7-Alkyldeoxyguanosine adduct detection by two-step HPLC and the 32P-postlabeling assay.
7-Alkyldeoxyguanosine DNA adducts may be a marker for some N-nitroso compound exposures and subsequent human cancer risk. A sensitive and highly specific assay for the detection of 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyldGp) and 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyldGp) has been developed by combining two different HPLC purification steps with the 32P-postlabeling assay. We previously reported that ion-pair reverse-phase (IP) chromatography coupled with the 32P-postlabeling assay detects 7-methyldGp in human lung, but have found that other nucleotides and unknown adducts co-elute. Thus, weak anion exchange (AE) HPLC was added in tandem with IP HPLC prior to the 32P-postlabeling assay. 2'-Deoxyguanosine-3'-monophosphate (dGp) is incorporated into the assay as an internal standard for the assessment of enzyme labeling efficiency and adduct recovery. The methodology was validated using radiolabeled DNA and liquid scintillation counting, which accounts for adduct loss from enzymatic digestion to detection. Levels of 7-ethyldGp also were correlated with accelerator mass spectrometry. The overall adduct recovery with this method was 58% for 7-methyldGp and 98% for 7-ethyldGp. The detection limit for both assays using 100 micrograms of DNA was one adduct in 10(8) unmodified dGp. 7-MethyldGp and 7-ethyldGp levels were determined in ten human lung samples at levels of 1.4-5.4 and 0.6-3.1 adducts per 10(7) dGp respectively, and in five human lymphocyte samples at levels of 5.0-8.3 and 0.3-1.4 adducts per 10(7) dGp respectively. Combining the two HPLC purification steps and the 32P-postlabeling assay attains chemical specificity, retains sufficient quantitative sensitivity and should be useful in human biomonitoring studies.
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH Bethesda, MD 20892.
S
S
Kato
S
S
Petruzzelli
E D
ED
Bowman
K W
KW
Turteltaub
B
B
Blomeke
A
A
Weston
P G
PG
Shields
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D003848:Deoxyguanine Nucleotides / Q000032:analysis*; D005038:Ethylnitrosourea / Q000494:pharmacology; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D008770:Methylnitrosourea / Q000494:pharmacology; D010761:Phosphorus Radioisotopes
D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C060943:7-methyl-2'-deoxyguanosine 3'-monophosphate; C080737:7-ethyl-2'-deoxyguanosine-3'-monophosphate; D008770:Methylnitrosourea; D006157:Guanosine Monophosphate; D004247:DNA; D005038:Ethylnitrosourea