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.

The clinical use of anthracyclines like doxorubicin and daunorubicin can be viewed as a sort of double-edged sword. On the one hand, anthracyclines play an undisputed key role in the treatment of many neoplastic diseases; on the other hand, chronic administration of anthracyclines induces cardiomyopathy and congestive heart failure usually refractory to common medications. Second-generation analogs like epirubicin or idarubicin exhibit improvements in their therapeutic index, but the risk of inducing cardiomyopathy is not abated. It is because of their janus behavior (activity in tumors vis-a-vis toxicity in cardiomyocytes) that anthracyclines continue to attract the interest of preclinical and clinical investigations despite their longer-than-40-year record of longevity. Here we review recent progresses that may serve as a framework for reappraising the activity and toxicity of anthracyclines on basic and clinical pharmacology grounds. We review 1) new aspects of anthracycline-induced DNA damage in cancer cells; 2) the role of iron and free radicals as causative factors of apoptosis or other forms of cardiac damage; 3) molecular mechanisms of cardiotoxic synergism between anthracyclines and other anticancer agents; 4) the pharmacologic rationale and clinical recommendations for using cardioprotectants while not interfering with tumor response; 5) the development of tumor-targeted anthracycline formulations; and 6) the designing of third-generation analogs and their assessment in preclinical or clinical settings. An overview of these issues confirms that anthracyclines remain "evergreen" drugs with broad clinical indications but have still an improvable therapeutic index.

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Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

Radicals are species containing one or more unpaired electrons. The oxygen radical superoxide (O 2 - ) and the non-radical oxidants hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are produced during normal metabolism and perform several useful functions. Excessive production of O 2 - and H2O2 can result in tissue damage, which often involves generation of highly reactive hydroxy 1 radical (· OH) and other oxidants in the presence of “catalytic” iron or copper ions. A major form of antioxidant defence is the storage and transport of iron or copper ions in forms that will not catalyze formation of reactive radicals. Tissue injury, e. g., by ischaemia or trauma, can cause increased iron availability and accelerate free radical reactions. This may be especially important in the brain, since areas of this organ are rich in iron and cerebrospinal fluid cannot bind released iron ions. Oxidative stress upon nervous tissue can produce damage by several interacting mechanisms, including rises in intracellular free Ca2+ and, possibly, release of excitatory amino acids. Recent suggestions that free radical reactions are involved in the neurotoxicity of aluminium and in damage to the substantia nigra in Parkinson’s disease are reviewed. Finally, the nature of antioxidants is discussed, with a suggestion that antioxidant enzymes and chelators of iron ions may be more generally useful protective agents than chain-breaking antioxidants. Careful precautions must be taken in the design of antioxidants for therapeutic use.

Reactive Oxygen Species and the Central Nervous System

1. A new method has been developed for measuring the total antioxidant capacity of body fluids and drug solutions, based on the absorbance of the ABTS.+ radical cation. 2. An automated method for use on a centrifugal analyser, as well as a manual method, is described. 3. The procedure has been applied to physiological antioxidant compounds and radical-scavenging drugs, and an antioxidant ranking was established based on their reactivity relative to a 1.0 mmol/l Trolox standard. 4. The Trolox equivalent antioxidant capacity of plasma from an adult reference population has been measured, and the method optimized and validated. 5. The method has been applied to investigate the total plasma antioxidant capacity of neonates and how this may be compromised in prematurity.

A Novel Method for Measuring Antioxidant Capacity and its Application to Monitoring the Antioxidant Status in Premature Neonates

Generation of free radicals and lipid peroxidation by redox cycling of adriamycin and daunomycin.

ion. The tendency to cyclize to compounds bearing five-membered rings also requires that a nonconjugated 1T system reside [3-"'{ to the peroxy radical (67, 68). Hence, such reactions require that the fatty acid possess three or more unsaturated bonds. The lower yields of internal hydroperoxide isomers of linoleic acid, for example, has been ascribed to the preferred cyclization of the 12and 13-

Mechanisms and Consequences of Lipid Peroxidation in Biological Systems

Generation of hydrogen peroxide in erythrocytes by hemolytic agents.

We briefly review current concepts with regard to the nature of oxygen-derived oxidants in biological systems. Of these substances, hydroxyl radicals derived from hydrogen peroxide seem most likely to be involved in the various stages of carcinogenesis. Hydrogen peroxide detoxification, primarily through glutathione activity, is essential in preventing hydroxyl-radical formation. Transittion metals such as iron play a central role in this latter process. Alternations in cellular macromolecules are most likely to take place if hydroxyl-radical formation is directed toward specific intranmolecular sites by appropriately sequestered metals. For this reason, repair and turnover events are apt to be more important protective devices than are the actions of molecules which scavenge hydroxyl radicals. Although many cellular constituents are potential targets in free-radical and oxidant attacks leading to carcinogenesis, nucleic acids have been most extensively studied in this connection. On the basis of these investigations, it is a facile conclusion that oxidants might be involved in the early events of carcinogenesis as well as in transformation or promotion. The literature on antioxidants in chemoprevention in animals is supportive of such a role. However, other biochemical effects of antioxidants should raise a note of caution in the interpretation of animal experiments.

The nature of oxidants and antioxidant systems in the inhibition of mutation and cancer

Publisher Summary DT-diaphorase is a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzes the oxidation of nicotine adenine dinucleotide, reduced nicotine adenine dinucleotide hydrogenase (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH) at equal rates This chapter discusses the purification, properties, and function of DT-diaphorase Most of the present knowledge about DT-diaphorase has been obtained in studies of the cytosolic enzyme, which was first purified from both rat and beef liver 44 by employing conventional methods The introduction of affinity chromatography for the purification of DTdiaphorase reduced the number of purification steps considerably In the method presented in the chapter for the purification of DT-diaphorase, the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, which is a potent competitive inhibitor of the enzyme with respect to NAD(P)H A gel with both a high affinity and a high binding capacity for DT-diaphorase is obtained by coupling dicoumarol through an azo linkage to divinyl sulfonate (DVS)-activated Sepharose 6B In the assay, DT-diaphorase activity is measured routinely with NADH or NADPH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) or 2-methyl-l,4-naphthoquinone (menadione) as the electron acceptor The standard assay system contains 50 m M Tris-HC1, pH 75, 008% Triton X-100, 05 m M NADH or NADPH, and 40 μM DCPIP or 10 μM menadione

Paul Hochstein

Papers

Generation of free radicals and lipid peroxidation by redox cycling of adriamycin and daunomycin.

Mechanisms and Consequences of Lipid Peroxidation in Biological Systems

Generation of hydrogen peroxide in erythrocytes by hemolytic agents.

The nature of oxidants and antioxidant systems in the inhibition of mutation and cancer

[30] DT-diaphorase : purification, properties, and function