NanoHPLC-nanoESI⁺-MS/MS Quantitation of Bis-N7-Guanine DNA–DNA Cross-Links in Tissues of B6C3F1 Mice Exposed to subppm Levels of 1,3-Butadiene

Abstract

1,3-butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA-DNA cross-links. We have previously employed capillary HPLC-ESI+-MS/MS methods to quantify DEB-induced DNA-DNA conjugates, e.g. 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N6-HMHP-dA), in tissues of laboratory mice exposed to 6.25 – 625 ppm BD (Goggin et al. Cancer Research 69(6), 2479–2486, 2009). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1– 2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI+-MS/MS method was developed for ultra-sensitive, accurate, and precise quantitation of bis-N7G-BD in tissues of laboratory mice treated with low ppm and sub-ppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 109 nucleotides. Bis-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 109 nucleotides, respectively, suggesting that N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and sub-ppm exposure to BD.

NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

“…To achieve detection limits in the low fmol to amol range (1 per 10 8 –1 per 10 9 nucleotides) as required for our in vivo studies, we employed nanoLC/ESI + -HRMS 3 methodology on an Orbitrap Velos mass spectrometer. We and others have recently reported the use of high resolution mass spectrometry for DNA adduct analysis in complex samples and have shown that it dramatically reduces the matrix background, leading to greatly improved signal to noise ratios [37, 45, 46]. …”

Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene

“…34–38 These adducts represent the biologically relevant carcinogen dose following exposure to BD, and appear to correlate with cancer development in experimental animal models. 12,35 We recently reported mass-spectrometry based quantitation of N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts induced by EB (a major genotoxic metabolite of BD) in DNA isolated from peripheral blood leukocytes of smokers, nonsmokers and occupationally exposed BD workers.…”

Quantitation of DNA Adducts by Stable Isotope Dilution Mass Spectrometry

“…63 The LOD value of the method was 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify bis -N7G-BD adducts present at concentrations of 3 per 10 9 nucleotides. Using this methodology, bis -N7G-BD was quantified in tissues of laboratory mice treated with low ppm and sub-ppm concentrations of BD.…”

“…24 More recently, we reported nanoHPLC-nanoMS analysis of bis -N7G-BD in tissues of mice treated with sub-ppm concentrations of BD that approach occupational exposure limits. 63 …”