5-chlorocytosine and 5-chlorouracil

Recent studies have shown that reactive chlorine species, derived from myeloperoxidase-mediated inflammation responses, can modify DNA bases, generating 5-chloropyrimidines. The chlorinated adducts could be mutagenic or perturb DNA-protein interactions; however, the biological significance of these adducts is as yet unknown. We report here a method for the synthesis of 5-chlorocytosine- (ClC-) containing oligonucleotides that will be used in subsequent biochemical and biophysical studies to determine the consequences of pyrimidine chlorination. The ClC-phosphoramidite synthon is obtained by chlorination of 2'-deoxyuridine followed by conversion to the O(4)-ethyl analogue. The amino group needed to form the corresponding cytosine derivative is added by displacement of the O(4)-ethyl group during ammonia deprotection. A battery of methods, including mass spectrometry, has been used to characterize oligonucleotides containing ClC. Following oligonucleotide synthesis and deprotection, only trace amounts of the deamination product 5-chlorouracil can be detected by enzymatic cleavage of duplex oligonucleotides with the mispaired uracil glycosylase, MUG. In contrast to previous reports, we find that ClC is more stable in DNA than anticipated. Approximately 20% ClC is lost under standard formic acid hydrolysis conditions (88% formic acid, 140 degrees C, 30 min), while only 5% is recovered as 5-chlorouracil (ClU).

Topics: Cytosine; Deamination; Formates; Hydrolysis; Mutagens; Oligonucleotides; Uracil

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.

Topics: Animals; Cattle; Chlorine; Cytosine; DNA; DNA Damage; Dose-Response Relationship, Drug; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Hydrogen Peroxide; Hypochlorous Acid; Nitrogen; Oxidative Stress; Oxygen; Peroxynitrous Acid; Reactive Oxygen Species; Thymus Gland; Uracil