Free radicals and other oxidants have gained importance in the field of biology due to their central role in various physiological conditions as well as their implication in a diverse range of diseases. The free radicals, both the reactive oxygen species (ROS) and reactive nitrogen species (RNS), are derived from both endogenous sources (mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells etc.) and exogenous sources (pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation). Free radicals can adversely affect various important classes of biological molecules such as nucleic acids, lipids, and proteins, thereby altering the normal redox status leading to increased oxidative stress. The free radicals induced oxidative stress has been reported to be involved in several diseased conditions such as diabetes mellitus, neurodegenerative disorders (Parkinson’s disease-PD, Alzheimer’s disease-AD and Multiple sclerosis-MS), cardiovascular diseases (atherosclerosis and hypertension), respiratory diseases (asthma), cataract development, rheumatoid arthritis and in various cancers (colorectal, prostate, breast, lung, bladder cancers). This review deals with chemistry, formation and sources, and molecular targets of free radicals and it provides a brief overview on the pathogenesis of various diseased conditions caused by ROS/RNS.

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Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

https://downloads.hindawi.com/journals/jna/2010/592980.pdf

Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

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.

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents including UVA and ionizing radiations. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.

Oxidative damage to DNA: formation, measurement and biochemical features.

Free radical-induced double lesions in DNA.

Free Radical-Induced Tandem Base Damage in DNA Oligomers

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.

Singlet oxygen-induced DNA damage.

Free radicals interact with DNA bases to produce secondary radicals. The secondary radicals are reactive species and tend to interact with neighboring bases, resulting in DNA lesions with two adjacent modified bases. In this study the DNA oligomers d(CpApTpG) and d(CpGpTpA) were exposed to free radicals generated in anoxic aqueous solution by X irradiation. Four new lesions were identified in which adjacent guanine and pyrimidine bases are covalently bonded. One of the tandem lesions formed in d(CpGpTpA) has the C5 carbon atom of cytosine covalently bonded to the C8 carbon atom of guanine. Interestingly, the same bond is formed between the terminal bases in d(CpApTpG), resulting in a cyclized molecule.

Tandem Lesions and Other Products in X-Irradiated DNA Oligomers

Box, H. C., Patrzyc, H. B., Dawidzik, J. B., Wallace, J. C., Freund, H. G., Iijima, H. and Budzinski, E. E. Double Base Lesions in DNA X-Irradiated in the Presence or Absence of Oxygen. Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in ...

https://bioone.org/journals/radiation-research/volume-153/issue-4/0033-7587(2000)153[0442:DBLIDX]2.0.CO;2/Double-Base-Lesions-in-DNA-X-Irradiated-in-the-Presence/10.1667/0033-7587(2000)153[0442:DBLIDX]2.0.CO;2.pdf

Jean B. Dawidzik

Papers

Free radical-induced double lesions in DNA.

Free Radical-Induced Tandem Base Damage in DNA Oligomers

Singlet oxygen-induced DNA damage.

Tandem Lesions and Other Products in X-Irradiated DNA Oligomers

Double base lesions in DNA X-irradiated in the presence or absence of oxygen.