Accurate and sensitive quantitation of N7-methyldeoxyguanosine-3'- monophosphate by 32P-postlabeling and storage-phosphor imaging
article
As N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) is the major, persistent DNA lesion generated by methylating agents, a combined HPLC/32P- postlabeling assay has been developed to quantitate this adduct in human DNA. N7-MedGp was purified from normal nucleotides by anion-exchange chromatography followed by reverse-phase HPLC procedures. The adduct was then 32P-postlabeled and resolved by two-dimensional TLC far detection and quantitation by storage-phosphor imaging. The effect of conditions used for DNA purification and digestion on the recovery of N7-MedGp has been investigated. Extended, raised temperature incubations normally employed during DNA purification were demonstrated to result in considerable loss of adduct through depurination after 22 h at 65 and 37 °C (82% and 20% loss, respectively), but depurination was reduced to 5% if the incubation was performed at either 4 or 22 °C. Similarly, close to optical recovery (83%) of N7-MedGp was achieved after DNA digestion by incubating at 4 °C, pH 7.4, for 18 h in the presence of micrococcal nuclease and calf spleen phosphodiesterase from Sigma and Boehringer Mannheim, respectively. Overall, the recovery of N7-MedGp was 40%, resulting in a detection limit of 1.3 fmol which is equivalent to 0.16 μmol of adduct/mol of 2'-deoxyguanosine-3'- monophosphate (dGp) when analyzing 10 μg of DNA. The N7-MedGp content of DNA that had been methylated in vitro using 0, 16, and 80 μM N-methyl-N- nitrosourea (NMU) was determined by 32P-postlabeling to be 12, 112, and 671 μmol of N7-MedGp/mol of dGp. Electrochemical detection of N7-methylguanine (N7-MeG) after HPLC purification measured approximately 2-fold higher levels, i.e., 25, 225, and 1080 μmol of N7-MeG/mol of Gua, at each NMU concentration, respectively. The levels of N7-MedGp in the white blood cell (WBC) DNA of patients receiving a single dose of 5-(3,3-dimethyl-1- triazeno)imidazole-4-carboxamide (DTIC) chemotherapy were determined by 32P-postlabeling. Maximum levels were found 4-6 h after treatment, and in two out of four individuals adduct levels were decreased by 21 h. Prior to treatment, N7-MedGp was detectable in WBC DNA in two out of the four individuals indicating that nontherapeutic exposure to methylating agents had occurred.
Topics
DacarbazineDeoxyguanosine derivativeAnion exchange chromatographyDNA adductDNA determinationDNA methylationDNA purificationfluorescencehigh performance liquid chromatographyhumanhuman cellprotein degradationreversed phase high performance liquid chromatographythin layer chromatographyAnimalsAutoradiographyChromatography, High Pressure LiquidDeoxyguanine NucleotidesDNADNA AdductsHumansImage Processing, Computer-AssistedIsotope LabelingPhosphorus RadioisotopesRadiographic Image EnhancementReproducibility of ResultsSensitivity and Specificity
TNO Identifier
233943
ISSN
0893228X
Source
Chemical Research in Toxicology, 10(6), pp. 660-666.
Pages
660-666
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