Detection and quantification of 5-chlorocytosine in DNA by stable isotope dilution and gas chromatography/negative ion chemical ionization/mass spectrometry.
Hypochlorous acid (HOCl) is generated from activated phagocytes during infections and inflammation. One of the major products of HOCl reaction with DNA was 5-chlorocytosine (5Cl-Cyt). In this report, a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay with stable isotope dilution was developed for detection and quantification of 5Cl-Cyt in DNA. During hydrolysis of DNA, 5Cl-Cyt undergoes spontaneous deamination quantitatively forming 5-chlorouracil (5Cl-Ura). The stable isotope of 5Cl-Ura with six mass units higher than the normal 5Cl-Ura was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selected ion monitoring at [M - 181](-) fragments of bispentafluorobenzylated 5Cl-Ura and its isotope analogue. The limit of detection was 20 amol (S/N = 8) of bispentafluorobenzylated 5Cl-Ura injected on column with selective ion monitoring mode and the limit of quantification for the entire assay was 14 fmol of 5Cl-Cyt. Analysis of hypochlorous acid-treated calf thymus DNA by both GC/NICI/MS and HPLC/UV detection provided similar adduct levels and thus verified this new GC/NICI/MS assay. Using this highly specific and ultrasensitive GC/NICI/MS method, the levels of 5Cl-Cyt in untreated calf thymus DNA and human placental DNA were determined as 0.6 and 6.6 adducts per 10(7) normal cytosine, respectively. Peroxynitrite also contributed to 5Cl-Cyt formation in DNA. Level of 5Cl-Cyt in DNA treated with peroxynitrite in the presence of chloride was higher than that without addition of chloride. Thus, quantification of 5Cl-Cyt in DNA by this isotope dilution GC/NICI/MS assay may facilitate research on the role of DNA chlorination in carcinogenesis and in cancer development.
Chen
Hauh-Jyun Candy
HJ
Department of Chemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan. chehjc@ccunix.ccu.edu.tw
Row
Shin-Wei
SW
Hong
Chia-Liang
CL
D000818:Animals; D002247:Carbon Isotopes; D002417:Cattle; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007201:Indicator Dilution Techniques; D009587:Nitrogen Isotopes
D002247:Carbon Isotopes; D018736:DNA Adducts; D009587:Nitrogen Isotopes; D003596:Cytosine; C401182:5-chlorocytosine