Mutagenic spectrum resulting from DNA damage by oxygen radicals.

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.     

Cell damage by oxygen free radicals

The exposure of isolated and cultured cells to oxygen free radicals generated extracellularly or intracellularly during the metabolism of foreing compounds results in the development of damage that eventually lead to cell death. Multiple mechanisms are involved in these cytopathological processes, including direct attack of free radicals to macromolecules essential for cell life, as well as indirect activation of catabolic processes such as proteases, endonucleases and phospholipases. A key role in triggering these indirect events is played by Ca²⁺ whose cytosolic concentration during oxidative stress raises well above the physiological limits.     

Hydroxyl radicals and DNA base damage

Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction.     

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.     

Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals.

The metal chelators 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical formation from hydrogen peroxide and Cu+ or F2(2+) (Fenton reaction) as determined by electron spin resonance. o-Phenanthroline prevented both Fe- and Cu-mediated Fenton reactions whereas neocuproine only prevented the Cu-mediated Fenton reaction. Because only 1,10-phenanthroline but not neocuproine prevented DNA strand-break formation in hydrogen peroxide-treated mammalian fibroblasts it appears that the Fe-mediated, as compared to the Cu-mediated, intranuclear Fenton reaction is responsible for DNA damage.     

Role of oxygen free radicals in carcinogenesis and brain ischemia

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin-trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage-induced loss of glutamine synthetase activity, may be a key event in stroke-induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8-hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin-traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.     

DNA damage by oxygen radicals and excited state species: a comparative study using enzymatic probes in vitro

Repair enzyme-containing extracts from a variety of cell types are used to analyse and compare DNA damage induced by oxygen radicals and excited molecules. The differing potentials of these extracts for recognising DNA damage leads to characteristic DNA damage profiles after treatment with superoxide (xanthine/xanthine oxidase), gamma-rays, chemically generated singlet oxygen, photosensitizers (rose bengal, methylene blue), UV254 and a 1,2-dioxetane. Three different types of damage profiles are distinguished and assigned to the predominant action of hydroxyl radicals, singlet oxygen or to the photoexcitation of thymine residues. The method applied in this study allows the analysis of DNA damage and the identification or exclusion of the participation of different ultimate reactive species without chemical identification of the lesions.     

Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa)

The effect of aluminium (Al) on seedlings of two rice cultivars, Pusa Basmati and Vikas was investigated after different hours of exposure to 80 mol/L of external Al supply. With increasing time of exposure, the growing seedlings readily absorbed Al and its localization was greater in roots than shoots. Prolonged exposure to Al intensified lipid peroxidation, changed the activities of SOD and peroxidase and caused DNA damage. However, differential responses were observed between the seedlings of two rice cultivars under Al stress. A close inverse relationship existed between decreased root growth and increased Al accumulation, lipid peroxidation, SOD, peroxidase activities and DNA damage. The results demonstrate that roots are the major sites of Al localization and accumulation of Al promoted oxygen free radicals mediated peroxidation of membranes as evidenced by increased MDA levels and the activities of SOD and peroxidase. Our results for the first time showed that Al can cause DNA damage in rice.     

Reactive oxygen and DNA damage in mitochondria.

During the last decade the importance of reactive oxygen species as major contributors to various types of cancer, heart diseases, cataracts, Parkinson's and other degenerative diseases that come with age, and to natural aging has become apparent. Mitochondria are the most important intracellular source of reactive oxygen. Mitochondrial DNA is heavily damaged by reactive oxygen at the bases, as indicated by the high steady-state level of 8-hydroxydeoxyguanosine, the presence of which causes mispairing and point mutations. Mitochondrial DNA is also oxidatively fragmented to a certain extent. Conceivably, such fragmentation relates to deletions found in mitochondrial DNA. Point mutations and deletions have recently been shown to be etiologically linked to several human diseases and natural aging. Future studies should address the causal relationship between mitochondrial dysfunction, production of reactive oxygen species, and aging.     

Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals

Semicarbazide, a hydrazine derivative, is carcinogenic to mice but shows no or little mutagenicity in the Salmonella-microsome test. To clarify whether or not the genotoxic mechanism contributes to the non-mutagenic carcinogenicity of semicarbazide, we investigated DNA damage induced by semicarbazide using 32P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Semicarbazide caused DNA damage frequently at the thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine partially inhibited DNA damage, suggesting that hydrogen peroxide plus Cu(I) participates in DNA damage. When a high concentration of semicarbazide was used in the presence of catalase, DNA damage was induced, especially at G in 5'-AG and slightly at 5'-G in GG and GGG sequences. An electron paramagnetic resonance (EPR) spectroscopic study has confirmed that the reaction of semicarbazide with Cu(II) produces carbamoyl radicals (z.rad;CONH(2)), possibly generated via the nitrogen-centered radicals of semicarbazide. Azodicarbonamide also produced carbamoyl radicals and induced DNA damage frequently at 5'-G in GG and GGG sequences, suggesting that carbamoyl radicals participate in this sequence-specific DNA damage by semicarbazide. On the basis of our previous reports, we consider that the sequence-specific DNA damage at G in 5'-AG in the present study is due to the nitrogen-centered radicals. This study has shown that semicarbazide induces DNA damage in the presence of Cu(II) through the formation of hydrogen peroxide and Cu(I). In addition, semicarbazide-derived free radicals participate in DNA damage. DNA damage induced by these reactive species may be relevant to the carcinogenicity of semicarbazide.     

DNA strand scission by enzymically generated oxygen radicals

Col E1 DNA suffers strand scission when exposed to xanthine oxidase acting aerobically on xanthine. Strand scission was prevented by low levels of superoxide dismutase or of catalase. Mannitol, benzoate, or histidine, which scavenge OH · but which react with neither O₂^? nor H₂O₂, also prevented strand scission. Replacement of 0.1 mm ethylenediaminetetraacetate by 0.1 mm diethylenetriaminepentaacetate prevented strand scission. Three mechanisms for the production of OH ·, or of a comparably powerful oxidant, by metal-catalyzed interaction of O₂^? with H₂O₂, are proposed.     

Immunochemical detection of oxidative DNA damage in cancer and aging using anti-reactive oxygen species modified DNA monoclonal antibody

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions and ischemia. These species have been implicated in various disorders, mutagenesis, carcinogenesis and aging. Of many macromolecules, DNA is the most susceptible to hydroxyl radical, the most reactive of the ROS. The present study is designed to detect oxidative DNA damage in cancer patients and healthy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified calf thymus DNA fragments (approximate size 400 bp) were modified with ·OH, generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline sucrose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in T_m, modification of thymine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was characterized for antigen binding specificity by competition ELISA. Monoclonal antibody showed strong binding to ROS-modified DNA, its modified fragments, polynucleotides and bases. With the exception of native DNA, binding of unmodified polynucleotides and bases was much lower. The mAb distinctly recognized DNA samples from lymphocytes of healthy aged humans and gave maximum inhibitions of 49, 53, 64 and 70%, while not reacting with DNA from young population. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as negative control. The studies demonstrate that the mAb, although cross-reactive, preferentially binds ROS-modified epitopes on DNA. High reactivity of mAb to DNA samples from cancer patients and healthy aged humans indicates increased oxidative stress leading to DNA damage.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.     

Role of deubiquitinases in DNA damage response

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.     

Role of leukocyte accumulation and oxygen radicals in ischemia-reperfusion-induced injury in skeletal muscle

The role of leukocytes and nonleukocyte-derived reactive oxygen metabolites (ROMs) in reperfusion-induced skeletal muscle injury was determined. Male rats received 2 h no-flow hindlimb ischemia-reperfusion (I/R, n = 6) or were rendered neutropenic via antineutrophil serum (ANS) before I/R (I/R + ANS, n = 5). Oxygen radicals in the absence of neutrophils were tested by administration of dimethylthiourea (DMTU) (I/R + ANS + DMTU, n = 5). Perfused capillaries (CD(per)) and rolling (L(r)), adherent (L(a)), and extravasated leukocytes (L(e)) in the extensor digitorum longus muscle were measured every 15 min during 90 min of reperfusion using intravital microscopy. The vital dyes bisbenzimide (BB) and ethidium bromide (EB) provided direct measures of tissue injury (EB/BB). CD(per) decreased immediately on reperfusion in the I/R and I/R + ANS groups. CD(per) in the I/R + ANS + DMTU group remained at baseline throughout reperfusion. L(a) increased in the I/R group; however, EB/BB was the same between I/R and I/R + ANS groups. Injury in the I/R + ANS + DMTU group did not differ from other groups > or =60 min, after which EB/BB became significantly lower. L(e) did not differ between groups and was highly correlated to tissue injury. The results suggest that L(e) lead to parenchymal injury, and ROMs lead to perfusion deficits during the early reperfusion period after ischemia.     

Protein damage and degradation by oxygen radicals. I. general aspects

Aggregation, fragmentation, amino acid modification, and proteolytic susceptibility have been studied following exposure of 17 proteins to oxygen radicals. The hydroxyl radical (.OH) produced covalently bound protein aggregates, but few or no fragmentation products. Extensive changes in net electrical charge (both + and -) were observed. Tryptophan was rapidly lost with .OH exposure, and significant production of bityrosine biphenol occurred. When incubated with cell-free extracts of human and rabbit erythrocytes, rabbit reticulocytes, or Escherichia coli, most .OH-modified proteins were proteolytically degraded up to 50 times faster than untreated proteins. The exceptions were alpha-casein and globin, which were rapidly degraded without .OH modification. ATP did not stimulate the degradation of .OH-modified proteins, but alpha-casein was more rapidly degraded. Leupeptin had little effect under any condition, and degradation was maximal at pH 7.8. The data indicate that proteins which have been denatured by .OH can be recognized and degraded rapidly and selectively by intracellular proteolytic systems. In both red blood cells and E. coli, the degradation appears to be conducted by soluble, ATP-independent (nonlysosomal) proteolytic enzymes. In contrast with the above results, superoxide (O2-) did not cause aggregation or fragmentation, tryptophan loss, or bityrosine production. The combination of .OH + O2- (+O2), which may mimic biological exposure to oxygen radicals, induced charge changes, tryptophan loss, and bityrosine production. The pattern of such changes was similar to that seen with .OH alone, although the extent was generally less severe. In contrast with .OH alone, however, .OH + O2- (+O2) caused extensive protein fragmentation and little or no aggregation. More than 98% of the protein fragments had molecular weights greater than 5000 and formed clusters of ionic and hydrophobic bonds which could be dispersed by denaturing agents. The results indicate a general sensitivity of proteins to oxygen radicals. Oxidative modification can involve direct fragmentation or may provide denatured substrates for intracellular proteolysis.     

Role of senataxin in DNA damage and telomeric stability

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin (SETX), a putative DNA/RNA helicase. The presence of the helicase domain led us to investigate whether SETX might play a role in DNA damage repair and telomere stability. We analyzed the response of AOA2 lymphocytes and lymphoblasts after treatment with camptothecin (CPT), mitomycin C (MMC), H?O? and X-rays by cytogenetic and Q-FISH (quantitative-FISH) assays. The rate of chromosomal aberrations was normal in AOA2 cells after treatment with CPT, MMC, H?O? and X-rays. Conversely, Q-FISH analysis showed constitutively reduced telomere length in AOA2 lymphocytes, compared to age-matched controls. Furthermore, CPT- or X-ray-induced telomere shortening was more marked in AOA2 than in control cells. The partial co-localization of SETX with telomeric DNA, demonstrated by combined immunofluorescence-Q-FISH and chromatin immunoprecipitation, suggests a possible involvement of SETX in telomere stability.