Oxygen derived species such as superoxide radical, hydrogen peroxide, singlet oxygen and hydroxyl radical are well known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases, including cancer. Various carcinogens may also partly exert their effect by generating reactive oxygen species (ROS) during their metabolism. Oxidative damage to cellular DNA can lead to mutations and may, therefore, play an important role in the initiation and progression of multistage carcinogenesis. The changes in DNA such as base modification, rearrangement of DNA sequence, miscoding of DNA lesion, gene duplication and the activation of oncogenes may be involved in the initiation of various cancers. Elevated levels of ROS and down regulation of ROS scavengers and antioxidant enzymes are associated with various human diseases including various cancers. ROS are also implicated in diabtes and neurodegenerative diseases. ROS influences central cellular processes such as proliferation a, apoptosis, senescence which are implicated in the development of cancer. Understanding the role of ROS as key mediators in signaling cascades may provide various opportunities for pharmacological intervention.

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Reactive oxygen species: role in the development of cancer and various chronic conditions

Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite.

In living cells reactive oxygen species (ROS) are formed continuously as a consequence of metabolic and other biochemical reactions as well as external factors. Some ROS have important physiological functions. Thus, antioxidant defense systems cannot provide complete protection from noxious effects of ROS. These include oxidative damage to DNA, which experimental studies in animals and in vitro have suggested are an important factor in carcinogenesis. Despite extensive repair oxidatively modified DNA is abundant in human tissues, in particular in tumors, i.e., in terms of 1–200 modified nucleosides per 105 intact nucleosides. The damaged nucleosides accumulate with age in both nuclear and mitochondrial DNA. The products of repair of these lesions are excreted into the urine in amounts corresponding to a damage rate of up to 104 modifications in each cell every day. The most abundant of these lesions, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), is also the most mutagenic, resulting in GT transversions which are frequently found in tumor relevant genes. A series of other oxidative modifications of base and sugar residues occur frequently in DNA, but they are less well studied and their biological significance less apparent. The biomarkers for study of oxidative DNA damage in humans include urinary excretion of oxidized nucleosides and bases as repair products and modifications in DNA isolated from target tissue or surrogate cells, such as lymphocytes. These biomarkers reflect the rate of damage and the balance between the damage and repair rate, respectively. By means of biomarkers a number of important factors have been studied in humans. Ionizing radiation, a carcinogenic and pure source of ROS, induced both urinary and leukocyte biomarkers of oxidative DNA damage. Tobacco smoking, another carcinogenic source of ROS, increased the oxidative DNA damage rate by 35–50% estimated from the urinary excretion of 8-oxodG, and the level of 8-oxodG in leukocytes by 20–50%. The main endogenous source of ROS, the oxygen consumption, showed a close correlation with the 8-oxodG excretion rate although moderate exercise appeared to have no immediate effect. So far, cross-sectional study of diet composition and intervention studies, including energy restriction and antioxidant supplements, have generally failed to show an influence on the oxidative DNA modification. However, a diet rich of Brussels sprouts reduced the oxidative DNA damage rate, estimated by the urinary excretion of 8-oxodG, and the intake of vitamin C was a determinant for the level of 8-oxodG in sperm DNA. A low-fat diet reduced another marker of oxidative DNA damage in leukocytes. In patients with diseases associated with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study of biobank material using a nested case control design. In addition, oxidative damage may be important for the aging process, particularly with respect to mitochondrial DNA and the pathogenesis of inflammatory diseases.

Cancer risk and oxidative DNA damage in man

THE possibility has been considered for almost forty years that the DNA double helix, which contains a π-stacked array of heterocyclic base pairs, could be a suitable medium for the migration of charge over long molecular distances. This notion of high charge mobility is a critical consideration with respect to DNA damage. We have previously found that the DNA double helix can serve as a molecular bridge for photo-induced electron transfer between metallointercalators, with fast rates (≥10^(10) S^(−1))^(10) and with quenching over a long distance (>40A)^8. Here we use a metallointercalator to introduce a photoexcited hole into the DNA π-stack at a specific site in order to evaluate oxidative damage to DNA from a distance. Oligomeric DNA duplexes were prepared with a rhodium inter-calator covalently attached to one end and separated spatially from 5′-GG-3′ doublet sites of oxidation. Rhodium-induced photo-oxidation occurs specifically at the 5′-G in the 5′-GG-3′ doublets and is observed up to 37 A away from the site of rhodium intercalation. We find that the yield of oxidative damage depends sensitively upon oxidation potential and π-stacking, but not on distance. These results demonstrate directly that oxidative damage to DNA may be promoted from a remote site as a result of hole migration through the DNA π-stack.

Oxidative DNA damage through long-range electron transfer

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance

Metabolism studies of the antitumor drug etoposide show the formation of metabolites in the lactone ring, which are probably not important for the drug's mechanism of action, and oxidative transformations in the dimethoxyphenol ring (E ring), which lead to products that can cause DNA damage and may play a role in the drug's mechanism of action. The cytotoxicity of etoposide is caused by the induction of DNA damage. The occurrence of the DNA lesions can be explained by the capacity of the drug to interfere with the scission-reunion reaction of mammalian topoisomerase II by stabilizing a cleavable complex.

Mechanism of Action of Antitumor Drug Etoposide: A Review

In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (.OH) and by exposure to singlet oxygen (1O2), released by thermo-dissociation of the endoperoxide 3,3′-(1,4-naphthalidene) dipropionate (NDPO2). We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ α gene. This 144 bp insert was used as a mutational target sequence. When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were γ-irradiated in the presence of oxygen (O2; 100% .OH) or nitrous oxide (N2O; 90% .OH, 10% .H), very specific mutation spectra were found. Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type. Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one. Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed. Under N2O-1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present. To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to γ-rays in phosphate buffer under nitrogen (55% oH, 45% .OH). Under these conditions a remarkable shift was observed from C/G → G/C to C/G → A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no - 1 bp deletions were detected. These results strongly suggest that H radicals do not cause - 1 bp deletions, but may be responsible for the observed C/G to A/T transversions. The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated. When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 bp sequence is part of M13mp10 DNA.

Mutational specificity of oxidative DNA damage

The antitumor agent VP-16-213 is oxidatively O-demethylated by rat liver microsomes and purified rat liver microsomal cytochrome P-450. 3-Methylcholanthrene can quantitatively induce O-demethylation of VP-16-213. The Km and Vmax values for O-demethylation by noninduced, phenobarbital-, and 3-methylcholanthrene-induced rat liver microsomes were found to be 130, 600, and 160 microM and 8.5, 11.8, and 15.6 nmol H2CO/min X mg protein, respectively. Mass spectrometric comparison of the product of O-demethylation of VP-16-213 with the synthetic metabolite resulted in identification of the orthodihydroxy derivative. In studies on the biological activity of the orthodihydroxy derivative, it was found to inactivate single- and double-stranded phiX174 DNA, to bind to calf thymus DNA and to be highly toxic against chinese hamster ovary cells.

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Cytochrome P 450 mediated O-demethylation: a route in the metabolic activation of etoposide (VP 16 213)

The catechol metabolite of the antitumor agent VP-16-213 and the ortho-quinone of VP-16-213--a secondary metabolite formed from the catechol--easily undergo auto-oxidation into a free radical at pH greater than or equal to 7.4. By elevation of the pH from 7.4 to 10, an increase in the production of the free radical was observed, which was accompanied by the formation of products with higher hydrophylicity than the catechol and ortho-quinone, as found by HPLC-analysis. The hyperfine structure of the free radical indicates that it is the semi-quinone radical of VP-16-213. At pH 12.5 a secondary radical is formed from the catechol and the ortho-quinone of VP-16-213 besides the semi-quinone radical. One-electron oxidation of the catechol with horseradish peroxidase/hydrogen peroxide resulted in the formation of the same radical as observed under alkaline conditions and subsequent oxidation to the ortho-quinone. If the ortho-quinone was incubated with NADPH cytochrome P-450 reductase, a free radical was detected by spin-trapping with POBN, but not without spin-trapping. Studies on inactivation of phi X174 DNA by the system ortho-quinone of VP-16-213/NADPH cytochrome P-450 reductase suggest that the semi-quinone radical may play a role in the process of inactivation of DNA.

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Semi-quinone formation from the catechol and ortho-quinone metabolites of the antitumor agent VP-16-213.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

J. Retel

Papers

Mechanism of Action of Antitumor Drug Etoposide: A Review

Mutational specificity of oxidative DNA damage

Cytochrome P 450 mediated O-demethylation: a route in the metabolic activation of etoposide (VP 16 213)

Semi-quinone formation from the catechol and ortho-quinone metabolites of the antitumor agent VP-16-213.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).