The iron-chelating drug triapine causes pronounced mitochondrial thiol redox stress

Triapine (Tp) is an iron chelator with activity against several types of cancer. Iron-Tp [Fe(III)(Tp)₂] can be redox-cycled to generate reactive oxygen species that may contribute to its cytotoxicity. However, evidence for this mechanism in cells is limited. The cytosolic and mitochondrial thioredoxins (Trx1 and Trx2, respectively) are essential for cell survival. They are normally maintained in the reduced state, and support the function of many intracellular proteins including the peroxiredoxins (Prxs). Their redox status can indicate oxidant stress in their respective subcellular compartments. Tp treatment of human lung A549 cells caused almost complete oxidation of Trx2 and its dependent peroxiredoxin (Prx3), but there was no effect on Trx1 redox status. Significant inhibition of total TrxR activity did not occur until Tp levels were 4-fold above those needed to cause Trx2 oxidation. While Tp caused a 36-45% decline in reduced glutathione (GSH) levels, GSH accounted for >99% of the total glutathione in the absence and presence of Tp. In vitro studies demonstrated that cysteine reduces Fe(III)(Tp)₂ to Fe(II)(Tp)₂, and cysteine was faster and more efficient than reduced glutathione (GSH) in this regard. Fe(III)(Tp)₂ also mediated the oxidation of purified Trx2 in vitro. Thus, Fe(III)(Tp)₂ itself, and/or various reactive species that may result from its redox cycling, could account for Trx2 and Prx3 oxidation in Tp-treated cells. The striking difference between the effects on Trx2 and Trx1 implies a pronounced thiol redox stress that is largely directed at the mitochondria. These previously unrecognized effects of Tp could contribute to its overall cytotoxicity.     

Intracellular generation of reactive oxygen species by mitochondria

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.     

Different antioxidant effects of polyphenols on lipid peroxidation and hydroxyl radicals in the NADPH-, Fe-ascorbate- and Fe-microsomal systems

Antioxidant and pro-oxidant effects of 14 naturally occurring polyphenols (PP) on rat liver microsomal lipid peroxidation (LP) and hydroxyl radical (*OH) production were studied in NADPH-dependent, 50 microM Fe(2+)-500 microM ascorbate (Fe-AA) or 50 microM Fe(2+) system, respectively. LP determined by the thiobarbituric acid method was inhibited in the NADPH system by flavonols and trans-resveratrol that were more effective than other flavonoids and derivatives of benzoic and cinnamic acid and were mostly more efficient than in the Fe-AA system. Inhibition of LP in the Fe system was higher by one order of magnitude than in the Fe-AA system. *OH production in the NADPH system, measured by formaldehyde production, was decreased by myricetin, fisetin and quercetin, but increased by kaempferol, morin and trans-resveratrol, indicating that z.rad;OH played a minor role in LP, which all of these PP inhibited. None of these PP at up to 40 microM concentration quenched *OH in the Fe-AA system. All tested PP, except trans-resveratrol and gentisic acid, spectrally interacted with Fe(2+) or Fe(3+), indicating formation of complexes or oxidation of PP. In contrast to the NADPH system we found no correlation between Fe(2+) chelation and inhibition of Fe-AA- or Fe-dependent LP indicating that iron chelation did not play a significant role in the two latter systems. It is concluded that the inhibition of LP by PP was apparently due to their hydrogen donating properties rather than chelation of iron.     

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO?) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO? formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450. Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO(.-) formation. HO(.-) formation in liver microsomes was inhibited by the HO(.-) radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO(.-). Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.     

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.     

Mitochondria,reactive oxygen species and cadmium toxicity in the kidney

The heavy metal cadmium accumulates in kidney cells, particularly those of the proximal tubular epithelium, and the damage this causes is associated with development of chronic kidney disease. One of the causative mechanisms of chronic kidney disease is thought to be oxidative stress. Cadmium induces oxidative stress, but the molecular mechanisms involved in the cell damage from oxidative stress in cadmium-induced chronic kidney disease are not well understood. Mitochondrial damage is likely, given that dysfunctional mitochondria are central to the formation of excess reactive oxygen species (ROS), and are known key intracellular targets for cadmium. Normally, ROS are balanced by natural anti-oxidant enzymes. When mitochondria become dysfunctional, for example, through long term exposure to environmental toxicants like cadmium, they produce less cell energy and more ROS. The imbalance between these ROS and the natural anti-oxidants creates the condition of oxidative stress. The outcomes of mitochondrial injury are manyfold: injured mitochondria perpetuate oxidative stress; the loss of mitochondrial membrane potential causes release of cytochrome-c and activation of caspase pathways that lead to apoptotic deletion of renal cells; and attempts by cells to remove dysfunctional mitochondria through autophagy lead to “autophagic cell death” or apoptosis. Three pathways of mitochondrial regulation (upstream signalling pathways, direct mitochondrial targeting, and downstream cell death effector pathways) are therefore all promising targets for effective anti-oxidant treatment of cadmium toxicity in the kidney.     

Selective iNOS inhibition reduces renal damage induced by cisplatin

Cisplatin is a chemotherapeutic agent used in the treatment of several cancer tumors; however, nephrotoxicity has restricted its use. Reactive oxygen species and peroxynitrite, which is formed by the reaction between superoxide anion and nitric oxide (NO*), are implicated in cisplatin-induced nephrotoxicity. In contrast, both toxic and beneficial effects of NO* have been suggested in cisplatin-induced nephrotoxicity. Therefore, nowadays the role of NO* in this experimental model remains controversial. The aim of the present work was to elucidate the role of NO* in cisplatin-induced renal damage using N-[3-(aminomethyl)benzyl]acetamidine (1400W), a selective and irreversible inhibitor of iNOS. The mRNA levels of iNOS were increased in cisplatin-treated rats. The administration of 1400W reduced the cisplatin induced histological damage, renal dysfunction (increase in proteinuria and kidney injury molecule expression and decrease in creatinine clearance), tubulointerstitial infiltration, oxidative stress (increase in renal malondialdehyde and inmmunostaining for 4-hydroxy-2-nonenal) and nitrosative stress (immunostaining for 3-nitrotyrosine). In addition, the administration of 1400W was unable to modify systolic blood pressure in control rats. Our data demonstrate that selective iNOS inhibition reduces the cisplatin-induced nephrotoxicity and nitrosative stress which strongly suggest that in this experimental model (1) the NO* production is toxic and (2) iNOS is the main source of NO*.     

Protective effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on copper-induced hydroxyl radical generation in the rat heart

The present study was examined the effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Cu(II)-induced hydroxyl radical generation (OH) in the extracellular fluid of rat myocardium. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. When Cu(II) was infused through the microdialysis probe, Cu(II) increased in OH formation trapped as 2,3-DHBA in the dialysate. When fluvastatin (100 microM) was administered to Cu(II) (50 microM)-pretreated animals, the levels of 2,3-DHBA at 300 min after administration of fluvastatin significantly decreased. In cumulative dose dependent experiments, three concentrations of Cu(II), 10, 25 and 50 microM, were infused through the microdialysis probe in the rat myocardium. A positive linear correlation between Cu(II) and the formation of 2,3-DHBA (R(2)=0.980) was observed. However, when corresponding experiments were performed with fluvastatin (100 microM) pretreated animals, the level of 2,3-DHBA decreased. These results suggest that blocking LDL oxidation by fluvastatin may attenuate Cu(II)-induced OH formation in the rat heart.     

Antioxidative function and biodistribution of [Gd@C82(OH)22]n nanoparticles in tumor-bearing mice

Oxidative stress is considered to be one of the important mechanisms involved in carcinogenesis. In our previous study, gadolinium endohedral metallofullerenol ([Gd@C82(OH)22]n nanoparticles) have shown high inhibitory activity on hepatoma cell (H22) growth in mice. To explore the antioxidative functions of nanoparticles, we investigated the biodistribution of [Gd@C82(OH)22]n nanoparticles, the changes of blood coagulation profiles, the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice as well as the possible relationships between nanoparticles treatment and ROS production in this paper. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and catalase (CAT) as well as the levels of reduced glutathione (GSH), protein-bound thiols and malondialdehyde (MDA) were compared between the tumor-bearing mice and normal mice. Transplanted tumors were grown in mice by subcutaneous injection of murine hepatoma cells in the mice. The comparison of the above parameters between nanoparticles and cyclophosphamide (CTX) therapy were also investigated. [Gd@C82(OH)22]n administration can efficiently restore the damaged liver and kidney of the tumor-bearing mice. All the activities of enzymes and other parameters related to oxidative stress were reduced after [Gd@C82(OH)22]n treatment and tended closely to the normal levels. The results suggest that [Gd@C82(OH)22]n nanoparticle treatment could regulate ROS production in vivo.     

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.     

Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: Focus on atherosclerosis

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several coronary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu²⁺-induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxidation and decreasing in the oxidation rate (V_max), moreover, the protein moieties from LDL were protected against Cu²⁺-induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr^−/−), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxidative stress in endothelial cells and improve endothelial function in mice.     

Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro.

The flaxseed lignan secoisolariciresinol diglucoside (SDG) and mammalian lignans enterodiol (ED) and enterolactone (EL) were previously shown to be effective antioxidants against DNA damage and lipid peroxidation. Others reported inhibition of activated cell chemiluminescence by supra-physiological concentrations of secoisolariciresinol (SECO), ED and EL. Thus, we evaluated the antioxidant efficacy of potential physiological concentrations of SDG, SECO, ED and EL against 1,1-diphenyl-2-picrylhydrazyl (DPPH()), and 2,2'-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-initiated peroxyl radical plasmid DNA damage and phosphatidylcholine liposome lipid peroxidation. SDG and SECO were effective (p<0.01) antioxidants against DPPH() at 25-200muM; whereas, ED and EL were inactive. Efficacy of lignans and controls against AAPH peroxyl radical-induced DNA damage was: SDG>SECO=17alpha-estradiol>ED=EL>genistein>daidzein. Lignan efficacy against AAPH-induced liposome lipid peroxidation was: SDG>SECO=ED=EL. Plant lignan antioxidant activity was attributed to the 3-methoxy-4-hydroxyl substituents of SDG and SECO, versus the meta mono-phenol structures of ED and EL. Benzylic hydrogen abstraction and potential resonance stabilization of phenoxyl radicals in an aqueous environment likely contributed to the antioxidant activity of the mammalian lignans. These represent likely extra- and intracellular antioxidant activities of flax-derived lignans at concentrations potentially achievable in vivo.     

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS⁺ assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts.     

Antioxidant properties of Thonningianin A,isolated from the African medicinal herb,Thonningia sanguinea

The antioxidant properties of Thonningianin A (Th A), an ellagitannin, isolated from the methanolic extract of the African medicinal herb, Thonningia sanguinea were studied using the NADPH and Fe2+/ascorbate-induced lipid peroxidation (LPO), electron spin resonance spectrometer and the deoxyribose assay. Th A at 10 microM inhibited both the NADPH and Fe2+/ascorbate-induced LPO in rat liver microsomes by 60% without inhibitory effects on cytochrome P450 activity. Th A was similar to the synthetic antioxidant, tannic acid, as an inhibitor of both the NADPH and Fe2+/ascorbate-induced LPO but potent than gallic acid, vitamin C and vitamin E. While Th A poorly scavenged the hydroxyl radical generated by the Fenton reaction it dose-dependently scavenged 1,1-diphenyl-2-picrylhydrazyl, superoxide anion and peroxyl radicals with IC50 of 7.5, 10 and 30 microM, respectively. Furthermore, Th A showed inhibitory effects on the activity of xanthine oxidase with an IC50 of 30 microM. In the deoxyribose assay both T. sanguinea and its methanolic component Th A showed only site-specific (Fe3+ + H2O2) but not non-site-specific (Fe3+ + EDTA + H2O2) hydroxyl radical scavenging suggesting chelating ability for iron ions. Spectroscopic studies showed that Th A enhanced absorbance in the visible region in the presence of Fe2+ ions. These results indicate that the antioxidant properties of Th A involve radical scavenging, anti-superoxide formation and metal chelation.     

Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India

Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, (ABTS⁺)], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe²⁺) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH radical scavenging activity (>400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (<125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS⁺ and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r² > 0.95) with phenolic contents, in DPPH, ABTS⁺ and FRAP assays, whereas poor correlation (r² = 0.297) was observed between Fe²⁺ chelating activity of the legumes and phenolic contents.     

Effects of (-)-nicotine and (-)-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson's disease

In view of the apparent controversial properties of (-)-nicotine (NIC) in relation to both oxidative stress and neuroprotection, we studied the effects of NIC on hydroxyl radical (*OH) formation, oxidative stress production by 6-hydroxydopamine (6-OHDA) autoxidation in the presence and absence of ascorbate, and 6-OHDA neurotoxicity. Both NIC and (-)-cotinine (COT) exhibited increased *OH production during 6-OHDA autoxidation. Although the same effect was observed in *OH generation by the Fenton reaction (H2O2 + Fe2+), this reaction was completely prevented with the previous incubation of Fe2+ with NIC or COT. Furthermore, both NIC and COT demonstrated a capacity to be able to reduce the TBARS formation provoked in rat brain mitochondrial preparations by 6-OHDA autoxidation. This effect is assumed as a consequence of the action of NIC and COT on lipid peroxidation propagation. We treated with NIC (1mg/kg, i.p.) two 6-OHDA-induced rat models of Parkinson's disease. However, only in one of these models did we obtain clear evidence of a neuroprotective effect of NIC on nigrostriatal terminals, as revealed by immunohistochemistry against tyrosine hydroxylase. Thus, the antioxidant properties of both NIC and COT in relation to the lipid peroxidation induced by 6-OHDA autoxidation, together with their reported capacity to prevent the Fenton reaction, probably by sequestration of Fe2+, may contribute to an understanding of its neuroprotective properties. In addition, the reported capacity of both NIC and COT to increase the production of *OH by 6-OHDA autoxidation might help explain the controversial observation found under different experimental conditions.     

Prooxidant action of knipholone anthrone: Copper dependent reactive oxygen species generation and DNA damage

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological activities including in vitro antioxidant and cytotoxic properties. By using DNA damage as an experimental model, the comparative Cu(II)-dependent prooxidant action of these two compounds were studied. In the presence of Cu(II) ions, the antioxidant KA (3.1–200 μM) but not KP (6–384 μM) caused a concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by KA could be abolished by reactive oxygen species scavengers, glutathione and catalase as well as EDTA and a specific Cu(I) chelator bathocuproine disulfonic acid. In addition to Cu(II) chelating activity, KA readily reduces Cu(II) to Cu(I). Copper-dependent generation of reactive oxygen species and the subsequent macromolecular damage may be involved in the antimicrobial and cytotoxic activity of KA.