Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection.
7-Methylguanine (7-meG) could be a useful marker of recent past exposure to environmental methylating agents for use in epidemiological studies. A method is described that is appropriate for such an application. 7-meG was released from DNA by thermal hydrolysis under conditions (pH 9, 70 degrees C, 8 h) that preferentially released the base from DNA rather than RNA and, following immunopurification using antibodies specific for this DNA adduct, quantification was achieved either by HPLC with electrochemical detection (ECD) or by ELISA. The detection limits of the two approaches were 0.5 and 2 pmol 7-meG/DNA sample respectively. 7-meG was analysed in DNA samples contaminated with known amounts of RNA to test the possible interference in the analysis by the minor modified nucleoside 7-methylguanine, which is present as a normal component of RNA. 7-meG levels measured in human pancreas and untreated rat liver DNA were between 2 and 7 pmol 7-meG/mumol guanosine and this level could not be explained by RNA contamination. The combination of immunoaffinity purification and HPLC with ECD provides a method that is sensitive and specific for 7-meG and suitable for integration into molecular epidemiological studies.
Bianchini
F
F
International Agency for Research on Cancer, Lyon, France.
Montesano
R
R
Shuker
D E
DE
Cuzick
J
J
Wild
C P
CP
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis / Q000302:isolation & purification; D004563:Electrochemistry / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006151:Guanosine / Q000031:analogs & derivatives / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D006868:Hydrolysis; D008745:Methylation; D010179:Pancreas / Q000737:chemistry; D012313:RNA / Q000032:analysis; D051381:Rats; D015203:Reproducibility of Results
D006151:Guanosine; C016578:7-methylguanosine; C048746:7-methyl-2-deoxyguanosine; D012313:RNA; D004247:DNA; D003849:Deoxyguanosine