HPLC-ESI+-MS/MS analysis of N7-guanine-N7-guanine DNA cross-links in tissues of mice exposed to 1,3-butadiene

Chem Res Toxicol. 2007 May;20(5):839-47. doi: 10.1021/tx700020q. Epub 2007 Apr 25.


1,3-butadiene (BD) is a major industrial chemical used in rubber and plastics production and is recognized as an animal and human carcinogen. Although the exact mechanism of BD-induced carcinogenesis is unknown, chemical reactions of epoxide metabolites of BD with DNA to form nucleobase adducts are likely to contribute to multistage carcinogenesis. Among BD-derived epoxy metabolites, 1,2:3,4-diepoxybutane (DEB) appears to be the most genotoxic and carcinogenic, probably because of its bifunctional nature. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine monoadducts, which can then be hydrolyzed to N7-(2',3',4'-trihydroxy-1'-yl)guanine or can react with another site in double-stranded DNA to form 1,4-bis(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links. While (2',3',4'-trihydroxy-1'-yl)guanine lesions have been previously quantified in vivo, they cannot be used as a biomarker of DEB because the same lesions are also formed by another, more prevalent BD metabolite, 1,2-epoxy-3,4-butanediol. In contrast, bis-N7G-BD can only be formed from DEB, potentially providing a specific biomarker of DEB formation. We have developed a quantitative HPLC-ESI+-MS/MS method for measuring racemic and meso forms of bis-N7G-BD in DNA extracted from tissues of BD-exposed laboratory animals. In our approach, bis-N7G-BD adducts are released from DNA as free bases by neutral thermal hydrolysis, purified by solid-phase extraction, and subjected to HPLC-ESI+-MS/MS analysis. Selected reaction monitoring is performed by following the loss of a guanine moiety from protonated molecules of bis-N7G-BD and the formation of protonated guanine under collision-induced dissociation. Quantitative analysis of racemic and meso forms of bis-N7G-BD is based on isotope dilution with the corresponding 15N-labeled internal standards. The lower limit of quantification of our current method is 10-20 fmol/0.1 mg of DNA. The accuracy and precision of the new method were determined by spiking control mouse liver DNA with racemic and meso forms of bis-N7G-BD (10 fmol each), followed by sample processing and HPLC-ESI+-MS/MS analysis. Calculated amounts of racemic and meso forms of bis-N7G-BD were within 20% of the theoretical value (9.7 +/- 2 and 9.2 +/- 1.9 fmol, respectively, N = 4). DNA extracted from liver and lung tissues of mice exposed to 625 ppm butadiene for 5 days contained 3.2 +/- 0.4 and 1.8 +/- 0.5 racemic adducts per 10(6) guanines, respectively, while the amounts of meso-bis-N7G-BD were below the detection limits of our method (1 per 10(7) guanines). Control animals did not contain either bis-N7G-BD lesion. Sensitive and specific quantitative methods for bis-N7G-BD analysis developed in this work provide a unique biomarker of DEB-induced DNA alkylation following exposure to BD.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Butadienes / chemistry
  • Butadienes / metabolism
  • Butadienes / toxicity*
  • Carcinogens, Environmental / chemistry
  • Carcinogens, Environmental / metabolism
  • Carcinogens, Environmental / toxicity*
  • Chromatography, High Pressure Liquid*
  • Cross-Linking Reagents / chemistry
  • DNA Adducts / chemistry
  • DNA Adducts / drug effects*
  • DNA Adducts / metabolism
  • Guanine / analogs & derivatives*
  • Guanine / chemistry
  • Guanine / metabolism
  • Liver / chemistry
  • Liver / drug effects
  • Liver / metabolism
  • Lung / chemistry
  • Lung / drug effects
  • Lung / metabolism
  • Mice
  • Reproducibility of Results
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • Butadienes
  • Carcinogens, Environmental
  • Cross-Linking Reagents
  • DNA Adducts
  • Guanine
  • 1,3-butadiene