Attenuation of radiation-induced genomic instability by free radical scavengers and cellular proliferation.

To investigate the mechanisms of radiation-induced chromosomal instability, cells were irradiated in the presence of the free radical scavengers DMSO, glycerol, or cysteamine, in the presence of DMSO while frozen, or held in confluence arrest post-irradiation to permit cells to repair potentially lethal DNA damage. Clones derived from single progenitor cells surviving each treatment were then analyzed for the subsequent development of chromosomal instability. The presence of scavengers (+/- freezing) during irradiation, and the recovery from potentially lethal damage after irradiation led to an increase in cell survival that was accompanied by a decrease in the initial yield of chromosomal rearrangements. Furthermore, analysis of over 400 clones and 80,000 metaphases indicates that these same treatments reduced the incidence of instability at equitoxic doses when compared to controls irradiated in the absence of scavengers at ambient temperature. Results suggest that preventing reactive species from damaging DNA, promoting chemical repair of ionized DNA intermediates, or allowing enzymatic removal of genetic lesions, represent measures that reduce the total burden of DNA damage and reduce the subsequent onset of radiation-induced genomic instability.     

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.     

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.     

Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells

Using siRNA technology, we down-regulated in human B-lymphoblastoid TK6 cells the two major oxidative DNA glycosylases/AP lyases that repair free radical-induced base damages, hNTH1 and hOGG1. The down-regulation of hOGG1, the DNA glycosylase whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, resulted in reduced radiation cytotoxicity and decreased double strand break (DSB) formation post-irradiation. This supports the idea that the oxidative DNA glycosylases/AP lyases convert radiation-induced clustered DNA lesions into lethal DSBs and is in agreement with our previous finding that overexpression of hNTH1 and hOGG1 in TK6 cells increased radiation lethality, mutant frequency at the thymidine kinase locus and the enzymatic production of DSBs post-irradiation [N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair (Amst) 3 (2004) 1323-1334]. Interestingly, cells deficient in hNTH1, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more radiosensitive but at the same time fewer DSBs were formed post-irradiation. These results indicate that hNTH1 plays two roles in the processing of radiation damages: repair of potentially lethal single lesions and generation of lethal DSBs at clustered damage sites. In contrast, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, the base excision repair pathway functioned to protect the cells. Here, overexpression of hNTH1 and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death.     

Radiation protection of in vitro mammalian cells: effects of hydroxyl radical scavengers on the slopes and shoulders of survival curves.

We have tested several chemical compounds, characterized and widely used as hydroxyl radical (.OH) scavengers, for their effects on the radiation sensitivity of Chinese hamster V79 cells irradiated in air or nitrogen. Our purpose is to reexamine the proposed relationship between the level of protection and the rates at which the scavengers react with .OH. We found that the additives can have two apparently independent effects on the shape of survival curves: a reduction in sensitivity (i.e., "protection," a decrease in the value of k) and an increase in the size of the shoulder of the survival curve (an increase in the value of Dq). We measured intracellular scavenger concentrations, and, using these values in our analysis, we found that neither of the two effects is correlated with the rates at which the scavengers react with .OH. Although these results could mean that .OH do not cause lethal damage, the interpretation we believe most probably correct is that these scavengers protect in multiple ways. The protection would occur in addition to or instead of simple .OH removal.     

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.     

Yield of DNA strand breaks after base oxidation of plasmid DNA

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.     

Site-specific oxidation at GG and GGG sequences in double-stranded DNA by benzoyl peroxide as a tumor promoter

Benzoyl peroxide (BzPO), a free-radical generator, has tumor-promoting activity. As a method for approaching the mechanism of tumor promoter function, the ability of oxidative DNA damage by BzPO was investigated by using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. BzPO induced piperidine-labile sites at the 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of Cu(I), whereas the damage occurred at single guanine residues of single-stranded DNA. Both methional and dimethyl sulfoxide (DMSO) inhibited DNA damage induced by BzPO and Cu(I), but typical hydroxyl radical ((*)OH) scavengers, superoxide dismutase (SOD) and catalase, did not inhibit it. On the other hand, H(2)O(2) induced piperidine-labile sites at cytosine and thymine residues of double-stranded DNA in the presence of Cu(I). Phenylhydrazine, which is known to produce phenyl radicals, induced Cu(I)-dependent damage at thymine residues but not at guanine residues. These results suggest that the BzPO-derived reactive species causing DNA damage is different from (*)OH and phenyl radicals generated from benzoyloxyl radicals. BzPO/Cu(I) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in double-stranded DNA more effectively than that in single-stranded DNA. Furthermore, we observed that BzPO increased the amount of 8-oxodG in human cultured cells. Consequently, it is concluded that benzoyloxyl radicals generated by the reaction of BzPO with Cu(I) may oxidize the 5'-guanine of GG and GGG sequences in double-stranded DNA to lead to 8-oxodG formation and piperidine-labile guanine lesions, and the damage seems to be relevant to the tumor-promoting activity of BzPO.     

Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation.

The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.     

Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide

Oxidative DNA damage has been implicated in diverse biological processes including mutagenesis, carcinogenesis, aging, radiation effects, and chemotherapy. We examined the in vitro effect of low concentrations of Cu(II) or H2O2 alone and in combination on supercoiled plasmid DNA. As much as 10(-2) M Cu(II) or 10(-2) M H2O2 alone did not break the DNA. However, a mixture of 10(-6) M Cu(II) plus 10(-5) M H2O2 produced strand breaks and inactivated transforming ability. Strand breakage was proportional to incubation time, temperature, and Cu(II) and H2O2 concentrations. Abasic sites were not detected. Strand breakage was inhibited by metal chelators, catalase, and by high levels of free radical scavengers implying that Cu(II), Cu(I), H2O2, and .OH were involved in the reaction. The extent of DNA strand breakage was not affected by superoxide dismutase indicating that superoxide was not a major contributor to the DNA damage. DNA sequence analysis demonstrated that hot piperidine-sensitive DNA lesions were produced preferentially at sites of 2 or more adjacent guanosine residues. This sequence specificity was observed with Cu(II) plus H2O2 but not with Cu(I) alone. Polyguanosine sequence specificity for DNA damage induction appears to be unique among simple chemical systems. This reaction may be important in mechanisms of oxidative damage in vivo.     

Split-dose recovery is due to the repair of DNA double-strand breaks

DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad 54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break.     

Recombinational repair of hydrogen peroxide-induced damages in DNA of phage T4

Recently, hydrogen peroxide and its free-radical product, the hydroxyl radical (OH.) have been identified as major sources of DNA damage in living organisms. They occur as ubiquitous metabolic by-products and, in humans, cause several thousand damages in a cell's DNA per day. They are thought to be a major source of DNA damage leading to aging and cancer in multicellular organisms. This raises two questions. First, what pathways are used in repair of DNA damages caused by H2O2 and OH.? Second, a new theory has been proposed that sexual reproduction (sex) evolved to promote repair of DNA in the germ line of organisms. If this theory is correct, then the type of repair specifically available during the sexual process should be able to deal with important natural lesions such as those produced by H2O2 and OH. . Does this occur? We examined repair of hydrogen peroxide damage to DNA, using a standard bacteriophage T4 test system in which sexual reproduction is either permitted or not permitted. Post-replication recombinational repair and denV-dependent excision repair are not dependent on sex. Both of these processes had little or no effect on lethal H2O2 damage. Also, an enzyme important in repair of H2O2-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H2O2 damage to the phage. However, multiplicity reactivation, a recombinational form of repair depending on the sexual interaction of two or more of the bacteriophage, was found to repair lethal H2O2 damages efficiently. Our results lend support to the repair hypothesis of sex. Also the homology-dependent recombinational repair utilized in the phage sexual process may be analogous to the homology-dependent recombination which is widespread in diploid eucaryotes. The recombinational repair pathway found in phage T4 may thus be a widely applicable model for repair of the ubiquitous DNA damage caused by endogenous oxidative reactions.     

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.     

Mechanism of metal-mediated DNA damage induced by metabolites of carcinogenic 2-nitropropane

2-Nitropropane (2-NP), a widely used industrial solvent, is carcinogenic to rats. To clarify the mechanism of carcinogenesis by 2-NP, we investigated DNA damage by 2-NP metabolites, N-isopropylhydroxylamine (IPHA) and hydroxylamine-O-sulfonic acid (HAS), using 32P-5'-end-labelled DNA fragments obtained from genes that are relevant to human cancer. In the presence of Fe(III) EDTA, both IPHA and HAS caused DNA damage at every nucleotide position without marked site preference. The damage was inhibited by free hydroxyl radical (-*OH) scavengers, catalase and deferoxamine mesilate, an iron chelating agent. These results suggest that the DNA damage was caused by -*OH generated via H(2)O(2) by both IPHA and HAS. In contrast, in the presence of Cu(II), IPHA frequently caused DNA damage at thymine. The Cu(II)-mediated DNA damage caused by IPHA was inhibited by catalase, methional and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that the DNA damage induced by IPHA in the presence of Cu(II) was caused by a reactive oxygen species like the Cu(I)-hydroperoxo complex. On the other hand, HAS most frequently induced DNA damage at 5'-TG-3', 5'-GG-3' and 5'-GGG-3' sequences. Catalase and methional only partly inhibited the Cu(II)-mediated DNA damage caused by HAS, suggesting that the reactive oxygen species and another reactive species participate in this process. Formation of 8-oxodG by IPHA or HAS increased in the presence of metal ions. This study suggests that metal-mediated DNA damage caused by 2-NP metabolites plays an important role in the mutagenicity and the carcinogenicity of 2-NP.     

Water radiolysis products and nucleotide damage in gamma-irradiated DNA

The radiation chemistry of nucleotide damage introduction into DNA gamma-irradiated in dilute aqueous solution has been studied with a damage-specific DNA binding protein. Irradiation in air, N2 or N2O in the presence and absence of free radical scavengers revealed that protein-recognizable lesions were introduced both by the hydrated electron and by the combination of the hydroxyl radical and superoxide anion radical, but not by the hydroxyl radical alone. In addition, nucleotide damage was introduced into DNA by an enzymatic superoxide-generating system.     

The crypto-OH radical in the damage of DNA by bleomycin-Fe2+?

1. Effects of various OH scavengers, superoxide dismutase and catalase on the formation of malondialdehyde-like products from DNA by bleomycin-Fe2+ were studied. In no case was a protective effect observed. 2. These results can be interpreted on the basis that a crypto-OH radical mediates the damage to DNA by bleomycin-Fe2+.     

Mechanism of site-specific DNA damage induced by methylhydrazines in the presence of copper(II) or manganese(III).

DNA damage induced by methylhydrazines (monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine) in the presence of metal ions was investigated by a DNA sequencing technique. 1,2-Dimethylhydrazine plus Mn(III) caused DNA cleavage at every nucleotide without marked site specificity. ESR-spin-trapping experiments showed that the hydroxyl free radical (.OH) is generated during the Mn(III)-catalyzed autoxidation of 1,2-dimethylhydrazine. DNA damage and .OH generation were inhibited by .OH scavengers and superoxide dismutase, but not by catalase. The results suggest that 1,2-dimethylhydrazine plus Mn(III) generates .OH, not via H2O2, and that .OH causes DNA damage. In the presence of Cu(II), DNA cleavage was caused by the three methylhydrazines frequently at thymine residues, especially of the GTC sequence. The order of Cu(II)-mediated DNA damage (1,2-dimethylhydrazine greater than monomethylhydrazine approximately 1,1-dimethylhydrazine) was not correlated with the order of methyl free radical (.CH3) generation during Cu(II)-catalyzed autoxidation (monomethylhydrazine greater than 1,1-dimethylhydrazine much greater than 1,2-dimethylhydrazine). Catalase and bathocuproine, a Cu(I)-specific chelating agent, inhibited DNA damage while catalase did not inhibit the .CH3 generation. The order of DNA damage was correlated with the order of ratio of H2O2 production to O2 consumption observed during Cu(II)-catalyzed autoxidation of methylhydrazines. These results suggest that the Cu(I)-peroxide complex rather than the .CH3 plays a more important role in methylhydrazine plus Cu(II)-induced DNA damage.     

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

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

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.