Simultaneous Quantitation of N super(2),3-Ethenoguanine and 1,N super(2)-Ethenoguanine with an Immunoaffinity/Gas Chromatography/High-Resolution Mass Spectrometry Assay

• Published in: Chemical research in toxicology 2001-03, Vol.14 (3), p.327-334
• Main Authors: Morinello, E J, Ham, A-JL, Ranasinghe, A, Sangaiah, R, Swenberg, JA
• Format: Article
• Language: English
• Subjects: Ethenoguanine; immunoaffinity chromatography
• ISSN: 0893-228X

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 super(2),3-ethenoguanine (N super(2),3- epsilon Gua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N super(2)-ethenoguanine (1,N super(2)- epsilon Gua), in the same DNA sample. 1,N super(2)- epsilon Gua and N super(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] super(-) fragments of 3,5-(PFB) sub(2)-N super(2),3- epsilon Gua and 3,5-(PFB) sub(2)-[ super(13)C sub(4), super(15)N sub(2)]-N super(2),3- epsilon Gua and the [M - 20] super(-) fragments of 3,5-(PFB) sub(2)-1,N super(2)- epsilon Gua and 3,5-(PFB) sub(2)-[ super(13)C sub(3)]-1,N super(2)- epsilon Gua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N super(2),3- epsilon Gua and 15 fmol of 1,N super(2)- epsilon Gua in similar to 250 mu g of DNA, which corresponded to 5.0 N super(2),3- epsilon Gua and 8.71,N super(2)- epsilon Gua adducts/10 super(8) unmodified Gua bases, respectively. 1,N super(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 super(2)- epsilon Gua to N super(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 super(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 super(2)- epsilon Gua plays a minor role relative to N super(2),3- epsilon Gua in VC-induced carcinogenesis, but that 1,N super(2)- epsilon Gua may be formed to a larger extent from endogenous oxidative processes.