Hypochlorous Acid-Induced Base Modifications in Isolated Calf Thymus DNA

Abstract

Exposure of calf thymus DNA to hypochlorous acid/hypochlorite leads to extensive DNA base modification. Large concentration-dependent increases in pyrimidine oxidation products [thymine glycol (cis/trans), 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxyhydantoin] but not purine oxidation products (8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine) were observed at pH 7.4. In addition, large increases in 5-chlorouracil (probably formed from 5-chlorocytosine during sample preparation), a novel chlorinated base, were observed. Addition of HOCl to DNA already damaged by .OH generated by a mixture of ascorbate, copper(II) chloride, and hydrogen peroxide showed that hypochlorous acid led to a loss of 8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine, and 5-hydroxycytosine in a concentration- and pH-dependent manner. Nevertheless, time course studies suggested that the formation of purine oxidation products in isolated DNA by hypochlorous acid was not a major oxidation pathway. If this pattern of damage, especially the production of 5-chlorocytosine, is unique to hypochlorous acid, it might act as a "fingerprint" of damage to DNA by HOCl.

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

“…1). Such a mutagenic signature would feature C→T mutations characteristic of 5ClC formation and also other mutations corresponding to the variety of oxidative and chlorinated lesions induced by HOCl (18). Some features of the mutagenic spectrum generated by chronic inflammation already have been reported in mouse models, and they include increased C→T mutations as well as small DNA base insertions and deletions (indels) (54).…”

“…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).…”

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

“…MPO also produces chlorinating species that react with DNA to form chlorinated adducts such as 5-chloro-2′-deoxycytidine (5-Cl-dC) (23), the presence of which was identified in colon tissue of H. hepaticus-infected Rag2 −/− mice (10). This modification of DNA may provide a mechanistic link between neutrophil activity and colitis-associated carcinoma (10,24,25).…”

DNA Methylation: Its Role in Cancer Development and Therapy

“…Mutagenesis resulting from the production of halogenated nucleotide precursors in activated neutrophils and eosinophils with subsequent incorporation into DNA has been demonstrated in vitro. 2,3 The link between inflammation-induced halogenated nucleotides and aberrant methylation stems from the demonstration that methyl-binding proteins cannot distinguish between methylated cytosine residues and chlorinated or brominated cytosine nucleotides. Furthermore, DNMT1 also could not distinguish between 5-methylcytosine and some 5-halocytosine products which could potentially result in mistaken methylation at sites of inflammation.…”