The effect of dichloroacetate on health- and lifespan in C. elegans.

Aging is associated with increased vulnerability to chronic, degenerative diseases and death. Strategies for promoting healthspan without necessarily affecting lifespan or aging rate have gained much interest. The mitochondrial free radical theory of aging suggests that mitochondria and, in particular, age-dependent mitochondrial decline play a central role in aging, making compounds that affect mitochondrial function a possible strategy for the modulation of healthspan and possibly the aging rate. Here we tested such a "metabolic tuning" approach in nematodes using the mitochondrial modulator dichloroacetate (DCA). We explored DCA as a proof-of-principle compound to alter mitochondrial parameters in wild-type animals and tested whether this approach is suitable for reducing reactive oxygen species (ROS) production and for improving organismal health- and lifespan. In parallel, we addressed the potential problem of operator bias by running both unblinded and blinded lifespan studies. We found that DCA treatment (1) increased ATP levels without elevating oxidative protein damage and (2) reduced ROS production in adult C. elegans. DCA treatment also significantly prolonged nematode health- and lifespan, but did not strongly impact mortality doubling time. Operator blinding resulted in considerably smaller lifespan-extending effects of DCA. Our data illustrate the promise of a "metabolic tuning" intervention strategy, emphasize the importance of mitochondria in nematode aging and highlight operator bias as a potential confounder in lifespan studies.

Vitamin C: poison, prophylactic or panacea?

Ascorbate is an essential enzyme cofactor but is often also regarded as an important antioxidant in vivo, protecting against cancer by scavenging DNA-damaging reactive oxygen species. Recent studies suggest that ascorbate sometimes increases DNA damage in humans. Although there is no evidence that any of these effects are deleterious to humans, we might need to change our thinking about the mechanisms of the antioxidant action of ascorbate in vivo.

The measurement and mechanism of lipid peroxidation in biological systems.

The basic chemistry of the propagation of lipid peroxidation reactions has been known for years, but the mechanism of initiation of this process in biological membrane systems is still uncertain. Currently available assays for measuring peroxidation are reviewed--the more specific the assay used, the less peroxide is found in healthy human tissues and body fluids. Lipid peroxidation can arise as a consequence of tissue injury in many disease states and may sometimes contribute significantly to worsening the tissue injury.

Proteasomal dysfunction: a common feature of neurodegenerative diseases? Implications for the environmental origins of neurodegeneration.

The neurodegenerative diseases that afflict humans affect different part of the nervous system and have different symptoms and prognoses, yet they have certain things in common. One of them is defects in the clearance of abnormal or other "unwanted" proteins, particularly affecting the proteasome system. In this review, I advance two concepts: (a) that defects in protein clearance can be a fundamental cause of neurodegeneration, and (b) that because proteasome inhibitors are widespread in nature, their ingestion may contribute to "spontaneous" neurodegeneration.

Nickel(II)- and cobalt(II)-dependent damage by hydrogen peroxide to the DNA bases in isolated human chromatin.

Nickel compounds are known to be carcinogenic to humans and animals. Cobalt compounds produce tumors in animals and are probably carcinogenic to humans. The mechanisms of the carcinogenicity of these metal compounds, however, have remained elusive. In the present work, we have investigated the ability of Ni(II) and Co(II) ions in the presence of H2O2 to cause chemical changes in DNA bases in chromatin extracted from cultured cells of human origin. Eleven modified DNA bases in chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry. 2-Hydroxyadenine (isoguanine), which has not previously been shown to occur DNA or chromatin, was also identified. Products identified were typical hydroxyl radical-induced products of DNA bases, suggesting that the hydroxyl radical was involved in their formation. This idea was supported by partial inhibition of product formation by typical scavengers of hydroxyl radical. Partial inhibition of product formation indicated a possible "site-specific" formation of hydroxyl radical by unchelated Ni(II) and Co(II) ions bound to chromatin. Although treatment of chromatin for 1 h with Co(II)/H2O2 caused formation of significant amounts of products, treatment with Ni(II)/H2O2 required incubation times of more than 5 h and an increase in Ni(II) concentration before increases in product amounts above background levels became detectable. In both cases, ascorbic acid did not increase product yields. Glutathione at a physiologically relevant concentration had little overall effect on DNA base modification. Superoxide dismutase increased the yields of most products. Chelation of Ni(II) and Co(II) ions with EDTA almost completely inhibited product formation. Ni(II) in the presence of H2O2 produced greater base damage to the DNA in chromatin than to isolated DNA, unlike other metal ions tested. DNA damage in chromatin caused by Ni(II) and Co(II) ions in the presence of H2O2 may contribute to the established genotoxicity and carcinogenicity of these metal ions.

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin.

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.

Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions.

Lead (Pb2+) ions accelerate the lipid peroxidation observed when Fe2+ ions are added to phospholipid liposomes at pH 5.5 or pH 7.4, although Pb2+ ions alone do not induce any peroxidation. Similarly, aluminium (Al3+) ions increase Fe2+-dependent liposomal peroxidation at pH 5.5. Both Pb2+ and Al3+ accelerate the peroxidation of erythrocytes induced by high concentrations of H2O2 in the presence of azide, and they also increase the peroxidation that occurs when Fe2+ or Fe2+-ADP is added to rat liver microsomes at pH 7.4. It is proposed that increased lipid peroxidation may contribute to the toxic actions of Pb2+ in humans.

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts.

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.

Cobalt(II) ion as a promoter of hydroxyl radical and possible 'crypto-hydroxyl' radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers.

Co(II) ions react with hydrogen peroxide under physiological conditions to form a 'reactive species' that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (.OH), their effectiveness being related to their second-order rate constants for reaction with .OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed 'in free solution' and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, .OH radical is formed in a 'site-specific' manner and is difficult to intercept by .OH scavengers. The relationship of these results to the proposed 'crypto .OH' radical is discussed.

The chemical modification of glycosaminoglycan structure by oxygen-derived species in vitro.

The effect of reactive oxygen species (ROS) on the chemical structure of glycosaminoglycans (GAG) was studied in order to consider their role in connective tissue damage during an inflammatory disease state such as periodontal disease. GAG were exposed to a radical generating system for 1 h and analysed by gel filtration for fragmentation and chemically with respect to uronic acid, hexosamine and sulfate content. Non-sulfated GAG, hyaluronan and chondroitin, were most susceptible to depolymerisation and chemical modification of uronic acid and hexosamine residues by ROS. Depolymerisation and chemical modification of sulfated GAG, chondroitin 4-sulfate, dermatan sulfate and heparan sulfate was significantly less than for non-sulfated GAG. The highly sulfated GAG heparin showed minimal depolymerisation by ROS, but uronic acid residues were readily modified. Analysis of the ROS-exposed residues suggests that uronic acid is capable of degrading to a 3-carbon aldehyde, malondialdehyde. Chondroitin sulfate exposed to ROS resulted in marginal desulfation. The results suggest that the presence of sulfate on the GAG chain may protect the molecule against ROS attack. However, chemical modification of GAG may affect proteoglycan function and be of importance in considering connective tissue destruction in a variety of pathological situations, including periodontal disease.

Enhancement by tamoxifen of the membrane antioxidant action of the yeast membrane sterol ergosterol: relevance to the antiyeast and anticancer action of tamoxifen.

The anticancer drug tamoxifen inhibits lipid peroxidation in ox-brain phospholipid liposomes, and is a good antiyeast agent, with clinical potential. We now report that the ergosterol-containing lipid fraction derived from yeast microsomal membranes (and the ergosterol separated from it) inhibited lipid peroxidation when introduced into ox-brain phospholipid liposomes. Inhibition of lipid peroxidation by the lipid fraction was greatly enhanced when yeast cell growth was inhibited with tamoxifen prior to lipid extraction. The ability of tamoxifen to enhance the membrane antioxidant ability of ergosterol is expressed in terms of a tamoxifen enhancement coefficient. Enhancement by tamoxifen of the membrane antioxidant action of ergosterol is discussed in relation to the antifungal and anticancer actions of tamoxifen.

Lipids and oxidised lipids in human atheroma and normal aorta.

Lipids and oxidised lipids were analysed by GC and GC-MS in samples of human atheroma (necrotic gruel from the interior of advanced atherosclerotic plaques in the aorta) and human normal aorta (lesion-free intima plus inner media) from necropsy subjects. Cholest-5-en-3 beta,26-diol and cholest-5-en-3 beta,7 beta-diol were detected in all the atheroma samples examined but not in significant amounts in normal aorta. In atheroma, cholest-5-en-3 beta,26-diol was approximately proportional to cholesterol. Several isomeric hydroxy-octadecadienoic acids were detected in atheroma, and, in smaller amounts, in normal aorta. Many of the components of atheroma showed a high degree of cross-correlation on linear regression analysis, whilst cross-correlations were somewhat weaker for normal aorta. Atheroma showed a vast accumulation of lipid, especially cholesterol, in comparison to normal aorta. The atheroma samples contained a larger proportion of linoleate relative to oleate than the normal aorta. Levels of fatty acids relative to cholesterol were lower for atheroma than for normal aorta. The chemical composition of atheroma appeared unrelated to the age of the subject, whereas age-related increases in linoleate, oleate and cholesterol content were seen in the samples of normal aorta.

Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice.

Serum from homozygous hypotransferrinaemic mice (a mixed group of males and females, aged 6-8 wk) was found to contain low levels of iron (mean 0.9 +/- 0.5 microM (SEM, n = 4), as assayed by conventional serum iron assays. Similarly, low levels of non-transferrin-bound iron were determined with a nitrilotriacetate chelation assay (1.3 +/- 0.4 microM, n = 4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undectable by electron paramagnetic resonance spectroscopy (EPR). Significantly larger quantities of iron (16 +/- 5 microM, n = 8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Halliwell, B. (1981) Biochemical Journal 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 microM iron. Haemoglobin iron was detectable at approximately 10 microM by spectrophotometry. Ferri-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641 +/- 128 micrograms/l (n = 14) in hypotransferrinaemic mice (wild-types 44 +/- 6 micrograms/l, n = 14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrinaemic mouse serum therefore contains large quantities of non-transferrin-bound iron which is unreactive in some assays used to detect such iron in human iron overload. Fractionation by Sephadex G200 chromatography revealed three distinct species with apparent molecular weights of > or = 150 kDa, 40-80 kDa and 1-5 kDa. The iron may be distinguished from known extracellular iron proteins and haem-proteins by its availability to hot acid extractions.

Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson's disease, Alzheimer's disease, traumatic injury or stroke?

Radicals are species containing one or more unpaired electrons. The oxygen radical superoxide (O2-) and the non-radical oxidant hydrogen peroxide (H2O2) are produced during normal metabolism and perform several useful functions. Excessive production of O2- and H2O2 can result in tissue damage, which often involves generation of highly-reactive hydroxyl radical (.OH) and other oxidants in the presence of "catalytic" iron ions. A major form of antioxidant defence is the storage and transport of iron ions in forms that will not catalyze formation of reactive radicals. Tissue injury, eg. 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. Oxidant 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 iron-dependent 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, it being suggested that antioxidant enzymes and chelators of iron ions may be more generally-useful protective agents than chain-breaking antioxidants.

Acidosis-induced ischemic brain damage: are free radicals involved?

Substantial evidence exists that reactive oxygen species participate in the pathogenesis of brain damage following both sustained and transient cerebral ischemia, adversely affecting the vascular endothelium and contributing to the formation of edema. One likely triggering event for free radical damage is delocalization of protein-bound iron. The binding capacity for some iron-binding proteins is highly pH sensitive and, consequently, the release of iron is enhanced by acidosis. In this study, we explored whether enhanced acidosis during ischemia triggers the production of reactive oxygen species. To that end, enhanced acidosis was produced by inducing ischemia in hyperglycemic rats, with normoglycemic ones serving as controls. Production of H2O2, estimated from the decrease in catalase activity after 3-amino-1,2,4-triazole (AT) administration, was measured in the cerebral cortex, caudoputamen, hippocampus, and substantia nigra (SN) after 15 min of ischemia followed by 5, 15, and 45 min of recovery, respectively (in substantia nigra after 45 min of recovery only). Free iron in cerebrospinal fluid (CSF) was measured after ischemia and 45 min of recovery. Levels of total glutathione (GSH + GSSH) in cortex and hippocampus, and levels of alpha-tocopherol in cortex, were also measured after 15 min of ischemia followed by 5, 15, and 45 min of recovery. The results confirm previous findings that brief ischemia in normoglycemic animals does not measurably increase H2O2 production in AT-injected animals. Ischemia under hyperglycemic conditions likewise failed to induce increased H2O2 production. No difference in free iron in CSF was observed between animals subjected to ischemia under hyper- and normoglycemic conditions. The moderate decrease in total glutathione or alpha-tocopherol levels did not differ between normo- and hyperglycemic animals in any brain region or at any recovery time. Thus, the results failed to give positive evidence for free radical damage following brief periods of ischemia complicated by excessive acidosis. However, it is possible that free radical production is localized to a small subcellular compartment within the tissue, thereby escaping detection. Also, the results do not exclude the possibility that free radicals are pathogenetically important after ischemia of longer duration.

The importance of free radicals and catalytic metal ions in human diseases.

The study of free radical reactions is not an isolated and esoteric branch of science. A knowledge of free radical chemistry and biochemistry is relevant to an understanding of all diseases and the mode of action of all toxins, if only because diseased or damaged tissues undergo radical reactions more readily than do normal tissues. However it does not follow that because radical reactions can be demonstrated, they are important in any particular instance. We hope that the careful techniques needed to assess the biological role of free radicals will become more widely used.

What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo?

Peroxynitrite (ONOO-) is a 'reactive nitrogen species' that can be formed (among other reactions) by combination of superoxide (O2.-) and nitric oxide (NO.) radicals. It is being increasingly proposed as a contributor to tissue injury in several human diseases. The evidence presented for peroxynitrite participation usually includes the demonstration of increased nitrotyrosine levels in the injured tissue. Indeed, this is often the only evidence presented: the assumption is that formation of nitrotyrosine is a biomarker specifically diagnostic of ONOO- production. The present article examines this assumption and concludes that nitrotyrosine is a biomarker for 'nitrating species' rather than being specific for ONOO-.

Protection against tissue damage in vivo by desferrioxamine: what is its mechanism of action?

Desferrioxamine (deferoxamine) is an inhibitor of iron-dependent free radical reactions that has been used to investigate the role of such reactions in several animal model systems for human disease. In vitro, desferrioxamine is not only an iron chelator but also binds other metal ions, reacts with superoxide and hydroxyl radicals, affects eicosanoid synthesis, can act as a substrate for peroxidases and can generate a reactive nitroxide radical. However, considerations of desferrioxamine concentration in vivo suggest that its ability to inhibit iron-dependent free radical reactions is the major factor that accounts for desferrioxamine's generally-protective action in animal models of human disease.

Thiourea and dimethylthiourea inhibit peroxynitrite-dependent damage: nonspecificity as hydroxyl radical scavengers.

Thiourea and, more recently, dimethylthiourea, have been used as hydroxyl radical (OH.) scavengers in experiments both in vitro and in vivo. We show that both compounds can inhibit nitration of the amino acid tyrosine on addition of peroxynitrite, and also the inactivation of alpha1-antiproteinase by peroxynitrite. Hence, protective effects of (dimethyl) thiourea could be due to inhibition of peroxynitrite-dependent damage as well as to OH. scavenging, and these compounds must not be regarded as specific OH. scavengers.