Mass spectrometry of structurally modified DNA.

Structural modifications of nucleobases within the deoxyribonucleic acid (DNA) of living cells can be induced as a result of actions of specialized DNA-modifying enzymes (creating epigenetic DNA modifications) or may result from exposure to reactive endogenous and exogenous electrophiles and oxidants (creating DNA adducts). Epigenetic DNA modifications such as 5-methylcytosine (MeC) are important regulators of cell function that influence chromatin structure and levels of gene expression. DNA methyltransferases (DMTs) catalyze the addition of the C-5 methyl group to cytosine nucleobases.1 DMTs preferentially recognize hypomethylated 5′-CG-3′ sequences, producing epigenetic modifications which preserve DNA methylation patterns. C-5 cytosine methylation controls gene expression by mediating the binding of specific proteins (methyl-CpG binding proteins) to MeCG sites, followed by the recruitment of histone-modifying enzymes that promote chromatin remodeling.2 Recent studies have discovered additional cytosine modifications, e.g. 5-hydroxymethyl-C, and 5-formyl-C, and 5-carboxyl-C; these modifications have been hypothesized to be demethylation intermediates or they may possess their own epigenetic functions within cells.3-5 In contrast to epigenetic modifications, chemical DNA damage including nucleobase alkylation, oxidation, deamination, and cross-linking occurs at a variety of sites, including the N-7, O-6, C-8, and N-2 of guanine; the N-1, N-3, and N-7 of adenine; the O-2 and O-4 of thymine; and the O-2 and N-4 of cytosine (Scheme 1 and Chart 1). 6 Some carcinogens are inherently reactive towards DNA, while others must first be metabolically activated to electrophilic intermediates (e.g. epoxides, quinone methides, diazonium ions, and nitrenium ions), which subsequently bind to DNA producing nucleobase adducts (Figure 1). 6 All living cells contain extensive DNA repair systems responsible for removing nucleobase lesions. If structurally modified DNA bases escape repair, they may induce base mispairing during DNA replication; thus, the chemical damage would be converted into permanent genetic damage (mutations). 6 Accumulation of mutations in genes controlling cell growth, proliferation, programmed cell death, and cell differentiation is likely to cause cancer.7-10 Figure 1 Central role of DNA adducts in chemical carcinogenesis. Scheme 1 DNA sites frequently modified by carcinogens and their metabolites. Chart 1 Structures of representative DNA adducts Due to their central role in chemical carcinogenesis, DNA adducts are considered the true mechanism-based biomarkers of carcinogen exposure. The presence of DNA adducts within a given tissue can be correlated to the formation of reactive intermediates available for binding to DNA and other biomolecules.11 Unlike hemoglobin adducts that reflect a cumulative exposure to carcinogens over time, DNA adducts provide information on the burden of DNA damage within a given tissue at a specific time. Adducts can be used to quantify the capacity of DNA repair systems and to assess the potential for genetic damage as a result of faulty replication. By employing these measurements, DNA adduct levels have been utilized to set human exposure limits for industrial and environmental chemicals and also to identify individuals and populations at risk for developing cancer.11-14 The concentrations of epigenetically modified DNA bases in vivo are relatively high (e.g. four MeC per 100 of total nucleobases), however, the amounts of chemically induced DNA adducts in animal and human tissues can be quite low, in the range of 0.01 - 10 adducts per 108 normal nucleotides. Therefore, analytical methods used for quantifying carcinogen-DNA adducts must be ultra-sensitive, accurate, and specific, allowing the quantitation of low abundance DNA lesions in the presence of a large molar excess of normal nucleosides. Early studies of DNA damage utilized radiolabel-based assays such as 32P-postlabeling methods to measure adduct levels.15-18 Recent developments in mass spectrometry instrumentation have offered an alternative approach that provides both accurate and sensitive quantitation and structural information for the damaged bases, without the need for radioactivity.12 DNA adduct structure can be established using tandem mass spectrometry experiments, while mass spectrometry in combination with stable isotope labeled internal standards (isotope dilution HPLC-ESI-MS-MS or IDMS) is considered a golden standard for DNA adduct analysis due to its high specificity, sensitivity, and accurate quantification.14 Furthermore, mass spectrometry can be used for sequencing native and structurally modified DNA. The present review is devoted to the applications of mass spectrometry to DNA adduct and epigenetic DNA modification identification, screening, and quantitation. We will also discuss the use of MS based approaches to map the distribution of DNA modifications along DNA duplexes and to establish the biological consequences of DNA adduct formation in cells. Taken together, this article provides an overview of the contributions of mass spectrometry to the field of chemical carcinogenesis and epigenetics, with a primary focus on the new developments and recent advances in the field.