Examination of Hypochlorous Acid-Induced Damage to Cytosine Residues in a CpG Dinucleotide in DNA

Abstract

Inflammation-mediated, neutrophil-derived hypochlorous acid can damage DNA and result in the chlorination damage products 5-chlorocytosine and 5-chlorouracil as well as the oxidation damage products 5-hydroxycytosine and 5-hydroxyuracil. While 5-chlorocytosine could potentially perturb epigenetic signals if formed at a CpG dinucleotide, the remaining products are miscoding and could result in transition mutations. In this article, we have investigated the reaction of hypochlorous acid with an oligonucleotide site-specifically enriched with 15N to probe the reactivity of cytosine at CpG. These experiments demonstrate directly the formation of 5-chlorocytosine at a CpG dinucleotide in duplex DNA. We observe that chlorination relative to oxidation damage is greater at CpG by a factor of approximately two, whereas similar amounts of 5-chlorocytosine and 5-hydroxycytosine are formed at two non-CpG sites examined. The relative amounts of deamination of the cytosine to uracil derivatives are similar at CpG and non-CpG sites. Overall, we observe that the reactivity of cytosine at CpG and non-CpG sites toward hypochlorous acid induced damage is similar (5-chlorocytosine > 5-hydroxycytosine > 5-hydroxyuracil > 5-chlorouracil), with a greater proportion of chlorination damage at CpG sites. These results are in accord with the potential of inflammation-mediated DNA damage to both induce transition mutations and to perturb epigenetic signals.

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

“…Although the average values in mouse colons were higher than those found in human tissues, the range of data were overlapping (Fig. 1), which suggests that 5-Cl-dC may be a contributing factor in the initiation of colon carcinogenesis through either mutational or gene silencing mechanisms (54)(55)(56).…”

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer

“…Although by itself this number seems relatively low, the impact of 5ClC and, by extension, the impact of chlorination damage on mammalian cell biology will depend on the total number of lesions present in the genome. The upper bound of the extent of 5ClC formation in DNA was reported in a study in which physiologically achievable concentrations of HOCl transformed in vitro about 1-10 of every 1,000 cytosines in 5ClC (19). At this level of incorporation, the mutagenesis of 5ClC would be catastrophic for a mammalian-sized genome.…”

“…The main biomarkers found in cells exposed to HOCl are chlorinated tyrosines (in proteins) and chlorinated nucleobases (in nucleic acids and nucleotide pools) (15,16). Among the chlorinated nucleobases, 5-chlorocytosine (5ClC) is the most abundant (16)(17)(18)(19) and recently has been recognized as a biomarker associated with chronic inflammation (20,21). In one study, the levels of 5ClC in both DNA and RNA were elevated in the colon of Rag2 −/− mice infected with Helicobacter hepaticus, a model of chronic inflammation in the gut (20).…”

The Role of Inflammation in Brain Cancer

“…Upon activation, both initiate an oxidative burst that generates H 2 O 2 , HOBr, and HOCl (Lonkar and Dedon 2011). DNA damage products include both 5-chlorocytosine (5ClC) and 5-bromocytosine (5BrC) (Whiteman et al 1997;Winterbourn and Kettle 2000;Kang and Sowers 2008). Both 5ClC and 5BrC have been shown in in vitro studies to mimic 5mC and to act as fraudulent epigenetic signals (Valinluck et al 2005;Lao et al 2009Lao et al , 2010Valinluck and Sowers 2007).…”