Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2′-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Spinal cord injury increases iron levels: catalytic production of hydroxyl radicals

This study used a weight drop impact injury model to explore the role of iron and the reality of iron-catalyzed hydroxyl radical ((*)OH) formation in secondary spinal cord injury (SCI). The time course of total extracellular iron was measured following SCI by microcannula sampling and atomic absorption spectrophotometry analysis. Immediately following SCI, the total iron concentration increased from an undetectable level to an average of 1.32 microM. The time course of SCI-induced (*)OH-generating catalytic activity in the cord was obtained by determining the ability of tissue homogenate to convert hydrogen peroxide to (*)OH and then measuring 2,3-dihydroxybenzoic acid, a hydroxylation product of salicylate. The concentration of 2,3-DHBA quickly and significantly increased (p <.001) and returned to sham level (p = 1) by 30 min post-SCI. Desferrioxamine (80 and 800 mg/kg body weight) significantly (p <.001) reduced the catalytic activity, suggesting that iron is the major contributor of the activity. Administering FeCl(3) (100 microM)/EDTA (0.5 mM) in ACSF into the cord through a dialysis fiber significantly increased SCI-induced (*)OH production in the extracellular space, demonstrating that Fe(3+) can catalyze (*)OH production in vivo. Our results support that iron-catalyzed (*)OH formation plays a role in the early stage of secondary SCI.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation

We have previously observed that both Fe(II) and Fe(III) are required for lipid peroxidation to occur, with maximal rates of lipid peroxidation observed when the ratio of Fe(II) to Fe(III) is approximately one (J. R. Bucher et al. (1983) Biochem. Biophys. Res. Commun. 111, 777-784; G. Minotti and S. D. Aust (1987) J. Biol. Chem. 262, 1098-1104). Consistent with the requirement for both Fe(II) and Fe(III), ascorbate, by reducing Fe(III) to Fe(II), stimulated iron-catalyzed lipid peroxidation but when the ascorbate concentration was sufficient to reduce all of the Fe(III) to Fe(II), ascorbate inhibited lipid peroxidation. The rates of lipid peroxidation were unaffected by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers. Exogenously added H2O2 also either stimulated or inhibited ascorbate-dependent, iron-catalyzed lipid peroxidation apparently by altering the ratio of Fe(II) to Fe(III). Thus, it appears that the prooxidant effect of ascorbate is related to the ability of ascorbate to promote the formation of a proposed Fe(II):Fe(III) complex and not due to oxygen radical production. The antioxidant effect of ascorbate on iron-catalyzed lipid peroxidation may be due to complete reduction of iron.     

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( •OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 μM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of •OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced •OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with •OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular •OH in damage associated with intracerebral iron injection.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on ⋅OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as β-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against ^⋅OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ^⋅OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of ^⋅OH from H₂O₂. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H₂O₂. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on ^⋅OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Effects of a Direct Injection of Liposoluble Iron into Rat Striatum. Importance of the Rate of Iron Delivery to Cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( &#148 OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 &#119 M FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of &#148 OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced &#148 OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with &#148 OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular &#148 OH in damage associated with intracerebral iron injection.     

The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation

Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions.     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.     

渗透胁迫下稻苗中铁催化的膜脂过氧化作用

在－０．７ＭＰａ渗透胁迫下,水稻幼苗体内和Ｈ２Ｏ２大量产生,Ｆｅ２＋积累,膜脂过氧化作用加剧。水稻幼苗体内Ｆｅ２＋含量与膜脂过氧化产物ＭＤＡ含量呈极显著的正相关。外源Ｆｅ２＋、Ｆｅ３＋、Ｈ２Ｏ２、Ｆｅ２＋＋Ｈ２Ｏ２、ＤＤＴＣ均能刺激膜脂过氧化作用,而铁离子的螯合剂ＤＴＰＡ则有缓解作用。ＯＨ的清除剂苯甲酸钠和甘露醇能明显地抑制渗透胁迫下Ｆｅ２＋催化的膜脂过氧化作用。这都表明渗透胁迫下水稻幼苗体内铁诱导的膜脂过氧化作用主要是由于其催化Ｆｅｎｔｏｎ型Ｈａｂｅｒ－Ｗｅｉｓｓ反应形成ＯＨ所致。     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Peroxynitrite-Dependent Aromatic Hydroxylation and Nitration of Salicylate and Phenylalanine. Is Hydroxyl Radical Involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO^-)-derived species involves hydroxyl radicals (OH*). This was investigated by using salicylate and phenylalanine, attack of OH* upon which leads to the formation of 2, 3- and 2, 5-dihydroxybenzoates, and o-, m- and p-tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimation of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH* scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H₂O₂ and ascorbate. However, OH* scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO^- was added to phenylalanine. The amounts of these products again decreased at high pH and were decreased by addition of OH* scavengers. We conclude that although comparison with Fenton systems suggests OH* formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH* and NO₂ would not explain why OH* scavengers inhibit formation of nitration products.     

Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions

The autoxidation and monoamine oxidase (MAO)-mediated metabolism of dopamine (3-hydroxytyramine; DA) cause a continuous production of hydroxyl radical (*OH), which is further enhanced by the presence of iron (ferrous iron, Fe(2+) and ferric ion, Fe(3+)). The accumulation of hydrogen peroxide (H2O2) in the presence of Fe(2+) appears to discard the involvement of the Fenton reaction in this process. It has been found that the presence of DA significantly reduces the formation of thiobarbituric acid reagent substances (TBARS), which under physiological conditions takes place in mitochondrial preparations. The presence of DA is also able to reduce TBARS formation in mitochondrial preparations even in the presence of iron (Fe(2+) and Fe(3+)). However, DA boosted the carbonyl content of mitochondrial proteins, which was further increased in the presence of iron (Fe(2+) and Fe(3+)). This latter effect is also accompanied by a significant reduction in thiol content of mitochondrial proteins. It has also been observed how the pre-incubation of mitochondria with pargyline, an acetylenic MAO inhibitor, reduces the production of *OH and increases the formation of TBARS. Although, the MAO-mediated metabolism of DA increases MAO-B activity, the presence of iron inhibits both MAO-A and MAO-B activities. Consequently, DA has been shown to be a double-edged sword, because it displays antioxidant properties in relation to both the Fenton reaction and lipid peroxidation and exhibits pro-oxidant properties by causing both generation *OH and oxidation of mitochondrial proteins. Evidently, these pro-oxidant properties of DA help explain the long-term side effects derived from l-DOPA treatment of Parkinson's disease and its exacerbation by the concomitant use of DA metabolism inhibitors.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

Hydroxylation of Salicylate and Phenylalanine as Assays for Hydroxyl Radicals: a Cautionary Note Visited for the Third Time

Hydroxylation of salicylate to2, 3- and2, 5-dihydroxy-benzoates (DHBs) is widely used as an index of hydroxyl radical (OH) formation in vivo and in vitro. Several recent studies indicate that peroxynitrite can lead to generation of DHBs from salicylate and it is uncertain as to whether or not OH' is involved. A similar problem may occur in the use of phenylalanine as an OH' detector. Hence formation of hydroxylation products from salicylate (or phenylalanine) may not in itself be a definitive index of OH' generation, especially in cases where such generation in physiological systems is decreased by inhibitors of nitric oxide syn-thase. Determination of salicylate (or phenylalanine) nitration products can allow distinction between peroxynitrite-dependent aromatic hydroxylation and that involving “real” OH.     

Lipid peroxidation in mitochondria and microsomes from adult and fetal rat tissues

Pregnant female Wistar rats that received a control (100 ppm Zn) or a Zn-deficient diet (1.5 ppm Zn) from d 0 to 21, or nonpregnant normally fed female rats without or with five daily oral doses of 300 mg/kg salicylic acid were used for the experiments. In isolated mitochondria or microsomes from various maternal and fetal tissues, lipid peroxidation was determined as malondialdehyde formation measured by means of the thiobarbiturate method. Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria. Lipid peroxidation in fetal microsomes was very low. Zn deficiency produced a further reduction of lipid peroxidation in fetal liver microsomes. Salicylate increased lipid peroxidation in liver mitochondria and microsomes after addition in vitro and after application in vivo. The increase of lipid peroxidation by salicylate may be caused by two mechanisms: an increased cellular Fe uptake that, in turn, can increase lipid peroxidation and chelating Fe, in analogy to the effect of ADP in lipid peroxidation. The latter effect of salicylate is particularly expressed at increased Fe content.