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Comet Assay Coupled to Repair Enzymes for the Detection of Oxidative Damage to DNA Induced by Low Doses of γ-Radiation: Use of YOYO-1, Low-Background Slides, and Optimized Electrophoresis Conditions

In this review article, emphasis is placed on the critical survey of available data concerning modified nucleobase and 2-deoxyribose products that have been identified in cellular DNA following exposure to a wide variety of oxidizing species and agents including, hydroxyl radical, one-electron oxidants, singlet oxygen, hypochlorous acid and ten-eleven translocation enzymes. In addition, information is provided about the generation of secondary oxidation products of 8-oxo-7,8-dihydroguanine and nucleobase addition products with reactive aldehydes arising from the decomposition of lipid peroxides. It is worth noting that the different classes of oxidatively generated DNA damage that consist of single lesions, intra- and interstrand cross-links were unambiguously assigned and quantitatively detected on the basis of accurate measurements involving in most cases high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The reported data clearly show that the frequency of DNA lesions generated upon severe oxidizing conditions, including exposure to ionizing radiation is low, at best a few modifications per 10⁶ normal bases. Application of accurate analytical measurement methods has also allowed the determination of repair kinetics of several well-defined lesions in cellular DNA that however concerns so far only a restricted number of cases.

This mini-review focuses on the recent identification of several novel radiation-induced single and tandem modifications in cellular DNA. For this purpose accurate high-performance electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was applied allowing their quantitative measurement and unambiguous characterization. Exposure of human cells to gamma rays led to the formation of several modified bases arising from the rearrangement of the pyrimidine ring of thymine, cytosine and 5-methylcytosine subsequent to initial addition of an hydroxyl radical (^•OH) to the 5,6-ethylenic bond. In addition, 5-hydroxymethylcytosine, an novel epigenetic mark, and 5-formylcytosine, were found to be generated consecutively to ^•OH-mediated hydrogen abstraction from the methyl group of 5-methylcytosine. Relevant mechanistic information on one-oxidation reactions of cellular DNA was also gained from the detection of 5-hydroxycytosine and guanine-thymine intra-strand adducts whose formation is rationalized by the generation of related base radical cation. Attempts to search for the radiation-induced formation of purine 5′,8-cyclo-2′-deoxyribonucleosides were unsuccessful with the exception of trace amounts of (5′S)-5′,8-cyclo-2′-deoxyadenosine.

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC–ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC–MS and HPLC–MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and ³²P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.

An oligonucleotide chip assay was designed for direct quantification of single strand breaks (SSBs) induced by γ-ray irradiation. The oligonucleotides used were 20-mers, which were short enough to produce only a single strand break within a single oligonucleotide. The two ends of the oligonucleotides were labeled with fluorescein and biotin, respectively. The biotinylated ends of the oligonucleotides were immobilized on a silicon wafer chip treated with (3-aminopropyl)triethoxysilane (APTES), glutaraldehyde, and avidin. The DNA fragments cleaved by γ-ray irradiation were detected by a laser-induced fluorescence (LIF) detection system. The γ-ray-induced SSBs were quantified using a calibration curve (fluorescence intensity versus γ-ray dose) without the need for complicated mathematical calculation based on gel-based separation. The experimentally determined γ-ray-induced SSBs yield was almost equal to the theoretical value derived from gel electrophoresis of plasmid DNAs and DNA surface coverage.

Exposure to low-dose gamma radiation is common in certain occupations but the biological and health effects from such exposure remain to be determined. The aim of this study was to investigate the effects of low-dose gamma radiation on DNA damage, chromosomal aberration and DNA repair gene expressions in whole blood and peripheral lymphocytes. The study revealed a dose-dependent effect of gamma radiation on DNA damage. Significant increases in DNA strand breaks and oxidative base damage, determined as formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites, were observed at absorbed doses of 5 and 10 cGy, respectively. However, gamma radiation at doses up to 500 cGy did not significantly increase the level of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) determined by HPLC with electrochemical detection (HPLC-ECD). Gamma radiation as low as 5 cGy caused chromosomal aberrations determined as dicentric and deletion frequencies. This finding is significant since the genotoxic effects of gamma radiation can be observed even at a low dose of 5 cGy. Furthermore, gamma radiation decreased the mRNA expression of both hOGG1 and XRCC1 repair genes determined by reverse transcriptase-polymerase chain reaction (RT-PCR), with a significant decrease of expression being observed at 20 cGy. The expression levels of hOGG1 and XRCC1 mRNA were inversely correlated with the levels of FPG-sensitive sites and DNA strand breaks. The finding of decreased expression levels for hOGG1 and XRCC1 in gamma-irradiated lymphocytes has not been reported elsewhere. Our observations suggest that the genotoxic effects of gamma radiation may be due to a combination of DNA-damaging effects and reduced DNA repair capacity, and may explain the significant increase in health risk from high doses of ionizing radiation.

We applied the alkaline version of the single-cell gel electrophoresis (comet) assay to roots and leaves of tobacco (Nicotiana tabacum var. xanthi) seedlings or isolated leaf nuclei treated with: (1) the alkylating agent ethyl methanesulphonate, (2) necrotic heat treatments at 50 °C, and (3) DNase-I. All three treatments induced a dose-dependent increase in DNA migration, expressed as percentage of tail DNA. A comparison of the fluorochrome DNA dyes ethidium bromide, DAPI and YOYO-1 demonstrated that for the alkaline version of the comet assay in plants, the commonly used fluorescent dye ethidium bromide can be used with the same efficiency as DAPI or YOYO-1.