It is increasingly proposed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a key role in human cancer development [1–6], especially as evidence is growing that antioxidants may prevent or delay the onset of some types of cancer (reviewed in [7,8]). ROS is a collective term often used by biologists to include oxygen radicals [superoxide (O # J−), hydroxyl (OHJ), peroxyl (RO # J) and alkoxyl (ROJ)] and certain nonradicals that are either oxidizing agents and}or are easily converted into radicals, such as HOCl, ozone (O $ ), peroxynitrite (ONOO−), singlet oxygen ("O # ) and H # O # . RNS is a similar collective term that includes nitric oxide radical (NOJ), ONOO−, nitrogen dioxide radical (NO # J), other oxides of nitrogen and products arising when NOJ reacts with O # J−, ROJ and RO # J. ‘Reactive ’ is not always an appropriate term; H # O # , NOJ and O # J− react quickly with very few molecules, whereas OHJ reacts quickly with almost anything. RO # J, ROJ, HOCl, NO # J, ONOO− and O $ have intermediate reactivities. ROS and RNS have been shown to possess many characteristics of carcinogens [4] (Figure 1). Mutagenesis by ROS}RNS could contribute to the initiation of cancer, in addition to being important in the promotion and progression phases. For example, ROS}RNS can have the following effects. (1) Cause structural alterations in DNA, e.g. base pair mutations, rearrangements, deletions, insertions and sequence amplification. OHJ is especially damaging, but "O # , RO # J, ROJ, HNO # , O $ , ONOO− and the decomposition products of ONOO− are also effective [9–13]. ROS can produce gross chromosomal alterations in addition to point mutations and thus could be involved in the inactivation or loss of the second wild-type allele of a mutated proto-oncogene or tumour-suppressor gene that can occur during tumour promotion and progression, allowing expression of the mutated phenotype [4]. (2) Affect cytoplasmic and nuclear signal transduction pathways [14,15]. For example, H # O # (which crosses cell and organelle membranes easily) can lead to displacement of the inhibitory subunit from the cytoplasmic transcription factor nuclear factor κB, allowing the activated factor to migrate to the nucleus [14]. Nitration of tyrosine residues by ONOO− may block phosphorylation. (3) Modulate the activity of the proteins and genes that respond to stress and which act to regulate the genes that are related to cell proliferation, differentiation and apoptosis [4,14–17]. For example, H # O # can stimulate transcription of c-jun

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Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer.

A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation, shock, and ischemia/reperfusion injury. The aim of this review is to describe recent developments in the field of oxidative stress research. The first part of the review focuses on the roles of reactive oxygen species (ROS) in shock, inflammation, and ischemia/reperfusion injury. The second part of the review deals with the novel findings using recently identified pharmacological tools (e.g., peroxynitrite decomposition catalysts and selective superoxide dismutase mimetics (SODm) in shock, ischemia/reperfusion, and inflammation. 1) The role of ROS consists of immunohistochemical and biochemical evidence that demonstrates the production of ROS in shock, inflammation, and ischemia/reperfusion injury. ROS can initiate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATPase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation, and ischemia/reperfusion. 2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm, which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, have been shown to prevent in vivo the delayed vascular decompensation and the cellular energetic failure associated with shock, inflammation, and ischemia/reperfusion injury. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical, and hydrogen peroxide) are all potential reactants capable of initiating DNA single-strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase, leading to eventual severe energy depletion of the cells and necrotic-type cell death. Antioxidant treatment inhibits the activation of poly(ADP-ribose) synthetase and prevents the organ injury associated with shock, inflammation, and ischemia/reperfusion.

Antioxidant Therapy: A New Pharmacological Approach in Shock, Inflammation, and Ischemia/Reperfusion Injury

In this survey, emphasis was placed on the main photoreactions of nucleic acid components, involving both direct and indirect effects. The main UVB- and UVA-induced DNA photoproducts, together with the mechanisms of their formation, are described. Information on the photoproduct distribution within cellular DNA is also provided, taking into account the limitations of the different analytical methods applied to monitor the formation of the DNA damage. Thus, the formation of the main DNA dimeric pyrimidine lesions produced by direct absorption of UVB photons was assessed using a powerful HPLC-tandem mass spectrometry assay. In addition, it was found that UVA photooxidation damage mostly involves the guanine residues of cellular DNA as the result of singlet oxygen generation by still unknown endogenous photosensitizers.

Direct and indirect effects of UV radiation on DNA and its components.

Cells in tissues and organs are continuously subjected to oxidative stress and free radicals on a daily basis. This free radical attack has exogenous or endogenous (intracellular) origin. The cells withstand and counteract this occurrence by the use of several and different defense mechanisms ranging from free radical scavengers like glutathione (GSH), vitamins C and E and antioxidant enzymes like catalase, superoxide dismutase and various peroxidases to sophisticated and elaborate DNA repair mechanisms. The outcome of this dynamic equilibrium is usually the induction of oxidatively induced DNA damage and a variety of lesions of small to high importance and dangerous for the cell i.e. isolated base lesions or single strand breaks (SSBs) to complex lesions like double strand breaks (DSBs) and other non-DSB oxidatively generated clustered DNA lesions (OCDLs). The accumulation of DNA damage through misrepair or incomplete repair may lead to mutagenesis and consequently transformation particularly if combined with a deficient apoptotic pathway. In this review, we present the current status of knowledge and evidence on the mechanisms and involvement of intracellular oxidative stress and DNA damage in human malignancy evolution and possible use of these parameters as cancer biomarkers. At the same time, we discuss controversies related to potential artifacts inherent to specific methodologies used for the measurement of oxidatively induced DNA lesions in human cells or tissues.

Role of oxidative stress and DNA damage in human carcinogenesis.

The role of reactive oxygen and antioxidant species in periodontal tissue destruction.

It is shown that various radicals exhibiting diverse ESR and ENDOR spectral characteristics are nonetheless a closely related family of alkoxy radicals. The relationship is established by correlating the g tensor with crystal structure and by relating dihedral angles inferred from proton hyperfine couplings to dihedral angles inferred from the g tensor and crystal structure. The analysis also serves to demonstrate that an ESR absorption observed in x‐irradiated single crystals of uridine 5′‐monophosphate is due to an alkoxy radical.

The gamut of alkoxy radicals

The characteristics of proton couplings in primary radiation products in irradiated protein and nucleic acid constituents are described. The range of β proton couplings in carboxylic acid anions is shown to be much smaller than for typical β couplings in π‐electron radicals. A close correlation between σ proton coupling tensors in conjugated oxidation and reduction products and principal axes derived from crystal structure is demonstrated. Proton hyperfine coupling tensors in the primary oxidation product of aminoisobutyric acid and the 13C hyperfine coupling tensor in the primary reduction product are reported.

Hyperfine couplings in primary radiation products

Reactive oxygen species (ROS) generate a type of DNA damage called tandem lesions, two adjacent nucleotides both modified. A subcategory of tandem lesions consists of adjacent nucleotides linked by a covalent bond. Covalently linked tandem lesions generate highly characteristic liquid chromotography-tandem mass spectrometry (LC-MS/MS) elution profiles. We have used this property to comprehensively survey X-irradiated DNA for covalently linked tandem lesions. A total of 15 tandem lesions were detected in DNA irradiated in deoxygenated aqueous solution, five tandem lesions were detected in DNA that was irradiated in oxygenated solution.

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Covalently Linked Tandem Lesions in DNA

Radiation-Induced Formation of 3,4-Dihydroxyphenylalanine in Tyrosine-Containing Peptides and Proteins as a Function of X-Irradiation Dose

Certain quantitative cytological measurements can be made by the addition of relatively simple components to a television flying‐spot microscope. The flying‐spot system avoids ``distributional errors'' inherent in conventional microphotometry measurements. The microscope has been used to measure the desoxypentose nucleic acid in whole nuclei.

Harold G. Freund

Papers

The gamut of alkoxy radicals

Hyperfine couplings in primary radiation products

Covalently Linked Tandem Lesions in DNA

Radiation-Induced Formation of 3,4-Dihydroxyphenylalanine in Tyrosine-Containing Peptides and Proteins as a Function of X-Irradiation Dose

Flying‐Spot Microscope Adapted for Quantitative Measurements