Simultaneous Quantitation of N2,3-Ethenoguanine and 1,N2-Ethenoguanine with an Immunoaffinity/Gas Chromatography/High-Resolution Mass Spectrometry Assay

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Abstract
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 N2,3-ethenoguanine (N2,3-εGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N2-ethenoguanine (1,N2-εGua), in the same DNA sample. 1,N2-εGua and N2,3-εGua 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-N2,3-εGua and 3,5-(PFB)2-[13C4,15N2]-N2,3-εGua and the [M − 201]- fragments of 3,5-(PFB)2-1,N2-εGua and 3,5-(PFB)2-[13C3]-1,N2-εGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N2,3-εGua and 15 fmol of 1,N2-εGua in ∼250 μg of DNA, which corresponded to 5.0 N2,3-εGua and 8.7 1,N2-εGua adducts/108 unmodified Gua bases, respectively. 1,N2-εGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N2-εGua to N2,3-εGua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N2,3-εGua 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,N2-εGua plays a minor role relative to N2,3-εGua in VC-induced carcinogenesis, but that 1,N2-εGua may be formed to a larger extent from endogenous oxidative processes.

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