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

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 N(2),3-ethenoguanine (N(2),3-epsilonGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N(2)-ethenoguanine (1,N(2)-epsilonGua), in the same DNA sample. 1,N(2)-epsilonGua and N(2),3-epsilonGua 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-epsilonGua and 3,5-(PFB)(2)-[(13)C(4),(15)N(2)]-N(2),3-epsilonGua and the [M - 201](-) fragments of 3,5-(PFB)(2)-1,N(2)-epsilonGua and 3,5-(PFB)(2)-[(13)C(3)]-1,N(2)-epsilonGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N(2),3-epsilonGua and 15 fmol of 1,N(2)-epsilonGua in approximately 250 microg of DNA, which corresponded to 5.0 N(2),3-epsilonGua and 8.7 1,N(2)-epsilonGua adducts/10(8) unmodified Gua bases, respectively. 1,N(2)-epsilonGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N(2)-epsilonGua to N(2),3-epsilonGua 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-epsilonGua 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)-epsilonGua plays a minor role relative to N(2),3-epsilonGua in VC-induced carcinogenesis, but that 1,N(2)-epsilonGua may be formed to a larger extent from endogenous oxidative processes.

Interactions of Carcinogen-Bound DNA with Individual DNA Polymerases

“…[237][238][239][240] This lesion is also formed from the oxidation products of fatty acids, resulting from lipid peroxidation. 241 In E. coli cells, the main outcome remains coding for dCTP insertion, and the main misincorporation is insertion of T (G to A transition), with A following (G to T transversion). 77 However, with several processive polymerases and E. coli pol I (Klenow fragment exo -) and rat pol , the residues C, G, T, and A were all inserted to some extent and -1 and -2 frame shifts were detected.…”

Vinyl Chloride—A Classical Industrial Toxicant of New Interest

“…Similar to other "etheno" adducts (see section III.B), this lesion is promutagenic (Akasaka and Guengerich, 1990) and repaired by glycosylases. The particular lesion results also from lipid oxidation and may be more linked to this pathway than to that of vinyl chloride (Morinello et al, 2001).…”

1,N 2-Ethenoguanine, a Mutagenic DNA Adduct, Is a Primary Substrate of Escherichia coliMismatch-specific Uracil-DNA Glycosylase and Human Alkylpurine-DNA-N-Glycosylase

“…1) (12). While 1,N 2 -⑀G is a moderate DNA polymerase-blocking lesion in vitro (13) widely differing misincorporation frequencies opposite 1,N 2 -⑀G have been observed both with in vitro assays and in Escherichia coli (13)(14).…”