Quantitation of DNA Adducts by Stable Isotope Dilution Mass Spectrometry
Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota , Minneapolis, Minnesota 55455, United States. Chemical Research in Toxicology
(Impact Factor: 3.53).
07/2012; 25(10):2007-35. DOI: 10.1021/tx3002548
Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations and potentially contributing to the development of cancer. Because of their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples, including immunoassay, HPLC, and (32)P-postlabeling, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adduct concentrations in biological samples are between 0.01-10 adducts per 10(8) normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry, especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS, have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke.
Available from: Aswin Mangerich
- "Pre-purification of samples by HPLC, or lowering of the derivatization temperature or addition of antioxidants have been suggested to avoid this type of artifact (Halliwell and Dizdaroglu, 1992). At present, one of the most specific and sensitive methods for the detection of a wide spectrum of DNA oxidation products represents reverse phase HPLC coupled ESI tandem mass spectrometry (LC–MS/ MS) which does not require derivatization prior to mass spectrometric analysis (Cadet et al., 2002; Mangerich et al., 2012; Ravanat, 2012; Taghizadeh et al., 2008; Tretyakova et al., 2012). "
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ABSTRACT: The oxidation of guanine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks [Moreno-Villanueva et al. (2009) BMC Biotechnol, 9, 39]. The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC-MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e. uric acid, and a protein, i.e. manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.
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ABSTRACT: Mass spectrometric analyses of DNA adducts usually require enzymatic digestion of the DNA to nucleosides. The digestive enzymes used in our laboratory included a calf spleen phosphodiesterase, whose marketing was stopped recently. Using DNA adducted with bioactivated methyleugenol and 1-methoxy-3-indolylmethyl glucosinolate-each forming dA and dG adducts-we demonstrate that replacement of calf spleen phosphodiesterase (Merck) by bovine spleen phosphodiesterase (Sigma-Aldrich) leads to unchanged results. Enzyme levels used for DNA digestion are extremely variable in different studies. Therefore, we sequentially varied the level of each of the three enzymes used. All dose (enzyme) - response (adduct level) curves involved a long plateau starting below the enzyme levels employed previously. Thus, we could reduce the amounts of micrococcal nuclease, phosphodiesterase and alkaline phosphatase for quantitative DNA digestion by factors of 4, 2 and 333, respectively, compared to our previous protocols. Moreover, we observed significant phosphatase activity of both phosphodiesterase preparations used, which may affect the recovery of adducts with methods requiring digestion to 2´-deoxynucleoside-3´-monophosphates (e.g.(32)P-postlabelling). In addition, the phosphodiesterase from Sigma-Aldrich, but not that from Merck, deaminated dA. This was irrelevant for the dA adducts studied, involving bonding at N(6), but might complicate the analysis of other dA adducts.
Available from: Fabian Schumacher
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ABSTRACT: Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adduct. A second adduct, N(6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 10(8) nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50-5500 adducts per 10(8) nucleosides are present after repeated treatment at the TD(50) (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk.
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