Structural variety of copper(II)-peroxide adducts and its relevance to DNA cleavage

The reactivity of copper (II) compounds with several tetradentate ligands towards some spin-trapping reagents was studied in the presence of hydrogen peroxide. The compounds used in this study are roughly divided into two groups based on the reactivity towards 2,2,6,6-tetramethyl-4-piperidinol (and also 2,2,6,6-tetramethyl-4-piperidone), which are trapping agents for singlet oxygen. 1O2(1deltag); The A-group compounds exhibited a high activity to form the corresponding nitrone radical, which was detected by ESR spectroscopy, but corresponding activity of the B-group compounds was very low. The A-group compounds defined as above exhibited high activity for cleavage of DNA (supercoiled) Form I) in the presence of hydrogen peroxide, yielding DNA Form II (relaxed circular) or Form III (linear duplex) under our experimental conditions ([Cu (II)] = 0.1 approximately 0.5 mM). On the other hand, the B-group compounds effected complete degradation of the DNA (double-strand scission) under the same experimental conditions, formation of Form II or Form III DNA was negligible. Two different DNA cleavage patterns observed for A- and B-group compounds were elucidated by the different structural property of the copper (II)-peroxide adducts, which is controlled by the interaction through both DNA and the peripheral group of the ligand system.     

Vascular effects of singlet oxygen (1O2) generated by photo-excitation on adrenergic neurotransmission in isolated rabbit mesenteric vein

It has been suggested that reactive oxygen species (ROS) may be involved in the regulation of vascular tone. However, the nature of ROS effects on vascular sensitivity remains to be elucidated. The present study was designed to investigate the effects of ROS, especially 1O2, on neurotransmission at the sympathetic neurovascular junction. Basal noradrenaline (NA) release, release of NA induced by electrical stimulation (ES), and resting NA release at the sympathetic nerve terminals were determined using a superfusion technique. The amount of NA was determined by HPLC; isometric tension changes evoked by ES were also recorded simultaneously. 1O2 was generated from Rose Bengal by photo-activation. The generation of 1O2 in the superfusate was monitored by electron spin resonance (ESR) using the spin trap 2,2,6,6-tetramethyl-4-piperidinol throughout the experimental time course. The ESR results confirmed that 1O2 was generated by photo-activation of Rose Bengal via the formation of 2,2,6,6-tetramethyl-4-hydroxyl-piperidinyloxy. Exposure of helical strips of rabbit mesenteric vein to 1O2 induced a significant increase in tension and NA release during the basal period, but had no effect on ES-induced release. L-histidine, an 1O2 scavenger, significantly inhibited the observed effects on vascular tension and NA release in response to 1O2. These results suggest that 1O2 may induce NA-mediated vasoconstriction at the postjunctional site, and may be associated with Ca(2+)-independent NA release from the prejunctional site of adrenergic neurotransmission.     

Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion.

Although photoexcited TiO2 has been known to initiate various chemical reactions, such as the generation of reactive oxygen species, precise mechanism and chemical nature of the generated species remain to be elucidated. The present work demonstrates the generation of singlet oxygen by irradiated TiO2 in ethanol as measured by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) as a 1O2-sensitive trapping agent. Under identical conditions, the superoxide ion was also detected by spin trapping agent 5,5-dimethyl-pyrroline-N-oxide (DMPO). Kinetic analysis in the presence of both 4-oxo-TMP and DMPO revealed that singlet oxygen is produced directly at the irradiated TiO2 surface but not by a successive reaction involving superoxide anion. The basis for this view is the fact that DMPO added in the mixture increased the signals responsible for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), a reaction product of 4-oxo-TMP and 1O2. The detailed mechanism for the generation of 1O2 and superoxide ion by irradiated TiO2 and reactions between these species and DMPO are discussed.     

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity

AimsHigh-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.MethodsReconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL + LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.ResultsrHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.ConclusionsIncreasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.Non-standard abbreviations and acronyms: AAPH, 2,2′-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.     

Formation of phospholipid hydroperoxides and its inhibition by alpha-tocopherol in rat brain synaptosomes induced by peroxynitrite

Peroxynitrite resulted from the reaction of nitric oxide and superoxide anion has been implicated in the genesis of neurotoxicity. In this study, the oxidation of phospholipids in rat brain synaptosomes induced by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1) was studied in vitro. The formation and accumulation of phospholipid hydroperoxides, including phosphatidylcholine hydroperoxide (PCOOH) and phosphatidyl-ethanolamine hydroperoxide (PEOOH) in rat brain synaptosomes induced by peroxynitrite, were observed. PEOOH and PCOOH were formed rapidly and SIN-1 concentration-dependently. The hydroperoxides formed in synaptosomes were unstable and it was suggested that phospholipase A2 played a role in degradation of the hydroperoxides. The endogenous alpha-tocopherol acted as a potent antioxidant. It was oxidized very rapidly and concentration-dependently by SIN-1 to alpha-tocopheryl quinone. Furthermore, uric acid was found to be an effective antioxidant in inhibiting oxidative damage to synaptosomal lipids induced by SIN-1. The results provide direct evidence to show that peroxynitrite can not only deplete alpha-tocopherol, but also cause production of phospholipid hydroperoxides resulting in disrupted brain tissue.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label.

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

Sensitivity of mouse lung fibroblasts heterozygous for GPx4 to oxidative stress

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a member of the family of selenium-dependent enzymes that catalyze the reduction of cell membrane-bound phospholipid hydroperoxides in situ and thus protects against membrane damage. Overexpression of GPx4 protects cultured cells from phosphatidylcholine hydroperoxide (PCOOH)-induced loss of mitochondrial membrane potential and blocks cell death induced by treatment with various apoptotic agents. We have generated mice that are heterozygous for a GPx4 null allele (GPx4 +/-); the homozygous null genotype is embryonic lethal. We report that cultured lung fibroblasts (LFs) isolated from adult GPx4 +/- mice had approximately 50% of the GPx4 activity of LFs from GPx4 +/+ mice and were significantly more susceptible to H2O2, cadmium, and cumene hydroperoxide-induced cytotoxicity, as measured by neutral red assay. Both GPx4 +/+ and GPx4 +/- LFs were susceptible to PCOOH-induced cytotoxicity at a high PCOOH concentration. We also found that GPx4 +/- LFs have lower mitochondrial membrane potential, greater cardiolipin oxidation, and lower amounts of reduced thiols relative to GPx4 +/+ LFs, but are more resistant than GPx4 +/+ LFs to further decrements in these endpoints following PCOOH treatment. These results suggest that adult lung fibroblasts deficient in GPx4 may have upregulated compensatory mechanisms to deal with the highly oxidized environment in which they developed.     

Major differences in oxysterol formation in human low density lipoproteins (LDLs) oxidized by *OH/O2*- free radicals or by copper.

The aim of our study was to determine the oxysterol formation in low density lipoproteins (LDLs) oxidized by defined oxygen free radicals (*OH/O2*-). This was compared to the oxysterol produced upon the classical copper oxidation procedure. The results showed a markedly lower formation of oxysterols induced by *OH/O2*- free radicals than by copper and thus suggested a poor ability of these radicals to initiate cholesterol oxidation in LDLs. Moreover, the molecular species of cholesteryl ester hydroperoxides produced by LDL copper oxidation seemed more labile than those formed upon *OH/O2*(-)-induced oxidation, probably due to their degradation by reaction with copper ions.     

Photodynamic action of C-phycocyanins obtained from marine and fresh water cyanobacterial cultures: a comparative study using EPR spin trapping technique

C-phycocyanins, major biliproteins of blue green algae (cyanobacteria), widely used as colourants in food and cosmetics are known for their antioxidant as well as therapeutic potential. Recent claims indicating phycobiliproteins exert stronger photodynamic action on tumor cells than clinically approved hematoporphyrin derivatives motivate us to investigate the photodynamic action of two newly isolated C-phycocyanins from Phormidium [PHR] and Lyngbya [LY] spp, respectively in comparison with known C-phycocyanin from Spirulina sp. [SPI]. Photolysis of air saturated solutions of PHR, LY and SPI in the presence of 2,2,6,6-Tetramethyl piperidinol (TEMPL) generated three line EPR spectrum characteristic of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxyl (TEMPOL). The increase in intensity of the EPR spectrum with time of irradiation and decrease in intensity, in the presence of 1O2 quencher DABCO confirm the formation of 1O2. Photoirradiation in the presence of spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) generated EPR signal characteristic of O2(-) adduct. Efficiency of 1O2 generation is of the order LY > PHR> SPI. The yield of reactive oxygen species (ROS) generation is found to be 1O2>O2(-) indicating type II mechanism to be the prominent pathway for photosensitation by phycocyanins.     

Detection and time-course analysis of phospholipid hydroperoxide in soybean seedlings after treatment with fungal elicitor, by chemiluminescence-HPLC assay.

Direct evidence for the existence of phosphatidylcholine hydroperoxide (PCOOH) and the determination of the changes in PCOOH after treatment of soybean seedlings with fungal elicitor, were examined using chemiluminescence-HPLC of hydroperoxide specific assay. PCOOH was transiently accumulated within 6 h after elicitor treatment but such an event was not observed in control plants, thus suggesting that accumulation of PCOOH was a response to fungal elicitor in the host plant. The result suggested that singlet oxygen had participated in the lipid hydroperoxidation, since non-conjugated fatty acid hydroperoxide isomers due to the singlet oxygen oxidation were detected in the phospholipid fraction. The reaction of lipid hydroperoxidation, preceding the induction of a defense compound, may be involved in the triggering of a plant defense mechanism against fungal invasion.     

Rapid determination of internal volumes of membrane vesicles with electron spin resonance-stopped flow technique

We have developed an electron spin resonance (ESR)-stopped flow technique and employed it for the simple and rapid determination of internal volumes of biomembrane vesicles and liposomes. A vesicle suspension containing a neutral and membrane-permeable spin label, 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPONE), was mixed in the stopped-flow apparatus with an isotonic solution of relatively impermeable line broadening agents, potassium tris(oxalato)chromate(III) or potassium ferricyanide, and an ESR spectrum was recorded. From the relative intensity of the sharp triplet signal due to TEMPONE in the aqueous space within vesicles, the determination of the internal aqueous volume was straightforward. Using this technique, it is possible to measure intravesicular volumes in 0.1 s. The internal volume of sonicated phospholipid vesicles was approximately 0.3 microliter/mg lipid. The light fraction of sarcoplasmic reticulum membrane vesicles isolated from rabbit skeletal muscle was estimated to have an internal volume of 2.2-2.6 microliter/mg protein in its resting state. Activation of Ca2+ pumps in the membrane upon addition of ATP and Ca2+ ions decreased the internal volume by about 10%. This finding supports the hypothesis that the Ca2+ pump is electrogenic and that the efflux of potassium ions compensates for the influx of positive charges. The present technique is widely applicable to the simple and rapid determination of the internal volumes of membrane vesicles.     

ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(II) complex of glycylglycyl-L-histidine

ESR studies using spin traps, 5,5-dimethylpyrroline-N-oxide and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone, revealed that hydroxyl radical adducts are produced by the decomposition of hydrogen peroxide in the presence of nickel(II) oligopeptides. Order of catalytic activities of nickel(II) oligopeptides used in the production of hydroxyl radical adducts was tetraglycine greater than pentaglycine greater than triglycine greater than GlyGly, GlyHis. Ni(II) GlyGlyHis plus hydrogen peroxide produced superoxide in addition to hydroxyl radical adduct. Trapping experiments with 2,2,6,6-tetramethyl-4-piperidone suggested that singlet oxygen was generated by the reaction of hydrogen peroxide with Ni(II) GlyGlyHis, but not in the case of tetraglycine, pentaglycine, triglycine, GlyGly or GlyHis.     

Increased endogenous ascorbyl free radical formation with singlet oxygen scavengers in reperfusion injury: an EPR and functional recovery study in rat hearts.

The objective of this study was to investigate the effect of singlet oxygen ((1)O2) scavengers on functional recovery and ascorbyl free radical (AFR) formation in isolated ischemic rat hearts. Hearts were subjected to 40 min. of global ischemia followed by 30 min. of reperfusion. Hemodynamics were measured as heart rate (HR), coronary flow (CF), left ventricular developed pressure (LVDP) and contractility (dP/dt). Electron paramagnetic resonance (EPR) spectroscopy was used to measure AFR release in coronary perfusate during the first two min. of reperfusion as a function of ROS scavengers. Relative to ischemic controls the administration of the (1)O2 scavengers 2,2,6,6-tetramethyl-4-piperidone x HCl (4-oxo-TEMP), carnosine (beta-alanyl-L-histidine) or a combination of the two significantly improved functional recovery as measured by LVDP. While no AFR signal was detected in coronary perfusate collected during preischemic perfusion with and without (1)O2 scavengers, the AFR background signal due to ischemia was significantly increased with the (1)O2 and *O2- scavengers. No such increase was observed with the hydroxyl radical (*OH) scavenger mannitol. Besides the AFR increase with the (1)O2 and *O2- scavengers the functional recovery was only significantly improved with the (1)O2 scavengers. In contrast to previous AFR studies we found with endogenous AFR that an increased AFR formation is not necessarily only reflecting increased oxidative stress but can also report improved functional recovery. Combining the hemodynamic data with increased AFR formation in the presence of several different ROS scavengers gives supportive evidence for (1)O2 also being involved in reperfusion injury.     

Photochemical Properties of Camptothecin in the Presence of Copper(II) Ions: The Role of Radicals as Prospective Species in Photodynamic Therapy

Camptothecin (CPT) is an anticancer drug that inhibits topoisomerase I (Topo I) by forming a ternary DNA-CPT-Topo I complex. However, it has also been shown that UVA-irradiated CPT in the absence of Topo I produces significant DNA damage to cancer cells. In this work, we explored and identified free radicals generated in these processes. From the low-temperature EPR spectrum of Cu(II)-CPT complex, a proximity between Cu(II) ion and 20-hydroxy group of lactone E ring of CPT is proposed. Upon irradiation (λ = 365 nm) of the Cu(II)-CPT complex in de-oxygenated dimethylsulfoxide (DMSO), the EPR signal of Cu(II) measured in situ at room temperature shows formal first-order exponential decay with a formal half-life of 11 min. By the use of a specific Cu(I) chelating agent, neocuproine, it was shown that, during this process, Cu(II) is reduced to Cu(I). The loss in EPR signal intensity of the Cu(II)-CPT complex upon irradiation is accompanied by the appearance of a new EPR signal at g ≈ 2.0022. Application of the spin trap nitrosodurene (ND) revealed that the main radical product formed upon continuous irradiation of CPT in DMSO solutions is the hydroxyl radical (trapped in DMSO as the ^•CH₃ adduct) and superoxide radical. Application of 2,2,6,6-tetramethyl-4-piperidinol has revealed that irradiation of CPT in aerated DMSO solution also leads to formation of singlet oxygen (¹O₂). Our spectroscopic experiments indicate that CPT is a promising photosensitizer and that radicals and singlet oxygen generated upon illumination play a central role in DNA cleavage and in the induction of apoptosis in cancer cells.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

Singlet oxygen scavenging activity of plastoquinol in photosystem II of higher plants: Electron paramagnetic resonance spin-trapping study

Singlet oxygen (¹O₂) scavenging activity of plastoquinol in photosystem II (PSII) of higher plants was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that illumination of spinach PSII membranes deprived of intrinsic plastoquinone results in ¹O₂ formation, as monitored by TEMPONE EPR signal. Interestingly, the addition of exogenous plastoquinol (PQH₂-1) to PQ-depleted PSII membranes significantly suppressed TEMPONE EPR signal. The presence of exogenous plastoquinols with a different side-chain length (PQH₂-n, n isoprenoid units in the side chain) caused a similar extent of ¹O₂ scavenging activity. These observations reveal that plastoquinol exogenously added to PQ-depleted PSII membranes serves as efficient scavenger of ¹O₂.     

Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmitters and neurotoxins: Role of reactive oxygen species

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383±46 adducts/10(6) nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2'-deoxyguanosine (122±19 adducts/10(6) nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H(2)O(2), Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible.     

Pure forms of the singlet oxygen sensors TEMP and TEMPD do not inhibit Photosystem II

In a recent article (Hakala-Yatkin and Tyystj?rvi BBA 1807 (2011) 243-250) it was reported that the singlet oxygen spin traps 2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidone (TEMPD) inhibit Photosystem II (PSII), the water oxidizing enzyme. O? evolution, chlorophyll fluorescence and thermoluminescence were measured and were shown to be greatly affected by these chemicals. This work casts doubts over an earlier body of work in which these chemicals were used as spin traps for monitoring 1O? production when PSII was inhibited by high light intensities. Here we show that these spin probes hardly affect PSII. We show that the commercial batches of TEMPD and TEMP used by Hakala-Yatkin and Tyystj?rvi contained impurities and/or derivatives that inhibited PSII and caused the specific effects on fluorescence. Earlier work that used pure spin traps to measure 1O? during photoinhibition, thus remains valid. However, concern must be expressed towards using these spin traps without proper controls.