Quantification of O6-methyl and O6-ethyl deoxyguanosine adducts in C57BL/6N/Tk+/- mice using LC/MS/MS

The carcinogenicity of many alkylating agents is derived from their ability to form persistent DNA adducts that induce mutations. This paper presents and validates methodology, based on LC with tandem mass spectrometry, for the separate or concurrent quantification by isotope dilution of O(6)-methyl-2'-deoxyguanosine (O(6)Me-dG) and O(6)-ethyl-2'-deoxyguanosine (O(6)Et-dG) DNA adducts. The limits of quantification were estimated to be < or =0.2 adducts/10(8) nucleotides for either adduct. This sensitivity permitted evaluation of adduct levels in livers from separate groups of untreated adult C57BL/6N/Tk(+/-) and C57BL/6N X Sv129 mice (undetectable to 5.5+/-6.7 O(6)Me-dG/10(8) nucleotides; undetectable to 0.04 O(6)Et-dG/10(8) nucleotides). Treatment of adult C57BL/6N/Tk(+/-) mice with equimolar doses (342micromol/kg body weight) of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea produced adduct levels in liver of 1700+/-80 O(6)Me-dG/10(8) nucleotides and 260+/-60 O(6)Et-dG/10(8) nucleotides, respectively, when assessed 4h after dosing. These methods should be useful for evaluations of DNA adducts in relation to cellular processes that modify carcinogenic and toxicological responses in experimental animals and humans.