Jean B. Dawidzik

Free radical-induced double lesions in DNA

This review surveys the work that has been done on free radical-induced DNA double lesions. Double lesions consist of two modifications of the DNA in close proximity. Double lesions can be generated by a single free radical-initiating event and the mechanism of formation often involves the participation of guanine. The identification of double lesions in oligomer and polymer DNA is reviewed and possible mechanisms of formation are outlined. The potential biological significance of double lesions is discussed. Double lesions induced by UV light are outside the scope of this review.

Free radical-induced tandem base damage in DNA oligomers.

A new tandem base lesion has been identified in two DNA oligomers, namely d(GpT) and d(CpGpTpA), exposed to X-irradiation in deoxygenated aqueous solution. In this lesion the C6 carbon atom of thymine is hydroxylated and a covalent link is formed between the C5 carbon atom of thymine and the C8 carbon atom of the adjacent guanine base. In addition, further evidence in the form of mass spectrometric data is presented confirming the structures of previously reported tandem base lesions that are produced by ionizing radiation in the presence of oxygen. New data is presented on the prevalence of a previously reported tandem base lesion in which the methyl carbon atom of thymine is covalently linked to the C8 carbon atom of the adjacent guanine base. The free radical-initiated processes by which tandem base damages are generated are discussed. To date four different radiation-induced tandem base lesion have been identified. The evidence suggests that tandem base damage is a significant component of free radical-induced DNA damage.

Tandem lesions and other products in X-irradiated DNA oligomers

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.

Double Base Lesions in DNA X-Irradiated in the Presence or Absence of Oxygen

Abstract 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 a deoxygenated environment.

Singlet Oxygen-Induced DNA Damage

Abstract DeFedericis, H-C., Patrzyc, H. B., Rajecki, M. J., Budzinski, E. E., Iijima, H., Dawidzik, J. B., Evans, M. S., Greene, K. F. and Box, H. C. Singlet Oxygen-Induced DNA Damage. Radiat. Res. 165, 445–451 (2006). 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.

Isolation and characterization of the products of anoxic irradiation of d(CpGpTpA).

An improved method for separating the products of DNA oligomers irradiated in aqueous solution has been devised. Altogether 39 products were isolated from the tetramer d(CpGpTpA) X-irradiated in dilute anoxic solution. Of these, 16, including most of the major products, were identified through the use of proton nmr spectroscopy. The identified products fall into four categories: (1) base products, (2) strand scission products, (3) base modifications and (4) tandem lesion. A tandem lesion in which the methyl carbon atom of thymine is covalently linked to the guanine C8 carbon atom was produced in larger yield than any of the simple base modifications.

Double lesions are produced in DNA oligomer by ionizing radiation and by metal-catalyzed H2O2 reactions.

Abstract Patrzyc, H. B., Dawidzik, J. B., Budzinski, E. E., Iijima, H. and Box, H. C. Double Lesions are Produced in DNA Oligomer by Ionizing Radiation and by Metal-Catalyzed H2O2 Reactions. It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H2O2 reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8- dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.

A study of pyrimidine base damage in relation to oxidative stress and cancer

Background:A long-standing hypothesis is that oxidative stress is a risk factor for cancer. Support for this hypothesis comes from observations of higher levels of oxidative damage in the DNA of WBC of cancer patients compared with healthy controls.Methods:Two generally overlooked types of DNA damage, the formamide modification and the thymine glycol modification, both derived from pyrimidine bases, were assayed as markers of oxidative stress. Damage levels were measured in the DNA of WBC of ovarian cancer patients and of healthy controls.Results:The levels of both modifications were higher in ovarian cancer patients than in healthy controls although in the case of the formamide modification age could not be ruled out as a factor.Conclusion:Our results in combination with other published measurements of oxidative DNA damage support the hypothesis that oxidative damage, on average, is higher in WBC of cancer patients than in healthy controls.

DNA damage measured by liquid chromatography-mass spectrometry in mouse fibroblast cells exposed to oxidative stress.

Oxidative DNA damage can result from environmental factors, such as radiation, as well as from the untoward consequences of normal metabolic processes. It is of interest to assay oxidative DNA damage in cells and tissues because this damage has been implicated in human disease, particularly cancer. Eleven indicators of oxidative DNA damage have been measured by Liquid Chromatography-Mass Spectrometry (LC-MS) in DNA extracted from cells exposed to oxidative stress. Mouse fibroblast cells were exposed to hydrogen peroxide and to UVC light and to the combined action of both agents. Significant increases of the 8-oxo-7,8-dihydropurine lesions over background were detected. Significant increases of the formamido lesions resulting from breakdown of pyrimidine bases were also observed. Of special interest was the observation of double lesions, tandem combinations of both aforementioned lesions, in cells exposed to oxidative stress.

Assessment of DNA Damage at the Dimer Level: Measurement of the Formamide Lesion

Abstract Greene, K. F., Budzinski, E. E., Iijima, H., Davidzik, J. B., DeFedericis, H-C., Patrzyc, H. B., Evans, M. S., Bailey, D. T., Freund, H. F. and Box, H. C. Assessment of DNA Damage at the Dimer Level: Measurement of the Formamide Lesion. Radiat. Res. 167, 146–151 (2007). UVC-radiation-induced DNA damage was measured in mouse fibroblast cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with isotopically labeled internal standards. The thymine glycol and formamide lesions were assayed in the form of modified dinucleoside monophosphates. The 8-oxo-7,8-dihydroguanine lesion was measured as the modified nucleoside. DNA damage in cells treated with tirapazamine was also measured. Tirapazamine is a chemotherapeutic agent that acts via a free radical mechanism. The two agents, UVC radiation and tirapazamine, produce markedly different profiles of DNA damage, reflecting their respective mechanisms of action. Both agents produce significant amounts of thymine glycol and formamide damage, but only the former produced a measurable amount of the 8-oxo-7,8-dihydroguanine lesion. The merits of measuring DNA damage at the dimer level are discussed.