Miral Dizdaroglu

Oxidative DNA damage: mechanisms, mutation and disease.

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8‐oxo‐2′deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.—Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195–1214 (2003)

Free radical-induced damage to DNA: mechanisms and measurement.

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products. Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GC/MS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced for the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques.

Oxidative damage to DNA in mammalian chromatin

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivo by a variety of free radical-producing systems. Methodologies incorporating the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H2O2 and in chromatin of organs of animals treated with carcinogenic metal salts.

Chemical determination of free radical-induced damage to DNA

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.

DNA base modifications in chromatin of human cancerous tissues

Free radical‐induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady‐state level of free radical‐modified bases in cellular DNA. We have investigated endogenous levels of typical free radical‐induced DNA base modifications in chromatin of various human cancerous tissues and their cancer‐free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine‐derived and six purine‐derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected‐ion monitoring. These were 5‐hydroxy‐5‐methylhydantoin, 5‐hydroxyhydantoin, 5‐(hydroxymethyl)uracil, 5‐hydroxycytosine, 5,6‐dihydroxycytosine, 4,6‐diamino‐5‐formamidopyrimidine, 8‐hydroxyadenine, xanthine, 2‐hydroxyadenine, 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine, and 8‐hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack an DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified buses over the control levels. The results clearly indicate higher steady‐state levels of modified DNA bases in cancerous tissues than in their cancer‐free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.

Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer

We have investigated levels of antioxidant enzymes and free radical‐induced DNA base modifications in human cancerous lung tissues and in their cancer‐free surrounding tissues. Various DNA base lesions in chromatin of lung tissues were measured by gas chromatography‐mass spectrometry. Activities of superoxide dismutase, catalase and glutathione peroxidase were also measured in lung tissues. Higher levels of DNA lesions were observed in cancerous tissues than in cancer‐free surrounding tissues. Antioxidant enzyme levels were lower in cancerous tissues. The results indicate an association between decreased activities of antioxidant enzymes and increased levels of DNA lesions in cancerous tissues. Higher levels of DNA lesions suggest that free radical reactions may be increased in malignant tumor cells.

Copper Oxide Nanoparticle Mediated DNA Damage in Terrestrial Plant Models

Engineered nanoparticles, due to their unique electrical, mechanical, and catalytic properties, are presently found in many commercial products and will be intentionally or inadvertently released at increasing concentrations into the natural environment. Metal- and metal oxide-based nanomaterials have been shown to act as mediators of DNA damage in mammalian cells, organisms, and even in bacteria, but the molecular mechanisms through which this occurs are poorly understood. For the first time, we report that copper oxide nanoparticles induce DNA damage in agricultural and grassland plants. Significant accumulation of oxidatively modified, mutagenic DNA lesions (7,8-dihydro-8-oxoguanine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 4,6-diamino-5-formamidopyrimidine) and strong plant growth inhibition were observed for radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum) under controlled laboratory conditions. Lesion accumulation levels mediated by copper ions and macroscale copper particles were measured in tandem to clarify the mechanisms of DNA damage. To our knowledge, this is the first evidence of multiple DNA lesion formation and accumulation in plants. These findings provide impetus for future investigations on nanoparticle-mediated DNA damage and repair mechanisms in plants.

CHEMICAL DETERMINATION OF OXIDATIVE DNA DAMAGE BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Publisher Summary Oxidative DNA damage produced by free radicals or other DNA-damaging agents has been implicated to play a role in mutagenesis, carcinogenesis, reproductive cell death, and aging. Oxygen-derived species, such as superoxide radical (O 2 − ) and H 2 O 2 are generated in all aerobic cells. Excess generation of these species by endogenous sources or exogenous sources (for example, redox-cyclic drugs and ionizing radiation) may cause damage to cellular DNA by a variety of mechanisms. The gas chromatography–mass spectrometry (GC/MS) technique has been applied to a variety of in vitro and in vivo studies of oxidative DNA damage. The technique offers the sensitivity, selectivity, speed, and versatility to solve a wide range of important measurement problems in terms of DNA base and sugar damage and DNA-protein cross-links. It also allows studying enzymatic repair of DNA damage. It appears that the GC/MS technique will continue to find a major role in studies of oxidative DNA damage and its repair in the future.

Mechanisms of free radical-induced damage to DNA

Abstract Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.