Ascorbate/iron mediation of hydroxyl free radical damage to PBR322 plasmid DNA

Plasmid PBR322 DNA has been exposed to hydroxyl free radicals generated from an ascorbate/Fe system. Hydroxyl free radical scavengers as well as the iron chelator desferroxamine and catalase inhibit the DNA nicking which occurs, but superoxide dismutase had no effect. The DNA nicking was temperature dependent, occuring more rapidly at higher temperatures. The rate of DNA nicking was accelerated by the addition of hydrogen peroxide. There was an early lag phase in DNA nicking, even though the rate of hydroxyl free radical generation, as assessed by salicylate hydroxylation, showed no lag phase. It is considered that the early hydroxyl free radical damage to DNA may be biologically very important in mutagenic and carcinogenic processes.     

Hydrogen peroxide and hydroxyl radical formation by methylene blue in the presence of ascorbic acid

Summary Using ESR we have demonstrated the formation of the ascorbate free radical from sodium ascorbate, methylene blue and light. In oxygen uptake experiments we have observed the production of hydrogen peroxide while spin trapping experiments have revealed the iron catalyzed production of the hydroxyl free radical in this system. The presence of this highly reactive radical suggests that it could be the radical that initiates free radical damage in this photodynamic system.     

A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCE) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats

Hydroxyl free radicals react with salicylate to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Utilizing the technique of high pressure liquid chromatography with electrochemical detection (LCED), it is possible to detect DHBAs at the level of femtomoles. Since salicylate is relatively non-toxic, we have administered it as a trapping agent in a first attempt to examine the use of the LCED method as a sensitive measure of in vivo OH production. Utilizing adriamycin administration as a model to induce oxygen free radical tissue damage, we found that the level of DHBAs present in drug treated rats versus controls was increased 100-fold in heart and muscle, 30-fold in lung, and 3- and 4-fold in brain and blood, respectively. These first observations support the theory that adriamycin induced OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo.     

In vivo formation of hydroxyl radicals following intragastric administration of ferrous salt in rats

Accidental poisoning by oral iron preparations is a serious problem in young children. We investigated the formation of hydroxyl radicals (.OH) in rats after intragastric instillation of ferrous sulfate. .OH was detected via its reaction with intragastrically administered 2-keto-4-methylthiobutyrate to generate ethylene gas. Ascorbic acid is typically present in oral iron preparations in order to facilitate absorption by maintaining iron in the reduced state. However, ascorbate possesses two properties that can affect .OH, recycling of oxidized iron to the ferrous state augments .OH production, while ascorbate in high concentration scavenges .OH. In experiments conducted in vitro, both actions were evident, depending upon the concentration of ascorbate. In parallel experiments conducted in vivo, the scavenging action of ascorbate was more prominent. Experiments in vitro with .OH-scavengers (dimethylsulfoxide, ethanol) and with the enzyme, catalase, confirmed both the presence of .OH and its dependence upon generated hydrogen peroxide during the oxidation of ferrous salt by molecular oxygen. Hydroxyl radicals (and/or reactive higher oxidation states of iron) may play a role in tissue damage after accidental overdose of oral iron.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species

Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o-phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o-phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H2O2----Fe(III) + OH- + OH.), it is proposed that most of the cell death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions.     

Methylene blue plus light mediates 8-hydroxy 2''-deoxyguanosine formation in DNA preferentially over strand breakage.

Methylene blue (MB) plus light, in the presence of oxygen, mediates formation of 8-hydroxyguanine in DNA. The yield of 8-hydroxyguanine may be as much as from 2 to 4% of the guanines present. The results presented here show that treatment of supercoiled plasmid DNA with methylene blue plus light causes single-stranded nicks. However, single-stranded nicking occurs approximately 17-fold less frequently than does formation of 8-hydroxyguanine. The nicking rate is reduced in the presence of Mg ion but is not prevented by inhibitors of the iron-catalyzed Fenton reaction or by scavengers of hydroxyl free radicals. Extensive exposure of DNA to light in the presence of MB produces no detectable thiobarbital reactive material thus implicating that single strand nicking does not occur by hydroxyl free radical attack on deoxyribose. Formation of 8-hydroxyguanine is apparently not dependent upon intercalative binding of MB to DNA, since it is formed in polydeoxyguanylic acid.     

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.     

Nucleosomal histone protein protects DNA from iron-mediated damage.

Iron promotes DNA damage by catalyzing hydroxyl radical formation. We examined the effect of chromatin structure on DNA susceptibility to oxidant damage. Oxygen radicals generated by H2O2, ascorbate and iron-ADP (1:2 ratio of Fe2+:ADP) extensively and randomly fragmented protein-free DNA, with double-strand breaks demonstrable even at 1 microM iron. In contrast, polynucleosomes from chicken erythrocytes were converted to nucleosome-sized fragments by iron-ADP even up to 250 microM iron. Cleavage occurred only in bare areas where DNA is unassociated with histone. In confirmation, reassembly of nucleosomes from calf thymus DNA and chicken erythrocyte histone also yielded nucleosomes resistant to fragmentation. Protection of DNA by histone was dependent on nucleosome assembly and did not simply reflect presence of scavenging protein. In contrast to this specific cleavage of internucleosomal linker DNA by iron-ADP, iron-EDTA cleaved polynucleosomes indiscriminately at all sites. The hydroxyl radical scavenger thiourea completely inhibited the random cleavage of polynucleosomes by iron-EDTA but inhibited the nonrandom cleavage of polynucleosomes by iron-ADP less completely, suggesting the possibility that the lower affinity iron-ADP chelate may allow association of free iron with DNA. Thus, oxygen radicals generated by iron-ADP indiscriminately cleaved naked DNA but cleaved chromatin preferentially at internucleosomal bare linker sites, perhaps because of nonrandom iron binding by DNA. These findings suggest that the DNA-damaging effects of iron may be nonrandom, site-directed and modified by histone protein.     

Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes.

Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.     

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.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O₂^-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.     

Fenton反应及其可能的活性产物

活性氧对许多生物分子,如脂质、蛋白质和DNA等均可引起损伤,它与许多疾病过程相联系.由超氧阴离子自由基和过氧化氢所引起的许多损伤被认为与它们转变为反应活性更强的组分有关,这些组分包括羟自由基及可能的高价铁组分.实验材料及理论结果表明,当铁盐与过氧化氢混合时,除羟自由基产生以外,高价铁组分也被认为同时产生.Fenton试剂的活性中间体是一亲核加合物,其反应活性及其产物不同于游离态羟自由基的反应活性及产物.Fenton试剂的产物分布依赖于不同的过渡金属离子、不同的配位体、不同的反应底物以及不同的溶剂基体效应.     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.