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

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 (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA–DNA conjugates, e.g. 1,4-<i>bis</i>-(guan-7-yl)-2,3-butanediol (<i>bis</i>-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and <i>1,N</i>^<i>6</i>-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (<i>1,N</i>^<i>6</i>-HMHP-dA), in tissues of laboratory mice exposed to 6.25–625 ppm BD (Goggin et al. <i>Cancer Res.</i> <b>2009</b>, <i>69</i>(6), 2479–2486). 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 ultrasensitive, accurate, and precise quantitation of <i>bis</i>-N7G-BD in tissues of laboratory mice treated with low ppm and subppm 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 <i>bis</i>-N7G-BD adducts present at concentrations of 3 per 10⁹ nucleotides. <i>Bis</i>-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 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 <i>bis</i>-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 subppm exposure to BD

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

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI⁺-HRMS³ methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0–200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1–2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (<i>p</i> = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers’ urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, <i>p</i> = 3.1 × 10^–7). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared in a genotyped multiethnic smoker cohort