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

Abstract

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.

Occurrence, Biological Consequences, and Human Health Relevance of Oxidative Stress-Induced DNA Damage

“…The lesion-containing dinucleotide was thus utilized for the quantification of thymidine glycol in DNA using LC-MS/MS coupled with the stable isotope-dilution method. 47,299,300 This method has been applied for probing oxidative stress in white blood cell DNA of ovarian cancer patients 301 and BRCA mutation carriers 302 as well as for examining the effect of smoking cessation 303 and antioxidant usage 304 on levels of oxidatively induced DNA damage. Rather than being highly mutagenic, thymidine glycol strongly inhibits DNA replication.…”

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

“…In this study two ROS-induced pyrimidine modifications of DNA were studied, namely the formamide remnant derived from pyrimidine bases and the thymine glycol modification of thymine base. Both modifications are produced in cells exposed to ionising radiation or to UVC (Bailey et al, 2006;Greene et al, 2007). However, the damage profiles are different depending on the ROS source, illustrating the advantage of evaluating oxidative stress through the simultaneous measurement of more than one lesion.…”

“…Consequently, these lesions appear in a nuclease P1 (plus alkaline phosphatase) hydrolysate of DNA predominately as modified dinucleoside monophosphates; for example as in Figure 1A and B. Measuring these lesions at the dimer level has advantages: (1) The sensitivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, using electrospray ionisation and operating in the negative ion mode, is better for dinucleoside monophosphates than for nucleosides (Bailey et al, 2006); (2) Isotopically labelled internal standards for quantifying these lesions can be obtained by labelling the nucleoside 3 0 to the modified nucleoside. As the inhibition of nuclease P1 by the modified nucleoside is not absolute, it would seem that quantification at the dimer level is problematical.…”

“…As modified dinucleoside monophosphate derived from the DNA from the sample and from the internal standard are chemically identical, loss of product due to partial hydrolysis is accounted for in the final quantification step. The measurement of DNA damage at the dimer level has been described previously (Bailey et al, 2006;Greene et al, 2007). Samples contained 100 mg of DNA.…”

“…The base modifications of interest were the thymine glycol modification of thymine and the formamide modification derived from the breakdown of pyrimidine bases (Bailey et al, 2006;Greene et al, 2007). These modifications were examined in two aspects: (1) Production in vitro by radiation, both ionising and UVC; (2) The relationship of DNA base damage to cancer, particularly ovarian cancer.…”