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.     

Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

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.     

Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention

Epidemiological studies have suggested that the use of aspirin is associated with a decreased incidence of human malignancies, particularly colorectal cancer. Since reactive oxygen species (ROS) are critically involved in multistage carcinogenesis, this study was undertaken to examine the ability of aspirin to inhibit ROS-mediated DNA damage. Hydrogen peroxide (H2O2)+Cu(II) and hydroquinone (HQ) + Cu(II) were used to cause oxidative DNA strand breaks in phiX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.5-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a marked inhibition of oxidative DNA damage induced by either H2O2/Cu(II) or HQ/Cu(II). The inhibition of oxidative DNA damage by aspirin was exhibited in a concentration-dependent manner. Moreover, aspirin was found to be much more potent than the hydroxyl radical scavengers, mannitol and dimethyl sulfoxide, in protecting against the H2O2/Cu(II)-mediated DNA strand breaks. Since the reduction of Cu(II) to Cu(I) is crucially involved in both H2O2/Cu(II)- and HQ/Cu(II)-mediated formation of hydroxyl radical or its equivalent, and the subsequent oxidative DNA damage, we examined whether aspirin could inhibit this Cu(II)/Cu(I) redox cycle. It was observed that aspirin at concentrations that showed the inhibitory effect on oxidative DNA damage did not alter the Cu(II)/Cu(I) redox cycle in either H2O2/Cu(II) or HQ/Cu(II) system. In addition, aspirin was not found to significantly scavenge H2O2. This study demonstrates for the first time that aspirin potently inhibits both H2O2/Cu(II)- and HQ/Cu(II)-mediated oxidative DNA strand breaks most likely through scavenging the hydroxyl radical or its equivalent derived from these two systems. The potent inhibition of oxidative DNA damage by aspirin may thus partially contribute to its anticancer activities observed in humans.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.     

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.     

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.     

Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening.

Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H2O2 plus Cu(II) caused predominant DNA damage at the 5' site of 5'-GGG-3' in the telomere sequence. Furthermore, H2O2 plus Cu(II) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in telomere sequences more efficiently than that in non-telomere sequences. NO plus O2- efficiently caused base alteration at the 5' site of 5'-GGG-3' in the telomere sequence. It is concluded that the site-specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

The mechanism of the enhanced antioxidant effects against superoxide anion radicals of reduced water produced by electrolysis

We reported that reduced water produced by electrolysis enhanced the antioxidant effects of proton donors such as ascorbic acid (AsA) in a previous paper. We also demonstrated that reduced water produced by electrolysis of 2 mM NaCl solutions did not show antioxidant effects by itself. We reasoned that the enhancement of antioxidant effects may be due to the increase of the ionic product of water as solvent. The ionic product of water (pKw) was estimated by measurements of pH and by a neutralization titration method. As an indicator of oxidative damage, Reactive Oxygen Species- (ROS) mediated DNA strand breaks were measured by the conversion of supercoiled phiX-174 RF I double-strand DNA to open and linear forms. Reduced water had a tendency to suppress single-strand breakage of DNA induced by reactive oxygen species produced by H2O2/Cu (II) and HQ/Cu (II) systems. The enhancement of superoxide anion radical dismutation activity can be explained by changes in the ionic product of water in the reduced water.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Paradoxical role of lipid metabolism in heart function and dysfunction

The naturally occurring flavonoid, quercetin, in the presence of Cu(II) and molecular oxygen caused breakage of calf thymus DNA, supercoiled pBR322 plasmid DNA and single stranded M13 phage DNA. In the case of the plasmid, the product(s) were relaxed circles or a mixture of these and linear molecules depending upon the conditions. For the breakage reaction, Cu(II) could be replaced by Fe(III) but not by other ions tested [Fe(II), Co(II), Ni(II), Mn(II) and Ca(II)]. Structurally related flavonoids, rutin, galangin, apigenin and fisetin were effective or less effecive than quercetin in causing DNA breakage. In the case of the quercetin-Cu(II) reaction, Cu(I) was shown to be essential intermediate by using the Cu(1)-sequestering reagent, bathocuproine. By using Job plots we established that, in the absence of DNA, five Cu(II) ions were reduced by one quercetin molecule; in contrast two ions were reduced per quercetin molecule in the DNA breakage reaction. Equally neocuproine inhibited the DNA breakage reaction. The involvement of active oxygen in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, iodide, mannitol, formate and catalase (the inhibition was complete in the last case). The strand scission reaction was shown to account for the biological activity of quercetin as assayed by bacteriophage inactivation. From these data we propose a mechanism for the DNA strand scission reaction of quercetin and related flavonoids.     

Clear evidence for the participation of OH in lambda DNA breakage induced by the enzymatic reduction of adriamycin in the presence of iron-ADP. Importance of local OH concentration for DNA strand cleavage

lambda DNA (a double-stranded DNA) was exposed to several adriamycin-mediated active oxygen generating systems (O2- and H2O2 generating, OH generating, and perferryl ion complex generating), extracted, and analyzed by gel electrophoresis on agarose gel. Only the DNA exposed to and subsequently isolated from the adriamycin-mediated OH generating system contained many DNA fragments of low molecular weight, indicating the breakage of DNA strands. Such a breakage was strikingly inhibited by catalase or 50 mM sodium benzoate, but not by superoxide dismutase. The local OH concentration near the DNA strand was considered to be important for DNA strand cleavage.     

Synergistic induction of hydroxyl radical-induced DNA single-strand breaks by chromium(VI) compound and cigarette smoke solution

Chromium(VI) compounds and cigarette smoke are known human carcinogens. We found that K2Cr2O7 and cigarette smoke solution synergistically induced DNA single-strand breaks (0.23+/-0.04 breaks per DNA molecule) in pUC118 plasmid DNA. K2Cr2O7 alone or cigarette smoke solution alone induced much less strand breaks (0.03+/-0.01 or 0.07+/-0.02 breaks per DNA molecule, respectively). The synergistic effect was prevented by catalase and by hydroxyl radical scavengers such as deferoxamine, dimethylsulfoxide, d-mannitol, and Tris, but not by superoxide dismutase. Ascorbic acid enhanced the synergism. Glutathione inhibited strand breakage only at high concentrations. Electron spin resonance (ESR) studies using a hydroxyl radical trap demonstrated that hydroxyl radicals were generated when DNA was incubated with K2Cr2O7 and cigarette smoke solution. Hydroxyl radical adduct decreased dose-dependently when strand breakage was prevented by catalase, deferoxamine, dimethylsulfoxide, d-mannitol or Tris, but not significantly by superoxide dismutase. We also used ESR spectroscopy to study the effects of different concentration of ascorbic acid and glutathione. The results showed that hydroxyl radical, which is proposed as a main carcinogenic mechanism for both chromium(VI) compounds and cigarette smoke solution was mainly responsible for the DNA breaks they induced.     

Thiols can either enhance or suppress DNA damage induction by catecholestrogens

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.     

Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay

In our preceding papers [M. Wojewódzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: I. Strand breakage, Mutat. Res., 416 (1998) 21-35; M. Kruszewski, M. Wojewódzka, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: II. Base damage, Mutat. Res. , 416 (1998) 37-57.], we evaluated the DNA breakage and base damage with the use of comet assay in a group of 49 workers chronically exposed to low doses of ionizing radiation. There was a statistically significant difference in the damage levels between the hazard and control group. In this paper we describe a confounding lack of effect of the smoking habit on the DNA damage in the tested groups. The genotoxic effect of the smoking habit, as well as its modifying effect on genome damage inflicted by other agents, have been firmly established. However, no statistically significant effect of smoking was found in our study, neither in the control nor in the hazard group. This lack of effect was seen in all DNA damage determinations, both direct (DNA strand breakage and alkali-labile lesions) and enzyme-combined (base damage) and did not depend on the comet parameters, which were taken as damage indicators.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.     

Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex

Tetracyclines (TCs) in combination with Cu(II) ions exhibited significant DNA damaging potential vis a vis tetracyclines per se. Interaction of tetracyclines with DNA resulted in alkylation at N-7 and N-3 positions of adenine and guanine bases, and caused destabilization of DNA secondary structure. Significant release of acid-soluble nucleotides from tetracycline-modified DNA upon incubation with S(1) nuclease ascertained the formation of single stranded regions in the DNA. Also, the treatment of tetracycline-modified DNA with 0.1 and 0.5M NaOH resulted in 62 and 76% hydrolysis compared to untreated control. Comparative alkaline hydrolysis of DNA modified with tetracycline derivatives showed differential DNA damaging ability in the order as DOTC > DMTC > TC > OTC > CTC. Addition of Cu(II) invariably augmented the extent of tetracycline-induced DNA damage. The alkaline unwinding assay clearly demonstrated the formation of approximately six strand breaks per unit DNA at 1:10 DNA nucleotide/TC molar ratio in the presence of 0.1mM Cu(II) ions. At a similar Cu(II) concentration, a progressive transformation of covalently closed circular (CCC) (form-I) plasmid pBR322 DNA to forms-II and -III was noticed with increasing tetracycline concentrations. The results obtained with the free-radical quenchers viz. mannitol, thiourea, sodium benzoate and superoxide dismutase (SOD) suggested the involvement of reactive oxygen species in the DNA strand breakage. It is concluded that the tetracycline-Cu(II)-induced DNA damage occurs due to (i) significant binding of tetracycline and Cu(II) with DNA, (ii) methyl group transfer from tetracycline to the putative sites on nitrogenous bases, and (iii) metal ion catalyzed free-radical generation in close vicinity of DNA backbone upon tetracycline photosensitization. Albeit, the DNA alkylation and strand cleavage are repairable lesions, but any defect in the critical repair pathway may augment the damage accumulation and mutagenesis.     

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.