Simultaneous quantitation of N2,3-ethenoguanine and 1,N2-ethenoguanine with an immunoaffinity/gas chromatography/high-resolution mass spectrometry assay

We have previously described an immunoaffinity/gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay for quantitation of the promutagenic DNA adduct N-2,3-ethenoguanine (N-2,3-epsilon Gua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N-2-ethenoguanine (1,N-2-epsilon Gua), in the same DNA sample. 1,N-2-epsilon Gua and N-2,3-epsilon Gua were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect the [M - 181](-) fragments of 3,5-(PFB)(2)-N-2,3-epsilon Gua and 3,5-(PFB)(2)- [C-13(4),N-15(2)]-N-2,3-epsilon Gua and the [M - 201](-) fragments of 3,5-(PFB)(2)-1,N-2-epsilon Gua and 3,5-(PFB)(2)-[C-13(3)]-1,N-2-epsilon Gua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N-2,3-epsilon Gua and 15 fmol of 1,N-2-epsilon Gua in similar to 250 mug of DNA, which corresponded to 5.0 N-2,3-epsilon Gua and 8.7 1,N-2-epsilon Gua adducts/10(8) unmodified Gua bases, respectively. 1,N-2-epsilon Gua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N-2-epsilon Gua to N-2,3-epsilon Gua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N-2,3-epsilon Gua was detected in DNA treated with the vinyl chloride (VC) metabolite 2-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks (6 h/day for 5 days/week). These data suggest that 1,N-2-epsilon Gua plays a minor role relative to N-2,3-epsilon Gua in VC-induced carcinogenesis, but that 1,N-2-epsilon Gua may be formed to a larger extent from endogenous oxidative processes.