Typical Hydroxyl Radical Scavengers Research Articles

Prooxidant action of chebulinic acid and tellimagrandin I: Causing copper-dependent DNA strand breaks

The prooxidant activity of two hydrolysable tannins, chebulinic acid and tellimagrandin I, on plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts was assessed. The results revealed that both hydrolysable tannins in combination with Cu(II) induced DNA strand breaks in pBR322 plasmid DNA in a concentration-dependent manner. Chebulinic acid and tellimagrandin I also induced genomic DNA strand breaks of MRC-5 human embryo lung fibroblasts in the presence of Cu(II). After treatment with chebulinic acid or tellimagrandin I alone, the pBR322 plasmid DNA and genomic DNA in MRC-5 cells kept intact. In addition, addition of Cu(I) reagent bathocuproinedisulfonic acid or catalase markedly inhibited the copper-dependent DNA strand breaks by both tannins. However, three typical hydroxyl radical scavengers, DMSO, ethanol and mannitol, did not inhibit the DNA strand breaks. Both tannins were able to reduce Cu(II) to Cu(I). These results indicated that chebulinic acid and tellimagrandin I induced the copper-dependent strand breaks of pBR322 plasmid DNA and MRC-5 genomic DNA with prooxidant action, in which Cu(II)/Cu(I) redox cycle and H 2O 2 were involved and hydroxyl radical formation is important in the hypothetical mechanism by which DNA strand breaks are formed.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H 2O 2)-resistant clone HP100 cells, suggesting the involvement of H 2O 2. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using 32P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5′-ACG-3′ and 5′-CCG-3′ sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H 2O 2 with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H2O2 in the DNA damage. These findings suggest that H2O2 generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 μM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H2O2 production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.

Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol.

Catechol, a naturally occurring and an important industrial chemical, has been shown to have strong promotion activity and induce glandular stomach tumors in rodents. In addition, catechol is a major metabolite of carcinogenic benzene. To clarify the carcinogenic mechanism of catechol, we investigated DNA damage using human cultured cell lines and 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. Catechol increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is known to be correlated with the incidence of cancer, in a human leukemia cell line HL-60, whereas the amount of 8-oxodG in its hydrogen peroxide (H2O2)-resistant clone HP100 was not increased. The formation of 8-oxodG in calf thymus DNA was increased by catechol in the presence of Cu(2+). Catechol caused damage to 32P-labeled DNA fragments in the presence of Cu(2+). When NADH was added, DNA damage was markedly enhanced and clearly observed at relatively low concentrations of catechol (<1 microM). DNA cleavage was enhanced by piperidine treatment, suggesting that catechol plus NADH caused not only deoxyribose phosphate backbone breakage but also base modification. Catechol plus NADH frequently modified thymine residues. Bathocuproine, a specific Cu(+) chelator and catalase inhibited the DNA damage, indicating the participation of Cu(+) and H2O2 in DNA damage. Typical hydroxyl radical scavengers did not inhibit catechol plus Cu(2+)-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H2O2 with Cu(+) participates in catechol-induced DNA damage. Therefore, we conclude that oxidative DNA damage by catechol through the generation of H2O2 plays an important role in the carcinogenic process of catechol and benzene.

Requirement of glutathione and cysteine in guanine-specific oxidation of DNA by carcinogenic potassium bromate.

Potassium bromate (KBrO3), a food additive, induces renal-cell tumors in rats. KBrO3 induced 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human leukemia cell line HL-60 as well as in its H2O2-resistant clone, HP100, suggesting no involvement of H2O2. Depletion of GSH by buthionine sulfoximine (BSO) had a little inhibitory effect on KBrO3-induced 8-oxodG formation. However, the amount of 8-oxodG was still significantly higher than that in control, suggesting that intracellular Cys can affect KBrO3 to oxidize DNA, when GSH decreased. KBrO3 caused 8-oxodG in isolated DNA in the presence of GSH (tripeptide; gamma-GluCysGly), gamma-GluCys, CysGly, or Cys. Methional completely inhibited 8-oxodG formation induced by KBrO3 plus GSH, but typical hydroxyl radical scavengers, SOD and catalase, had little or no inhibitory effects. When bromine solution (BrO(-)) was used instead of BrO3(-), similar scavenger effects were observed. Experiments with 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene suggested that KBrO3 induced 8-oxodG formation at 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of GSH and that treatment of formamidopyrimidine-DNA glycosylase led to chain cleavages at the guanine residues. ESR spin-trapping studies showed that 1:2:2:1 quartet DMPO (5,5-dimethyl-1-pyrroline N-oxide) spectrum similar to DMPO/hydroxy radical (*OH) adduct, but the signals were not inhibited by ethanol. Therefore, the signal seemed not to be due to *OH but byproduct due to oxidation of DMPO by the reactive species. The signals were suppressed by the addition of dGMP, but not by other mononucleotides, suggesting the specific reactivity with guanine. On the basis of our results and previous literature, it is speculated that reduction of KBrO3 by SH compounds in renal proximal tubular cells yields bromine oxides and bromine radicals, which are the reactive species that cause guanine oxidation, leading to renal carcinogenesis of KBrO3.

Strategies for the spectroscopic characterization of irradiated proteins and other biomolecules

The molecular properties of proteins and other biomolecules have been investigated in aqueous solution, after preceding X- or UV-irradiation. Spectroscopic techniques (UV–Vis absorption, fluorescence, circular dichroism), size-exclusion chromatography, and other physicochemical techniques were applied to differentiate between various radiation effects. Numerous functional and structural changes of the irradiated samples could be monitored; the effects continued in the post-irradiation period. Modifications of radiation effects were achieved by various approaches. In particular, a variety of additives reduce the detrimental effects of radiation considerably. Typical hydroxyl radical scavengers act as potent protectives against X-irradiation, while compounds absorbing in the UV range protect biomolecules against UV light exposure. Thiols and ascorbate may provide a chemical repair of already damaged particles, restoring some of the original molecular features.

Enhancement by glucose of low density lipoprotein-oxidation by peroxynitrite.

Oxidation of low density lipoprotein (LDL) by copper ions is not influenced by glucose in a concentration range between 1 and 20 mM. LDL-oxidation by peroxynitrite or the simultaneous action of nitrogen monoxide and superoxide, produced by morpholino-sydnonimine (SIN-1) is considerably enhanced by typical hydroxyl-radical scavengers such as formate or mannitol and by glucose. Since both free radicals, nitrogen monoxide and superoxide, are produced by activated leukocytes and endothelial cells the presented reaction might represent a simple model for the cooperative function of reactive oxygen species (ROS) and glucose in certain diabetic blood vessel diseases such as atherogenesis and retinopathy.

N-acetylcysteine, a cancer chemopreventive agent, causes oxidative damage to cellular and isolated DNA

Although N-acetylcysteine is an antioxidant which has been expected to be a cancer chemopreventive agent, its safety and risk assessment have not been evaluated. N-acetylcysteine increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, whereas the amount of 8-oxodG in HP100, which is a hydrogen peroxide (H(2)O(2))-resistant cell line derived from HL-60, was not increased. To clarify the mechanism of cellular DNA damage, we investigated DNA damage and its site specificity induced by N-acetylcysteine, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. N-acetylcysteine induced extensive DNA damage in the presence of Cu(II). The DNA cleavage was enhanced by piperidine treatment, suggesting that N-acetylcysteine plus Cu(II) caused not only deoxyribose phosphate backbone breakage but also base modification. N-acetylcysteine plus Cu(II) frequently modified thymine and guanine residues. Bathocuproine, a specific Cu(I) chelator, and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. Typical hydroxyl radical scavengers did not inhibit N-acetylcysteine plus Cu(II)-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H(2)O(2) with Cu(I) participates in N-acetylcysteine plus Cu(II)-induced DNA damage. The content of 8-oxodG in calf thymus DNA was increased by N-acetylcysteine in the presence of Cu(II). The present study has demonstrated that N-acetylcysteine could induce metal-dependent H(2)O(2) generation and, subsequently, damage to cellular and isolated DNA. Therefore, it is reasonable to consider that N-acetylcysteine may have the dual function of carcinogenic and anti-carcinogenic potentials. This work requires further studies on safety and risk assessment of N-acetylcysteine.

Copper-mediated DNA damage by metabolites of p-dichlorobenzene.

p-Dichlorobenzene (p-DCB) has been reported to be carcinogenic for rodents, although it does not seem to be mutagenic in bacterial test systems. In this study, the mechanism of DNA damage by metabolites of p-DCB in the presence of metals was investigated by a DNA sequencing technique using 32P-labeled DNA fragments and by an electrochemical detector coupled to an HPLC. 2,5-Dichlorohydroquinone (DCHQ), one of the major metabolites, caused DNA damage in the presence of Cu(II). 2,5-Dichloro-p-benzoquinone (DCBQ) slightly induced DNA damage in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. DCHQ plus Cu(II) induced piperidine-labile sites at thymine residues at high frequency. A similar DNA cleavage pattern was observed with DCBQ plus Cu(II) in the presence of NADH. Both DCHQ and DCBQ plus NADH increased 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Typical hydroxyl radical scavengers showed no inhibitory effects on this Cu(II)-mediated DNA damage. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and hydrogen peroxide (H2O2) in the DNA damage. UV-visible and ESR spectroscopy has demonstrated that DCHQ is rapidly autoxidized to DCBQ via a semiquinone radical, even in the absence of metal ions, indicating that the semiquinone radical itself is not the main active species inducing DNA damage. These results suggest that a semiquinone radical produced by autoxidation of DCHQ and/or reduction of DCBQ by NADH reacts with O2 to form superoxide and subsequently H2O2. Consequently, it is considered that the active species derived from the reaction of H2O2 with Cu(I) participates in the DNA damage.

Peroxynitrite-dependent tryptophan nitration.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2.-) and nitric oxide (.NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pKa of 7.8. High concentrations of Fe(3+)-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 microM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal-independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 +/- 11 M-1 s-1 at 37 degrees C. The activation enthalpy was 9.1 +/- 0.3 kcal mol-1, and the activation entropy was -19 +/- 1 cal mol-1 K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.

Removal of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in water by oxidative, reductive, thermal or photochemical treatments

Removal of a highly potent Ames mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) with decrease of its mutagenic potency from an aqueous solution by oxidative (ozonation and addition of H 2O 2 with or without ferrous ion), reductive (addition of Na 2SO 3, Na 2S 2O 4 and ascorbic acid), thermal (heating in a boiling water bath) and photochemical (UV irradiation) treatments were investigated. More than 97% of removal of MX (initial amount of 1 mM) and its mutagenicy was achieved by the treatments with O 3 Fe 2+ and H 2 O 2 Fe 2+ . Hydroxyl radical was an important reactant in these treatments, since the removal of MX was strongly inhibited by the addition of typical hydroxyl radical scavengers, mannitol and L-tryptophan methyl ester. The removal of 1 mole of MX required 2 moles of hydroxyl radical accompanied by the release of 2 moles of chloride ion. In the thermal and photochemical treatments, 59% and 76% of MX and the same portions of its mutagenic potency were removed during 60 min, respectively. The release of 2 moles of chloride ion from 1 mole of MX were also observed in these treatments, but hydroxyl radical did not participitate in the decomposition. In the reductive treatments, 68% of MX was removed by the addition of Na 2SO 3 during 60 min. However, the decrease of mutagenic potency was 48%. It was strongly suggested that the lack of regularity between the amount of removed MX and the decrease of its mutagenic potency is due to weak addition of SO 3 2− to MX rather than reduction of MX.

Damage to the Bases in DNA Induced by Hydrogen Peroxide and Ferric Ion Chelates

Incubation of a number of ferric ion chelates with H2O2 at pH 7.4 generated a reactive species able to produce chemical modifications of the bases in DNA that are very similar to those produced in DNA by the hypoxanthine/xanthine oxidase system (Aruoma, O.I., Halliwell, B., and Dizdaroglu, M. (1989) J. Biol. Chem. 264, 13024-13028). Products were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring. Compared with other complexes used, ferric ion-nitrilotriacetic acid produced by far the largest amount of the base products. Typical hydroxyl radical scavengers and superoxide dismutase provided significant decreases in the yields of the products. On this basis, it is proposed that ferric ion complexes react with H2O2 to produce hydroxyl radical; this was also shown using the deoxyribose assay. Inhibition of product formation by superoxide dismutase suggests the involvement of superoxide radical in this reaction. It is likely that hydroxyl radical generated by reaction of the ferric ion-nitrilotriacetic acid complex with H2O2 contributes to the carcinogenicity and nephrotoxicity associated with this chelating agent.

NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. I. Oxidation of hydroxyl radical scavenging agents.

Isolated microsomes catalyze an NADPH-dependent oxidation of typical hydroxyl radical scavenging agents. To determine which microsomal components participate in the oxidation of the scavengers, experiments were carried out with purified NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from phenobarbital-treated rats. The production of ethylene from 4-ketothiomethylbutyrate or of formaldehyde from either dimethyl sulfoxide or tertiary butyl alcohol was measured in the presence of NADPH plus reductase or NADPH plus reductase plus cytochrome P-450. The reductase-dependent system itself catalyzed the oxidation of the three scavengers. Addition of cytochrome P-450 had no effect on the rates of oxidation of the scavengers. Relative rates of oxidation of the scavengers reflected the amount of the reductase in the assay system. Varying the amount of cytochrome P-450 at a fixed concentration of the reductase did not result in rates different from that observed in the absence of cytochrome P-450. Dilauroyl phosphatidylcholine appeared to have a stimulatory role in these oxidations, whereas cytochrome P-450 reductase and NADPH were obligatory components. The reductase-dependent oxidation of the scavengers was inhibited by superoxide dismutase, catalase, and competing hydroxyl radical scavenging agents suggesting that superoxide, hydrogen peroxide, and an oxygen radical with the oxidizing power of the hydroxyl radical played a role in these oxidations. Further, these oxidations were inhibited by the iron-chelator desferrioxamine but stimulated by either EDTA or by iron. These results suggest that an iron-catalyzed Haber-Weiss reaction might be involved in the mechanism by which purified cytochrome P-450 reductase mediates the oxidation of typical hydroxyl radical scavengers. The results demonstrated that NADPH-cytochrome P-450 reductase may represent an important locus of oxygen activation leading to the production of a highly oxidizing species characteristic of the hydroxyl radical.

Evidence for two ethanol oxidizing pathways in reconstituted mixed-function oxidase systems

The oxidation of ethanol and typical hydroxyl radical scavengers by NADPH-cytochrome P-450 reductase and cytochrome P-450 purified from phenobarbital-treated rats were studied. Ethanol and the scavengers could be oxidized by the reductase system itself. This system was inhibited by superoxide dismutase, competing hydroxyl radical scavengers and desferrioxamine, but stimulated by either EDTA or iron. These results suggest that an iron-catalyzed Haber-Weiss reaction might be involved in the mechanism by which the reductase mediates the oxidation of typical hydroxyl radical scavengers and ethanol. The addition of cytochrome P-450 had no effect on the oxidation of the scavengers, whereas the oxidation of ethanol was enhanced two-to three-fold over the reductase-dependent rate. The oxidation of ethanol was dependent on both the amount of reductase and P-450. There was no effect of competing scavengers, superoxide dismutase or desferrioxamine on the increased rate of ethanol oxidation produced upon addition of cytochrome P-450. Organic hydroperoxides supported the oxidation of ethanol, but not that of the scavengers when added directly to cytochrome P-450. These results suggest that two independent pathways are operative in supporting NADPH-dependent microsomal oxidation of ethanol. One pathway involves hydroxyl radicals which can be generated by the reductase, whereas the other pathway requires the combined presence of both the reductase and cytochrome P-450, and appears to be independent of oxygen radicals.