Reducing oxidative/nitrosative stress: a newly-discovered genre for melatonin

The discovery of melatonin and its derivatives as antioxidants has stimulated a very large number of studies which have, virtually uniformly, documented the ability of these molecules to detoxify harmful reactants and reduce molecular damage. These observations have clear clinical implications given that numerous age-related diseases in humans have an important free radical component. Moreover, a major theory to explain the processes of aging invokes radicals and their derivatives as causative agents. These conditions, coupled with the loss of melatonin as organisms age, suggest that some diseases and some aspects of aging may be aggravated by the diminished melatonin levels in advanced age. Another corollary of this is that the administration of melatonin, which has an uncommonly low toxicity profile, could theoretically defer the progression of some diseases and possibly forestall signs of aging. Certainly, research in the next decade will help to define the role of melatonin in age-related diseases and in determining successful aging. While increasing life span will not necessarily be a goal of these investigative efforts, improving health and the quality of life in the aged should be an aim of this research.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Chemical reactivity of the carbon-centered free radicals and ferrous iron in coals: Role of bioavailable Fe2+ in coal workers' pneumoconiosis

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe²⁺ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe²⁺ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe²⁺ with O₂ in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe²⁺, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe²⁺-induced oxidative stress and eventual CWP development.     

Generation of superoxide and singlet oxygen from α-tocopherolquinone and analogues

Three potential routes to generation of reactive oxygen species (ROS) from α-tocopherolquinone (α-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 10⁸ dm³/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10^{? 6} s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 10⁹ dm³/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^?₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^?₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^?₂. Second order kinetics for the decay of RNO^?₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^?₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

DNA-breaking versus DNA-protecting activity of four phenolic compounds in vitro

Given the paradoxical effects of phenolics in oxidative stress, we evaluated the relative pro-oxidant and antioxidant properties of four natural phenolic compounds in DNA nicking. The phenolic compounds differed dramatically in their ability to nick purified supercoiled DNA, with the relative DNA nicking activity in the order: 1,2,4-benzenetriol (100% nicking) > gallic acid > caffeic acid > gossypol (20% nicking). Desferrioxamine (0.02 mM) decreased DNA strand breakage by each phenolic, most markedly with gallate (85% protection) and least with caffeic acid (26% protection). Addition of metals accelerated DNA nicking, with copper more effective (～5-fold increase in damage) than iron with all four phenolics. Scavengers revealed the participation of specific oxygen-derived active species in DNA breakage. Hydrogen peroxide participated in all cases (23–90%). Hydroxyl radicals were involved (32–85%), except with 1,2,4-benzenetriol. Superoxide participated (81–86%) with gallic acid and gossypol, but not with caffeic acid or 1,2,4-benzenetriol. With 1,2,4-benzenetriol, scavengers failed to protect significantly except in combination. Thus, in the presence of desferrioxamine, catalase or superoxide dismutase inhibited almost completely. When DNA breakage was induced by Fenton's reagent (ascorbate plus iron) the two catechols (caffeic acid and gossypol) were protective, whereas the two triols (1,2,4-benzenetriol and gallic acid) exacerbated damage.     

Specific Deprotonation Reactions of the Pyrimidine Radical Cation Resulting from the Menadione Mediated Photosensitization of 2′-Deoxycytidine

Menadione(2-methyl-1, 4-naphthoquinone) was shown to sensitize 2′-deoxycytidine to near ultraviolet light according to two main mechanisms. Reaction of a water molecule with the initially photo-induced pyrimidine radical cation and subsequent addition of molecular oxygen leads to the preponderant formation of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine. Pyrimidine ring opening and rearrangement products are also generated through the intermediate 6-hydroxy-5,6-dihydro-2′-deoxyurid-5-yl radical. The competitive deprotonation reaction of the radical cation is likely to involve two sites. Loss of an amino group proton is the likely initial event to explain the formation of 2′-deoxyuridine which is resistant to further photooxidation. The second deprotonation reaction involves the osidic carbon C(1′). The resulting radical will further react with oxygen leading to the release of free cytosine with concomitant formation of 2-deoxy-D-ribono-1,4-lactone. This reaction which is not prevented by hydroxyl radical scavengers constitutes to our knowledge the first example of a pyrimidine radical which is able to initiate selective intramolecular reaction at position 1 within the sugar moiety.     

Resveratrol treatment protects against doxorubicin-induced cardiotoxicity by alleviating oxidative damage

The possible protective effects of resveratrol (RVT) against cardiotoxicity were investigated in Wistar albino rats treated with saline, saline+doxorubicin (DOX; 20 mg/kg) or RVT (10 mg/kg)+DOX. Blood pressure and heart rate were recorded on the 1^st week and on the 7^th week, while cardiomyopathy was assessed using transthoracic echocardiography before the rats were decapitated. DOX-induced cardiotoxicity resulted in decreased blood pressure and heart rate, but lactate dehydrogenase, creatine phosphokinase, total cholesterol, triglyceride, aspartate aminotransferase and 8-OHdG levels were increased in plasma. Moreover, DOX caused a significant decrease in plasma total antioxidant capacity along with a reduction in cardiac superoxide dismutase, catalase and Na⁺,K⁺-ATPase activities and glutathione contents, while malondialdehyde, myelopreoxidase activity and the generation of reactive oxygen species were increased in the cardiac tissue. On the other hand, RVT markedly ameliorated the severity of cardiac dysfunction, while all oxidant responses were prevented; implicating that RVT may be of therapeutic use in preventing oxidative stress due to DOX toxicity.