Development and application of a sensitive and rapid immunoassay for the quantitation of N7-methyldeoxyguanosine in DNA samples

N7-Methyldeoxyguanosine (N7-MedG) in DNA is a biomarker of exposure to envi ronmental and endogenous methylating agents and may be of use in epidemiolo gical studies. To quantitate N7-MedG in human samples, a sensitive assay sy stem that uses only small quantities of DNA (<10 <mu>g) is required. To thi s end, polyclonal antibodies against the imidazole ring-opened form of N7-M edG have been used to develop a highly sensitive immunoslot blot (ISB) assa y. The limit of detection of the assay is 0.10 mu mol of N7-MedG/mol of deo xyguanosine (dG) using 1 mug of DNA per analysis. The method was optimized using in vitro-methylated calf thymus DNA and then applied to a study of DN A methylation in liver and brain tissues of mice following a single iv dose of the antitumor agent Temozolomide. The amount of N7-MedG in both tissues was strictly proportional to dose over a range of 10-200 mg of Temozolomid e/kg of body weight. The ISB assay was then validated using pyloric DNA of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and DNA samples from human bladder tumors, for both of which N7-MedG levels had already been qu antitated by an HPLC/P-32-postlabeling method previously described. The res ults showed a high degree of correlation (r = 0.98) between the two assays. The ISB assay was then applied to a range of human samples. A series of pe ripheral blood mononuclear cell DNA samples from cancer patients following treatment with Temozolomide had levels of N7-MedG ranging from 0.22 to 320 mu mol/mol of dG. DNA samples from colon carcinoma and normal colorectal mu cosa from individuals not known to be exposed to methylating agents contain ed levels of 0.11-1.34 mu mol of N7-MedG/mol of dG. The ISB assay offers th e potential for the rapid and high-throughput analysis of DNA obtained from routine biopsies and blood samples, thus enabling the determination of the extent of human exposure to environmental and endogenous sources of methyl ating agents in large-scale biomonitoring studies.