The enzymatic one-electron reduction of porphyrins to their anion free radicals

The anaerobic enzymatic one-electron reduction of uroporphyrin I (in the absence of light) by the ferredoxin/ferredoxin:NADP+ oxidoreductase system was investigated using NADPH as the source of reducing equivalents. The porphyrin anion free radical metabolite formed by one-electron reduction of the parent molecule was detected with ESR spectroscopy. The ESR spectrum exhibited a singlet (g = 2.0021) with a 5.4-G peak-to-peak linewidth. The reduction process was also investigated under aerobic conditions. The reduction of molecular oxygen to superoxide anion radical by the porphyrin anion radical was demonstrated by using the ESR technique of spin trapping. The ESR spectra of the spin-trapped oxygen-derived radicals were superoxide dismutase-sensitive and catalase-insensitive, supporting the assignment of the trapped radical to the superoxide anion radical. These aerobic experiments demonstrate electron transfer from the porphyrin anion radical to molecular oxygen. The anaerobic reduction of Photofrin II by hepatic microsomes and the ferredoxin/ferredoxin:NADP+ oxidoreductase system to a porphyrin anion radical was also investigated. Free radical formation by ferredoxin: NADP+ oxidoreductase is totally dependent upon ferredoxin. The ESR spectrum of this porphyrin free radical also exhibited a singlet (g = 2.0026) with a 15-G peak-to-peak linewidth.     

Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions

The photoreduction of crystal violet to a carbon-centered radical was detected directly by electron spin resonance (ESR) spectroscopy under anaerobic conditions. The linewidth (0.9 G) of this radical was less broad than the linewidth (11.0 G) of the free radical obtained in Trypanosoma cruzi incubations. No crystal violet radical could be detected under aerobic conditions. However, crystal violet was found to convert oxygen to superoxide anion and hydrogen peroxide in the presence of light. This superoxide anion and hydrogen peroxide formation was greatly enhanced by reducing agents such as NAD(P)H. In addition, irradiation of crystal violet did not generate detectable amounts of singlet oxygen.     

Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants

Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here, we report the direct measurement of the rate constant between hydroethidine and superoxide radical anion using the pulse radiolysis technique. This reaction rate constant was calculated to be ca. 2 x 10(6) M(-1) s(-1) in water:ethanol (1:1) mixture. The spectral characteristics of the intermediates indicated that the one-electron oxidation product of HE was different from the one-electron reduction product of ethidium (E+). The HPLC-electrochemical measurements of incubation mixtures containing HE and the oxygenated Fenton's reagent (Fe2+/DTPA/H2O2) in the presence of aliphatic alcohols or formate as a superoxide generating system revealed 2-OH-E+ as a major product. Formation of 2-OH-E+ by the Fenton's reagent without additives was shown to be superoxide dismutase-sensitive and we attribute the formation of superoxide radical anion to the one-electron reduction of oxygen by the DTPA-derived radical. Addition of tert-butanol, DMSO, and potassium bromide to the Fenton's system caused inhibition of 2-OH-E+ formation. Results indicate that reducing and oxidizing radicals have differential effects on the formation of 2-OH-E+.     

ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.     

Cephaloridine-induced lipid peroxidation initiated by reactive oxygen species as a possible mechanism of cephaloridine nephrotoxicity

Rat kidney microsomes reduced cephaloridine when incubated anaerobically with NADPH. Superoxide anion was generated in a concentration- and time-dependent manner when cephaloridine was incubated with rat kidney microsomes. Cephaloridine increased the in vitro peroxidation of rat kidney microsomal lipids in a concentration- and time-dependent manner. Cephaloridine-induced lipid peroxidation was inhibited by a combination of superoxide dismutase and catalase, by the hydroxyl radical scavengers, mannitol, (+)-cyanidanol-3 and by the singlet oxygen scavenger histidine in a concentration-dependent manner. It is proposed that cephaloridine nephrotoxicity may occur through the transfer of an electron from reduced cephaloridine to oxygen and subsequent formation of the superoxide anion, hydrogen peroxide, the hydroxyl radical and singlet oxygen. These activated oxygen species then are very likely to react with membrane lipids to induce lipid peroxidation and nephrotoxicity.     

Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species

We previously reported that benzo[a]pyrene (BaP) and UVA radiation synergistically induced oxidative DNA damage via 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vitro. The present study shows that microsomal BaP metabolites and UVA radiation potently enhance 8-OHdG formation in calf thymus DNA about 3-fold over the parent compound BaP. Utilization of various reactive oxygen species scavengers revealed that singlet oxygen and superoxide radical anion were involved in the 8-OHdG formation induced by microsomal BaP metabolites and UVA. Two specific BaP metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/-) (anti) (BPDE) and BaP-7,8-dione, were further tested for synergism with UVA. BaP-7,8-dione showed an effect on 8-OHdG formation induced by UVA radiation that was similar to that of the parent BaP, whereas BPDE exhibited significantly higher induction of 8-OHdG than BaP. At as low as 0.5 microM, BPDE plus UVA radiation substantially increased 8-OHdG levels about 25-fold over the parent BaP. BPDE increased the formation of 8-OHdG levels in both BPDE concentration- and UVA dose-dependent manners. Additionally, singlet oxygen was found to play a major role in 8-OHdG induction by BPDE and UVA. These results suggest that BaP metabolites such as BPDE synergize with UVA radiation to produce ROS, which in turn induce DNA damage.     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Superoxide anion production by the autoxidation of cytochrome P450cam

Chemiluminescence occurs on autoxidation of oxygenated ferrous cytochrome P450_cam and is abolished by reagents that scavenge free radicals, by superoxide dismutase and singlet oxygen quenchers. We attribute the chemiluminescence to the decay of an excited singlet oxygen which arises from a superoxide anion radical precursor.     

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

Possible contribution of oxyhemoglobin to the iron-induced hemolysis simultaneous effect of iron and hemoglobin on lipid peroxidation

The mechanism of iron toxicity in iron overloaded patients is not well established. A hypothesis was put forward that free radical processes are involved. Our earlier study indicates that iron-induced hemolysis is preceded by peroxidation of the membrane lipids. In the present work the simultaneous effect of iron and hemoglobin on lipid peroxidation was studied. It was found that in hemoglobin-containing liposome suspensions Fe2+ in concentrations above 10(-5) M inhibits the peroxidation, while Fe3+ drastically potentiates it, with concomitant transformation of oxyhemoglobin to methemoglobin. The experiments with scavengers of activated oxygen indicate superoxide anion radical (O-.2), hydroxyl radical (OH.) and singlet oxygen (1O2) participation. The possible mechanism of the phenomenon is discussed. A conclusion is drawn that the toxic effect of Fe3+ may be associated not only with iron--membrane interaction, but also with increased methemoglobin formation and O-.2 release.     

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O₂^-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.     

Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga, Haematococcus pluvialis

In a green alga, Haematococcus pluvialis, a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe²⁺ to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe²⁺ than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe²⁺ even in the presence of the inhibitors. The Fe²⁺ -enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe²⁺ in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.     

NADPH2 and organic hydroperoxide-dependent oxidation of adrenaline to adrenochromes in liver and brain microsomes

It was shown that oxidation of adrenaline to adrenochrome in microsomal membranes of the brain and liver in the presence of NADP . H2 or NAD . H2 is mainly accounted for by the formation of a superoxide anion radical. The formation of adrenochrome from adrenalin was found to depend on organic hydroperoxides (natural and synthetic). The organic hydroperoxide-dependent oxidation of adrenochrome involves singlet oxygen. In microsomal fractions of the liver the organic peroxide-dependent oxidation of adrenalin was catalyzed by cytochrome P-450.     

Hydroxylation of Phenol to Hydroquinone Catalyzed by A Human Myeloperoxidase-Superoxide Complex: Possible Implications In Benzene-Induced Myelotoxicity

Benzene, a known human rnyelotoxin and leukemogen is metabolized by liver cytochrome P-450 mono-oxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide disniutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.     

Hydroxylation of Phenol to Hydroquinone Catalyzed by A Human Myeloperoxidase-Superoxide Complex: Possible Implications In Benzene-Induced Myelotoxicity

Benzene, a known human rnyelotoxin and leukemogen is metabolized by liver cytochrome P-450 mono-oxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide disniutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.     

Superoxide, hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system.

1. Xanthine oxidase acting aerobically upon acetaldehyde was found to cause the peroxidation of linolenate. This was demonstrated by increased absorbance at 233 nm due to diene conjugation and by the detection of a lipid peroxide spot on the thin layer chromatograms. 2. Superoxide dismutase inhibited this lipid peroxidation, as did catalase, thus indicating that both O2- and H2O2 were essential intermediates. Scavengers of singlet oxygen also inhibited the peroxidation of linolenate, whereas scavengers of hydroxyl radical did not. These effects, which were observed in the absence of iron salts, led to the proposal that O2- and H2O2 can directly give rise to a singlet oxygen, as follows: O2- + H2O2 leads to OH- + OH. + O2. 3. This proposal was further supported through the use of 2,5-dimethylfuran, as an indicating scavenger of singlet oxygen. Thus, when this compound was exposed to a known source of singlet oxygen, it gave a product which was detectable by thin layer chromatography. This product was also observed when 2,5-dimethylfuran was exposed to the xanthine oxidase system, in which case its accumulation was prevented by superoxide dismutase or by catalase, but not by scavengers of hydroxyl radical.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.