-treated rats, but not control^.^***^ FIGURE 1A shows that DMSO produces a type I1 spectrum (peak at 418-420 nm, trough at 380-385 nm) with microsomes from 4-MP-treated rats, but not saline controls. 2-Butanol produced a modified type I spectrum with 4-MP microsomes (peak at 408-410 nm, trough at 426-428 nm) but not with controls. FIGURE 1C shows that 4-MP itself interacted with P-450 to produce a type I1 binding spectrum (peak at 429 nm, trough at 392 nm). This interaction could be observed in

Increased Content of Cytochrome P‐450 and Microsomal Alcohol Oxidation after 4‐Methylpyrazole Treatment

370 S-adenosyl--methionine represses the responsiveness of the alpha 2(I) collagen promoter to CCL in transgenic mice

There is increasing evidence for the involvement of oxygen-free radicals in the toxicity associated with paraquat and several quinones.14 Studies were carried out to evaluate the effect of paraquat and menadione (as a representative quinone) on the production of hydroxyl radicals ('OH) by microsomes. Microsomal production of ethylene from 10 m M 2-keto-4-thiomethylbutyric acid (KMBA) was used as a measure of 'OH generation. Aminopyrine demethylase activity was assayed to reflect classical mixed-function oxidase activity. The oxidation of alcohols was also assayed since alcohols can be oxidized by a cytochrome P-450 dependent, or in the presence of iron-EDTA, by a 'OH-dependent pathway.'*6 All assays were carried out as previously described.'\

Arthur I. Cederbaum

Papers

A comparison of the effects of ethanol and acetaldehyde on glucose production from various precursors by isolated rat liver cells.

Pathways of Metabolism of Formaldehyde Produced from the Oxidation of Aminopyrine by Intact Rat Hepatocytes

Increased Content of Cytochrome P‐450 and Microsomal Alcohol Oxidation after 4‐Methylpyrazole Treatment

370 S-adenosyl--methionine represses the responsiveness of the alpha 2(I) collagen promoter to CCL in transgenic mice

Microsomal Interactions between Iron, Paraquat, and Menadione on Hydroxyl Radical Formation, Alcohol Oxidation, and Drug Metabolism