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 environmental and endogenous methylating agents and may be of use in epidemiological studies. To quantitate N7-MedG in human samples, a sensitive assay system that uses only small quantities of DNA (<10 microg) is required. To this end, polyclonal antibodies against the imidazole ring-opened form of N7-MedG have been used to develop a highly sensitive immunoslot blot (ISB) assay. The limit of detection of the assay is 0.10 micromol of N7-MedG/mol of deoxyguanosine (dG) using 1 microg of DNA per analysis. The method was optimized using in vitro-methylated calf thymus DNA and then applied to a study of DNA 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 Temozolomide/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 quantitated by an HPLC/(32)P-postlabeling method previously described. The results 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 peripheral blood mononuclear cell DNA samples from cancer patients following treatment with Temozolomide had levels of N7-MedG ranging from 0.22 to 320 micromol/mol of dG. DNA samples from colon carcinoma and normal colorectal mucosa from individuals not known to be exposed to methylating agents contained levels of 0.11-1.34 micromol of N7-MedG/mol of dG. The ISB assay offers the 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 methylating agents in large-scale biomonitoring studies.
School of Epidemiology and Health Sciences, Medical School, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
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Harrison
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Wood
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Lees
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Hall
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Margison
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Povey
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000009:adverse effects / Q000627:therapeutic use; D015415:Biomarkers / Q000032:analysis; D001921:Brain / Q000187:drug effects; D018736:DNA Adducts / Q000032:analysis*; D019175:DNA Methylation; D003606:Dacarbazine / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000627:therapeutic use; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D008099:Liver / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D000077204:Temozolomide; D013950:Thymus Gland
D018906:Antineoplastic Agents, Alkylating; D015415:Biomarkers; D018736:DNA Adducts; C048746:7-methyl-2-deoxyguanosine; D003606:Dacarbazine; D003849:Deoxyguanosine; D000077204:Temozolomide