5-chlorocytosine and Inflammation

During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase β replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.

Topics: Biomarkers, Tumor; Carcinogenesis; Chromatography, High Pressure Liquid; Cytosine; DNA Mutational Analysis; Humans; Hypochlorous Acid; Inflammation; Inflammatory Bowel Diseases; Models, Molecular; Mutagenesis; Mutagens; Mutation; Neoplasms; Oligonucleotides; Peroxidase; Sequence Analysis, DNA

Cytosine methylation patterns are essential for the proper control of gene expression in higher vertebrates. Although alterations in methylation patterns are frequently observed in human tumors, neither the mechanisms for establishing methylation patterns during normal development nor the mechanisms leading to pathological alterations of methylation patterns are currently known. While epidemiological studies have implicated inflammation in cancer etiology, a mechanistic link has yet to be established. Investigations of inflammation-mediated DNA damage may have provided important new insights. Our in vitro studies revealed that the inflammation-mediated DNA damage product, 5-chlorocytosine, could direct fraudulent methylation of previously unmethylated CpG sites. The purpose of this study was to recapitulate our in vitro findings by introducing 5-chlorocytosine residues into the DNA of replicating mammalian cells and to examine its impact on gene expression and cytosine methylation patterns. CHO-K1 cells hemizygous for the hprt gene were electroporated with the triphosphates of cytosine [2'-deoxycytidine-5'-triphosphate (dCTP)], 5-methylcytosine [5-methyl-2'-deoxycytidine-5'-triphosphate (MedCTP)] and 5'-chloro-2'-deoxycytidine-5'-triphosphate (CldCTP), and then selected with 6-thioguanine for silencing the hprt gene. Both modified nucleotides, MedCTP and CldCTP, but not unmodified dCTP, silenced hprt gene expression. Subsequent bisulfite pyrosequencing of CpG sites within the hprt promoter region of the selected cells confirmed hypermethylation, although global methylation levels as measured by gas chromatography-mass spectrometry did not change. Modified nucleotide-induced gene silencing could be reversed with 5-aza-2'-deoxycytidine indicating an epigenetic rather than mutagenic alteration. These results provide further evidence that the inflammation damage product 5-chlorocytosine could be a link between inflammation and cancer development.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Cytosine; DNA; DNA Damage; DNA Replication; Gene Silencing; Glycine; Humans; Inflammation; Mammals; Methylation; Neoplasms; Oligonucleotides

Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2'-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2'-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.

Topics: Adenine; Biomarkers; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytosine; Deoxyadenosines; Deoxycytidine; Deoxyguanosine; Guanine; Humans; Inflammation; Leukocytes; Nucleosides; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization