Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA.

Membrane lipid peroxidation processes yield products that may react with DNA to cause oxidative modifications. We have investigated this possibility and have found that calf thymus DNA exposed to autooxidized lipids causes the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 8-OH-dG formation in DNA was measured using high-pressure liquid chromatography with electrochemical detection. Methyl linolenate oxidized for different lengths of time was exposed to DNA. The amount of 8-OH-dG formed in DNA was proportional to the amount of lipid peroxidation as measured by the thiobarbituric reactive substances present. The formation of 8-OH-dG in DNA by autooxidized methyl linolenate was dependent on the presence of the transition metal ions Cu or Fe and was inhibited by various scavengers, including superoxide dismutase and catalase. This implicates the involvement of oxygen free radicals in the process. Liposomes formed from phosphatidylcholine (82%) and methyl arachidonate (18%) were peroxidized for different lengths of time and then exposed to DNA. 8-OH-dG was formed in DNA by exposure to Cu(II) and peroxidized liposomes. Under these conditions, Fe(III) was slightly less effective than Cu(II) in mediating 8-OH-dG formation. These observations clearly show that 8-OH-dG formation in DNA may result from processes that may occur during intracellular lipid peroxidation.     

Salsolinol, a naturally occurring tetrahydroisoquinoline alkaloid, induces DNA damage and chromosomal aberrations in cultured Chinese hamster lung fibroblast cells

Salsolinol (SAL) is a tetrahydroisoquinoline neurotoxin that has been speculated to contribute to pathophysiology of Parkinson's disease and chronic alcoholism. The compound is also found in certain beverages and food stuffs, including soy sauce, beer and bananas. Despite potential human exposure to SAL and its endogenous formation, little is known about the genotoxic or carcinogenic potential of this substance. In the present investigation, SAL induced DNA damage in cultured Chinese hamster lung (CHL) fibroblasts as assessed by single cell gel electrophoresis (Comet). CHL cells treated with SAL also exhibited higher frequencies of chromosomal aberrations than did vehicle-treated controls. Our recent study has revealed that SAL in combination with Cu(II) causes the strand scission in phiX174 supercoiled DNA [Neurosci. Lett. 238 (1997) 95]. In line with this notion, addition of cupric ion potentiated the DNA damaging and clastogenic activity of SAL. Antioxidant vitamins, such as Vitamin C and Vitamin E, and reduced glutathione inhibited clastogenicity of SAL, suggesting the involvement of reactive oxygen species (ROS) in SAL-induced DNA damage and genotoxicity in CHL cells.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his(+) reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H(2)O(2)/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H(2)O(2)/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H(2)O(2) formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

Oxidative DNA damage induced by aminoacetone, an amino acid metabolite

We investigated DNA damage induced by aminoacetone, a metabolite of threonine and glycine. Pulsed-field gel electrophoresis revealed that aminoacetone caused cellular DNA cleavage. Aminoacetone increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human cultured cells in a dose-dependent manner. The formation of 8-oxodG in calf thymus DNA increased due to aminoacetone only in the presence of Cu(II). DNA ladder formation was observed at higher concentrations of aminoacetone than those causing DNA cleavage. Flow cytometry showed that aminoacetone enhanced the generation of hydrogen peroxide (H2O2) in cultured cells. Aminoacetone caused damage to 32P-5'-end-labeled DNA fragments, obtained from the human c-Ha-ras-1 and p53 genes, at cytosine and thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. Analysis of the products generated from aminoacetone revealed that aminoacetone underwent Cu(II)-mediated autoxidation in two different pathways: the major pathway in which methylglyoxal and NH+4 are generated and the minor pathway in which 2,5-dimethylpyrazine is formed through condensation of two molecules of aminoacetone. These findings suggest that H2O2 generated by the autoxidation of aminoacetone reacts with Cu(I) to form reactive species capable of causing oxidative DNA damage.     

Copper-activated DNA photocleavage by a pyridine-linked bis-acridine intercalator

We report the synthesis of new photonuclease 4 consisting of two acridine rings joined by a pyridine-based copper binding linker. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of copper(II) (419 nm, 22 degrees C, pH 7.0). Viscometric data indicate that 4 binds to DNA by monofunctional intercalation, and equilibrium dialysis provides an estimated binding constant of 1.13 x 105 M-1 for its association with calf thymus DNA. In competition dialysis experiments, 4 exhibits preferential binding to GC-rich DNA sequences. When Cu(II) is added at a ligand to metal ratio of 1:1, electrospray ionization mass spectrometry demonstrates that compound 4 undergoes complex formation, while thermal melting studies show a 10 degrees C increase in the Tm of calf thymus DNA. Groove binding and intercalation are suggested by viscometric data. Finally, colorimetric and scavenger experiments indicate that the generation of Cu(I), H2O2, and superoxide contributes to the production of DNA frank strand breaks by the Cu(II) complex of 4. Whereas the strand breaks are distributed in a relatively uniform fashion over the four DNA bases, subsequent piperidine treatment of the photolysis reactions shows that alkaline labile lesions occur predominantly at guanine.     

Formation of 8-hydroxydeoxyguanosine in DNA treated with fecapentaene-12 and -14

Fecapentaene-12 and -14, direct-acting mutagens in human feces, were found to hydroxylate the C-8 position of guanine residues in DNA in vitro. Fecapentaene-12 or -14 was incubated with 0.5 mg of calf thymus DNA in 1 ml of reaction mixture at pH 7.4 for 2 h at 37 degrees C in the dark, and then 8-hydroxydeoxyguanosine (8-OH-dG) was analyzed. In these conditions 8-OH-dG was formed dose-dependently at levels of 1.1-4.6 residues/10(4) dG with concentrations of 0.5-3.0 mM of fecapentaene-12. Similar results were obtained with fecapentaene-14. The amount of 8-OH-dG in untreated DNA was 0.2-0.3 residue/10(4) dG.     

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his⁺ reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide (t-BOOH). DNA strand breaks by ROS derived from H₂O₂/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H₂O₂/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H₂O₂ formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.     

Distinct mechanisms of oxidative DNA damage by two metabolites of carcinogenic o-toluidine

Mechanisms of DNA damage by metabolites of carcinogenic o-toluidine in the presence of metals were investigated by the DNA sequencing technique using (32)P-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolite, caused DNA damage in the presence of Cu(II). Predominant cleavage sites were thymine and cytosine residues. o-Nitrosotoluene, a minor metabolite, did not induce DNA damage even in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. The DNA cleavage pattern was similar to that in the case of 4-amino-3-methylphenol. Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. Typical free hydroxyl radical scavengers showed no inhibitory effects on the DNA damage. o-Toluidine metabolites increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). UV-visible and ESR spectroscopic studies have demonstrated that 4-amino-3-methylphenol is autoxidized to form the aminomethylphenoxyl radical and o-nitrosotoluene is reduced by NADH to the o-toluolhydronitroxide radical in the presence and absence of Cu(II). Consequently, it is considered that these radicals react with O(2) to form O(-)(2) and subsequently H(2)O(2), and that the reactive species generated by the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. Metal-mediated DNA damage by o-toluidine metabolites through H(2)O(2) seems to be relevant for the expression of the carcinogenicity of o-toluidine.     

Formation of 8-hydroxydeoxyguanosine in DNA treated with fecapentaene- 12 and -14

Fecapentaene-12 and -14, direct-acting mutagens in human feces, were found to hydroxylate the C-8 position of guanine residues in DNA in vitro. Fecapentaene-12 or -14 was incubated with 0.5 mg of calf thymus DNA in 1 ml of reaction mixture at pH 7.4 for 2 h at 37°C in the dark, and then 8-hydroxydeoxyguanosine (8-OH-dG) was analyzed. In these conditions 8-OH-dG was formed dose-dependently at levels of 1.1–4.6 residues/10⁴ dG with concentrations of 0.5–3.0 mM of fecapentaene-12. Similar results were obtained with fecapentaene-14. The amount of 8-OH-dG in untreated DNA was 0.2–0.3 residue/10⁴ dG.     

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.     

Oxidative DNA damage by a metabolite of carcinogenic and reproductive toxic nitrobenzene in the presence of NADH and Cu(II)

The mechanism of DNA damage induced by metabolites of nitrobenzene was investigated in relation to the carcinogenicity and reproductive toxicity of nitrobenzene. Nitrosobenzene, a nitrobenzene metabolite, induced NADH plus Cu(II)-mediated DNA cleavage frequently at thymine and cytosine residues. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Typical free hydroxyl radical scavengers showed no inhibitory effects on DNA damage. Nitrosobenzene caused the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of NADH and Cu(II). ESR spectroscopic study has confirmed that nitrosobenzene is reduced by NADH to the phenylhydronitroxide radical even in the absence of Cu(II). These results suggest that nitrosobenzene can be reduced non-enzymatically by NADH, and the redox cycle reaction resulted in oxidative DNA damage due to the copper-oxygen complex, derived from the reaction of Cu(I) with H2O2.     

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.     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

Bilirubin-Cu(II) complex degrades DNA.

It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers.     

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4beta-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H2O2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H2O2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H2O2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) demonstrated that procyanidin B2 decreased the signal of M4PO-OH from H2O2 and Fe(II), whereas procyanidin B2 enhanced the signal from H2O2 and Cu(II). As an antioxidant mechanism, UV-visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H2O2 and metal ions.     

Metal ion-directed cooperative DNA binding of small molecules

Compound (1), which consists of an oxine and a pyridinium group, was synthesized as a metal-responsive DNA binding ligand. Two 1s coordinate to a Cu(II) to form a stable dimer (1(2)-Cu), even in the presence of DNA. The binding of 1 with sonicated calf thymus DNA was enhanced by ca. 10(3) times after forming the dimer; the binding constants were estimated to be 3.2 x 10(4)M(-1) and 2.4 x 10(7)M(-1) in the absence and the presence, respectively, of a half mole of Cu(II). The enormous acceleration of the binding is partly attributed to the generation of a dicationic charge by the formation of the dimer. High cooperativity between dimers could be also responsible; dimers would gather along the duplex as a template to form 1D spiral aggregates.     

Identification of Possible Reactive Oxygen Species Involved in Ultraviolet Radiation-Induced Oxidative DNA Damage

We have previously demonstrated that each region of the ultraviolet (UV) spectrum (UVA, UVB, and UVC) induces the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells in a fluence-dependent manner. In the present study, we further characterize the possible reactive oxygen species (ROS) that are involved in the induction of 8-oxodGuo by UV radiation. Sodium azide, a singlet oxygen (¹O₂) scavenger though its quenching effect on HO· was also reported, inhibited 8-oxodGuo production in calf thymus DNA exposed to UVA, UVB, or UVC in a concentration-dependent fashion with maximal quenching effect of over 90% at a concentration of 10 mM. Catalase, at a concentration of 50 U/ml, reduced the yields of UVA- and UVB-induced 8-oxodGuo formation by approximately 50%, but had little effect on UVC-induced 8-oxodGuo production. In contrast, 50 U/ml of superoxide dismutase (SOD) did not affect induction of 8-oxodGuo by any portion of the UV spectrum. Hydroxyl radical (HO·) scavengers mannitol and dimethylsulfoxide (DMSO) moderately reduced the levels of 8-oxodGuo induced by UVA and UVB, but not those by UVC. Instead, mannitol and DMSO enhanced the formation of 8-oxodGuo induced by UVC. These results suggest that certain types of ROS are involved in UV-induced 8-oxodGuo formation with ¹O₂ playing the predominant role throughout the UV spectrum. Except for UVC, other ROS such as hydrogen peroxide (H₂O₂) and HO· may also be involved in UVA- and UVB-induced oxidative DNA damage. Superoxide anion appears not to participate in UV-induced oxidation of guanosine in calf thymus DNA, as SOD did not display any quenching effects.     

Cytotoxicity and site-specific DNA damage induced by nitroxyl anion (NO(-)) in the presence of hydrogen peroxide. Implications for various pathophysiological conditions.

Nitroxyl anion (NO(-)), the one-electron reduction product of nitric oxide (NO(.)), is formed under various physiological conditions. We have used four different assays (DNA strand breakage, 8-oxo-deoxyguanosine formation in calf thymus DNA, malondialdehyde generation from 2'-deoxyribose, and analysis of site-specific DNA damage using (32)P-5'-end-labeled DNA fragments of the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene) to study the effects of NO(-) generated from Angeli's salt on DNA damage. It was found that strong oxidants are generated from NO(-), especially in the presence of H(2)O(2) plus Fe(III)-EDTA or Cu(II). NO(.) released from diethylamine-NONOate had no such effect. Distinct effects of hydroxyl radical (HO(.)) scavengers and patterns of site-specific DNA cleavage caused by Angeli's salt alone or by Angeli's salt, H(2)O(2) plus metal ion suggest that NO(-) acts as a reductant to catalyze the formation of the HO(.) from H(2)O(2) plus Fe(III) and formation of Cu(I)-peroxide complexes with a reactivity similar to HO(.) from H(2)O(2) and Cu(II). Angeli's salt and H(2)O(2) exerted synergistically cytotoxic effects to MCF-7 cells, determined by lactate dehydrogenase release assay. Thus NO(-) may play an important role in the etiology of various pathophysiological conditions such as inflammation and neurodegenerative diseases, especially when H(2)O(2) and transition metallic ions are present.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG 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, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.