Accurate and Sensitive Quantitation of N7-Methyldeoxyguanosine-3‘-monophosphate by 32P-Postlabeling and Storage-Phosphor Imaging

Abstract

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 for 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 degrees C (82% and 20% loss, respectively), but depurination was reduced to 5% if the incubation was performed at either 4 or 22 degrees C. Similarly, close to optical recovery (83%) of N7-MedGp was achieved after DNA digestion by incubating at 4 degrees 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 mumol of adduct/mol of 2'-deoxyguanosine-3'-monophosphate (dGp) when analyzing 10 micrograms of DNA. The N7-MedGp content of DNA that had been methylated in vitro using 0, 16, and 80 microM N-methyl-N-nitrosourea (NMU) was determined by 32P-postlabeling to be 12, 112, and 671 mumol 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 mumol 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.