Helen B. Patrzyc

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.

Vicinal Lesions in X-irradiated DNA?

Irradiation of the dinucleoside monophosphate d(GpT) in an oxygenated solution gives products characterized by damage on one or both guanine and thymine bases, the yields of which were proportional to radiation dose.

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.

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.

Modification of amino acids and bovine pancreatic ribonuclease a by kethoxal

Kethoxal (3-ethoxy-2-ketobutanal) reacts with the guanidino group of Nalpha-acetylarginine to produce four derivatives, reactive to periodate, stable at pH 7, with 15% reverting to arginine on acid hydrolysis. Other amino acids with blocked alpha-amino groups do not react, except the epsilon-amino of lysine (slowly). The pK of the mixed Kethoxal-Nalpha-acetylarginine derivatives is 5.8-6.1. Kethoxal reacts at neutral pH with arginyl residues of bovine pancreatic ribonuclease A. In the presence of an active-site ligand, arginine-39 and arginine-85 react at about equal rates. The loss of enzymic activity at pH 7 is proportional to the combined loss of these residues. The enzymic activity toward RNA is 20-25% of that of native RNAase at pH 7, and 90-100% at pH 5. In the absence of an active site ligand, arginine-10 is also modified with the loss of almost all enzymic activity, although arginine-10 is not an active-site residue. Arginine-33 is unreactive. Kethoxal-modified RNAase undergoes cross-linking in solution at pH 7 or in the freeze-dried state, Incubation at pH 9 in the presence of homoarginine results in partial regeneration of arginyl residues and activity at pH 7. Kethoxal modification of arginines-39 and -85 appears to raise the pK of lysine-41 by about 1 unit, as indicated ty the pH dependence of arylation by 2-carboxy-4,6-dinitrochlorobenzene. The claims of Patthy and Smith (J. Biol, Chem. (1975) 250, 565-569), and of Takahashi (J. Biol. Chem. (1968) 243, 6171-6179) that arginine-39 is a more important functional residue than is arginine-85 are questioned.

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.

A Novel Approach to DNA Damage Assessments: Measurement of the Thymine Glycol Lesion

Abstract Bailey, D. T., DeFedericis, H-C. C., Greene, K. F., Iijima, H., Budzinski, E. E., Patrzyc, H. B., Dawidzik, J. B. and Box, H. C. A Novel Approach to DNA Damage Assessments: Measurement of the Thymine Glycol Lesion. Radiat. Res. 165, 438– 444 (2006). A different approach to the measurement of DNA damage has been developed based on the fact that many lesions can be excised from DNA in the form of modified dinucleoside monophosphates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used in conjunction with isotopically labeled internal standards to quantify the lesion. The method has several advantages, including high sensitivity for the detection of dinucleoside monophosphates. The method was applied to the measurement of the 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) lesion in the DNA of mouse fibroblast cells exposed in culture to various treatments including ionizing radiation, UVC light and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. The application of the method to the measurement of other DNA lesions is discussed.

Covalently linked tandem lesions in DNA.

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.