The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

Egg Yolk Phosvitin Inhibits Hydroxyl Radical Formation from the Fenton Reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical (^?OH) formation from H₂O₂ in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited ^?OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the ^?OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H₂O₂. These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron

Hydrogen peroxide (H₂O₂) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H₂O₂ can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H₂O₂ by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that yaaA, an uncharacterized gene found in many bacteria, was induced by H₂O₂ in Escherichia coli as part of its OxyR regulon. Here we confirm that yaaA is a key element of the stress response to H₂O₂. In a catalase/peroxidase-deficient (Hpx⁻) background, yaaA deletion mutants grew poorly, filamented extensively, and lost substantial viability when they were cultured in aerobic LB medium. The results from a thyA forward mutagenesis assay and the growth defect of the yaaA deletion in a recombination-deficient (recA56) background indicated that yaaA mutants accumulated high levels of DNA damage. The growth defect of yaaA mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx⁻ yaaA cells had a higher hydroxyl radical (HO^•) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H₂O₂ stress the induction of YaaA is a critical device to suppress intracellular iron levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown.     

Inhibition of succinate-linked respiration and complex II activity by hydrogen peroxide

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is sensitive to H₂O₂-mediated inhibition. Nevertheless, the effects of H₂O₂ on succinate-linked respiration and complex II activity have not been examined in intact mitochondria. Results presented indicate that H₂O₂ inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner. H₂O₂ has no effect on complex II activity during state 2 respiration, but inhibits activity during state 3. It was found that conditions which prevent oxaloacetate accumulation during state 3 respiration, such as inclusion of rotenone, glutamate, or ATP, blunted the effect of H₂O₂ on succinate-linked respiration and complex II activity. It is concluded that H₂O₂ inhibits succinate-linked respiration indirectly by sustaining and enhancing oxaloacetate-mediated inactivation of complex II.     

Hyperoxia increases oxygen radical production in rat lung homogenates

Lung damage during hyperoxia has been postulated to be due to increased rates of local organ oxygen radical production. Lung homogenate respiration was inhibited with cyanide, and residual respiration was used as an indicator of electron diversion to O₂^? and H₂O₂. Cyanide-resistant respiration in lung homogenates, supplemented with 1 mm NADH, increased linearly with oxygen tension, and accounted for 7% of total respiration in air and for 17% of total respiration when homogenates were incubated in 80% oxygen. Exposure of rats to 85% oxygen for 7 days induces tolerance to the lethal effects of 100% oxygen. Rats which previously breathed 85% oxygen for 7 days had a greater CN^?-resistant respiration than control rats. This implies that adaptation to hyperoxia does not include decreased lung tissue oxygen radical production as indicated by CN^?-resistant respiration. One possible explanation for the increased CN^?-resistant respiration in oxygen tolerant rat lungs is that they contain increased cell mass. Lung homogenates of rats exposed to 85% oxygen for 7 days also had 2.5 times greater thiobarbituric acid positive material than controls, indicating that increased lung lipid peroxidation occurs as a consequence of hyperoxia. Incubation of normal rat lung homogenates under hyperoxic conditions also acutely increased lipid peroxidation, which could be inhibited by both superoxide dismutase and catalase. This confirms that hyperoxia enhances cellular production of O₂^? and H₂O₂ and implies an essential role for both O₂^? and H₂O₂ in hyperoxic lung damage.     

Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide

Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(II) catalyze hydroxyl free radical formation from H₂O₂. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of ?H produced from H₂O₂ as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from H₂O₂ over the low level obtained in the buffer system only. The cations, Mg²⁺ and Ca²⁺, even at much higher than physiological levels and much higher than the level of added Fe(II), did not cause a substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H₂O₂ to yield ?H. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated ?H formation from H₂O₂ demonstrated that Fe(II) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(II) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing ?H formation from H₂O₂; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H₂O₂ catalysis to yield ?H. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when H₂O₂ is present with ferrous-nucleotide complexes.     

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H₂O₂) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H₂O₂ around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H₂O₂-alone treatment kills catalase-deficient (katEG) mutants similar to H₂O₂+NO treatment. However, the Fenton reaction, in addition to H₂O₂, requires Fe(II), which H₂O₂ excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H₂O₂ is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H₂O₂-alone and H₂O₂+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H₂O₂-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H₂O₂ and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.     

Atmospheric hydrogen peroxide and Eoarchean iron formations

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean–Early Paleoproterozoic (2.7–2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H₂O₂), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H₂O₂ that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO₂, O₂, and CH₄ mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H₂O₂ rainout was calculated to be <10⁶ molecules cm^?2 s^?1. Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H₂O₂ was insufficient by several orders of magnitude to account for IF deposition (requiring ~10¹¹ H₂O₂ molecules cm^?2 s^?1). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)‐oxidizing micro‐organisms the most likely mechanism responsible for Earth's oldest IF.     

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.     

Impact of hydrogen peroxide-driven Fenton reaction on mouse oocyte quality

Here we show that hydroxyl radical ^(•OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH). Metaphase-II mouse oocytes, obtained commercially, were grouped as follows: control, hydrogen peroxide (H₂O₂), Fe(II), and combined (Fe(II) +H₂O₂) treatments. After 7–10 min of incubation at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson χ² test and Fisher exact test were used to compare outcomes between controls and treated groups and also among the treated groups. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with a combination of H₂O₂ and Fe(II) (p<0.001); oocytes treated with H₂O₂ alone or Fe(II) alone showed no or few changes compared to control. Comparison of oocyte groups that received increasing concentrations of H₂O₂ and a fixed amount of Fe(II) showed that 70–80% demonstrated poor scores in both MT and CH when pretreated with 5 μM H₂O₂, and this increased up to 90–100% when treated with 10–20 μM H₂O₂. Hydroxyl radical generated by H₂O₂-driven Fenton reaction deteriorates the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the ^•OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states.     

Mechanism of DNA Damage and Apoptosis Induced by Tetrahydropapaveroline, a Metabolite of Dopamine

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H₂O₂)-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to ³²P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical (^·OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to ^·OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5′-TG-3′ sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H₂O₂ and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H₂O₂ and metals plays an important role in cytotoxicity of L-DOPA.     

Iron oxidation stimulates organic matter decomposition in humid tropical forest soils

Humid tropical forests have the fastest rates of organic matter decomposition globally, which often coincide with fluctuating oxygen (O₂) availability in surface soils. Microbial iron (Fe) reduction generates reduced iron [Fe(II)] under anaerobic conditions, which oxidizes to Fe(III) under subsequent aerobic conditions. We demonstrate that Fe (II) oxidation stimulates organic matter decomposition via two mechanisms: (i) organic matter oxidation, likely driven by reactive oxygen species; and (ii) increased dissolved organic carbon (DOC) availability, likely driven by acidification. Phenol oxidative activity increased linearly with Fe(II) concentrations (P < 0.0001, pseudo R² = 0.79) in soils sampled within and among five tropical forest sites. A similar pattern occurred in the absence of soil, suggesting an abiotic driver of this reaction. No phenol oxidative activity occurred in soils under anaerobic conditions, implying the importance of oxidants such as O₂ or hydrogen peroxide (H₂O₂) in addition to Fe(II). Reactions between Fe(II) and H₂O₂ generate hydroxyl radical, a strong nonselective oxidant of organic compounds. We found increasing consumption of H₂O₂ as soil Fe(II) concentrations increased, suggesting that reactive oxygen species produced by Fe(II) oxidation explained variation in phenol oxidative activity among samples. Amending soils with Fe(II) at field concentrations stimulated short‐term C mineralization by up to 270%, likely via a second mechanism. Oxidation of Fe(II) drove a decrease in pH and a monotonic increase in DOC; a decline of two pH units doubled DOC, likely stimulating microbial respiration. We obtained similar results by manipulating soil acidity independently of Fe(II), implying that Fe(II) oxidation affected C substrate availability via pH fluctuations, in addition to producing reactive oxygen species. Iron oxidation coupled to organic matter decomposition contributes to rapid rates of C cycling across humid tropical forests in spite of periodic O₂ limitation, and may help explain the rapid turnover of complex C molecules in these soils.     

Hydrogen peroxide induced by modulated electromagnetic radiation protects the cells from DNA damage

It is believed that non-ionizing electromagnetic radiation (EMR) and low-level hydrogen peroxide (H₂O₂) may change nonspecific resistance and modify DNA damage caused by ionizing radiation. To check this assumption, the combined effects of extremely high-frequency EMR (EHF EMR) and X-rays on induction of DNA damage in mouse whole blood leukocytes were studied. The cells were exposed to X-rays with or without preliminary treatment with EHF EMR or low-level H₂O₂. With the use of enhanced chemiluminescence, it was shown for the first time that pulse-modulated EHF EMR (42.2 GHz, incident power density of 0.1 mW/cm², exposure duration of 20 min, modulation frequency of 1 Hz) induced H₂O₂ at a concentration of 4.6 ± 0.3 nM L^?1 in physiological saline. With the use of an alkaline comet assay, it was found that the exposure of cells to the pulse-modulated EHF EMR, 25 min prior to treatment with X-rays at a dose of 4 Gy reduced the level of ionizing radiation-induced DNA damage. Continuous EHF EMR was inefficient. In turn, it was shown that low-level H₂O₂ (30–500 nM L^?1) protected the cells against X-irradiation. Thus, the mechanisms of radiation protective effect of EHF EMR are connected with the induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated EHF EMR.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

The ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H₂O₂)-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H₂O₂-induced injury. In addition to cell death, pretreatment with PPT could also reduce H₂O₂-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD⁺. Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H₂O₂. The metabolism of glutathione was also changed. H₂O₂ could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H₂O₂-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells

Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H₂O₂). Interestingly, a bimodal H₂O₂ dose–response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells. Furthermore, it has been demonstrated that H₂O₂ causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose–response curve. Here we show that H₂O₂ produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H₂O₂ were much more efficient at inducing ADLs than higher concentrations. This dose–response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H₂O₂. Interestingly, serial treatment of submillimolar levels of H₂O₂ induced a massive accumulation of ADLs. The toxicity arising from H₂O₂ determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs. The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H₂O₂. These results suggest that ADLs induced by submillimolar levels of H₂O₂ may be due to a Fenton-type reaction between H₂O₂ and intracellular iron ions in mammalian cells.     

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

H₂O₂ production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H₂O₂ production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H₂O₂ production was dominated by complex II (44% from site II_F in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site I_F). With added carnitine, H₂O₂ production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site III_Qo) and 25% from site I_F. Thus complex II (site II_F in the forward reaction) is a major source of H₂O₂ production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H₂O₂ production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site II_F in the reverse reaction). About half the rest was from site I_F, but a significant portion, ∼40 pmol H₂O₂·min⁻¹·mg protein⁻¹, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200 pmol H₂O₂·min⁻¹·mg protein⁻¹ under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H₂O₂ productionduring fatty acid oxidation under appropriate conditions.     

Novel iron complexes and chelators based on cis,cis-1,3,5-triaminocyclohexane: iron-mediated ligand oxidation and biochemical properties

 The interaction of Fe(II) and Fe(III) with the novel Fe(II) chelator N,N′N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (referred to as tachpyr) gives rise to six-coordinate, low-spin, cationic complexes of Fe(II). Tachpyr also displays a cytotoxicity toward cultured bladder cancer cells that is believed to involve coordination of intracellular iron. The anaerobic reaction of tachpyr with Fe(II) salts affords the Fe(II)-tachpyr²⁺ complex, but in presence of oxygen, oxidative dehydrogenation of one or two of the aminomethylene group(s) of the ligand occurs, with formal loss of H₂: R—N(H)—C(H)₂—(2-py) → R—N=C(H)—(2-py)+H₂. The resulting mono- and diimino Fe(II) complexes (denoted as [Fe(tachpyr-H₂)]²⁺ and [Fe(tachpyr-2H₂)]²⁺) are an inseparable mixture, but they may be fully oxidized by H₂O₂ to the known tris(imino) complex Fe(II)[cis,cis-1,3,5-tris(pyridine-2-carboxaldimino)cyclohexane]²⁺ (or [Fe(tachpyr-3H₂)]²⁺). Cyclic voltammetry of the imino complex mixture reveals an irreversible anodic wave at +0.78 V vs. NHE. Tachpyr acts as a reducing agent toward Fe(IIII) salts, affording the same two Fe(II) imino complexes as products. Tachpyr also reductively removes Fe(III) from an Fe(III)(ATP)₃ complex (which is a putative form of intracellular iron), producing the two Fe(II) imino complexes. Novel N-alkylated derivatives of tachpyr have been synthesized. N-Alkylation has two effects on tachpyr: lowering metal affinity through increased steric hindrance, and preventing Fe(III) reduction because oxidative dehydrogenation of nitrogen is blocked. The N-methyl tachpyr derivative binds Fe(II) only weakly as a high-spin complex, and no complexation or reduction of Fe(III) is observed. Corresponding to their inability to bind iron, the N-alkylated chelators are nontoxic to cultured bladder cancer cells. A tach-based chelator with three N-propyleneamino arms is also synthesized. Studies of the chemical and biochemical properties of this chelator further support a relationship between intracellular iron chelation, iron reduction, and cytotoxicity. Received: 23 March 1998 / Accepted: 1 June 1998     

Electrochemical detection of natural DNA damage induced by ferritin/ascobic acid/H2O2 system and amplification of DNA damage by endonuclease Fpg

Oppositely charged natural DNA and chitosan (CS) were assembled into (CS/DNA)_n layer-by-layer films on electrode surface, and Ru(bpy)₃²⁺ (bpy = bipyridyl) in solution was used as electroactive catalyst to detect damage of DNA in the films after incubation of the films in ferritin/AA/H₂O₂ solutions (AA = ascorbic acid). The mechanism of DNA damage caused by the ferritin/AA/H₂O₂ system was similar to that of Fenton reaction, where the reaction of ferritin with AA would release some Fe(II) ions from ferritin and the following reaction between Fe(II) ions and H₂O₂ would produce hydroxyl radical, which could induce DNA oxidative damage. This system provided an in vitro model to imitate the DNA damage indirectly induced by ferritin in real bio-systems. In addition, formamidopyrimidine DNA glycosylase (Fpg), a key endonuclease enzyme in repair of oxidatively damaged DNA, was used to amplify the DNA damage caused by ferritin/AA/H₂O₂ system through conversion of oxidative purine bases into single-strand breaks. The high sensitivity of electrocatalytic method with Ru(bpy)₃²⁺ as the catalyst in detection of DNA damage and the magnification function of Fpg may provide a novel idea to detect natural DNA lesion sensitively.