Quantitative NanoLC/NSI+-HRMS Method for 1,3-Butadiene Induced bis-N7-guanine DNA-DNA Cross-Links in Urine

Abstract

1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI⁺-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

Keywords: 1,3-butadiene; LC-MS/MS; bis-N7G-BD; mercapturic acids; urinary DNA adducts.

Scheme 1

Partial metabolic scheme of 1,3-butadiene showing DNA adduct formation, hydrolysis and urinary excretion. ^aCR, carbonyl reductase; EH, epoxide hydrolase; EB-GII, N7-(1-hydroxy-3-buten-2-yl) guanine; MA, mercapturic acid; bis-N7G-BD, 1,4-bis-(gua-7-yl)-2,3-butanediol; bis-BDMA, 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol; MHBMA, 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene; DHBMA 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane.

Figure 1

NanoLC-NSI⁺-HRMS method validation: correlation analysis between the spiked and the observed amounts of bis-B7G-BD spiked into control mouse urine (10 μL). Spiked amounts were 0, 0.5, 1.0, 2.0, 5.0, or 10.0 fmol of bis-N7G-BD and 10.0 fmol of ¹⁵N₆- bis-N7G-BD (internal standard), followed by sample processing and nanoLC-NSI⁺-HRMS analysis.

Scheme 2

Experimental scheme for isotope dilution nanoLC-NSI⁺-HRMS analysis of bis-N7G-BD in urine.

Figure 2

Representative extracted ion chromatogram (A) and MS² spectra for bis-N7G-BD and ¹⁵N₆- bis-N7G-BD (B) obtained upon nanoLC-NSI⁺-HRMS² analysis of urine of laboratory mice exposed to 1,3-butadiene. Mouse urine (10 μL) was spiked with ¹⁵N₆-bis-N7G-BD (10.0 fmol) and subjected to sample processing and nanoLC-NSI⁺-HRMS analysis on an Obitrap QExactive mass spectrometer.

Figure 3

Quantitation of bis-N7G-BD DNA (A), bis-BDMA (B), MHBMA (C), and DHBMA (D) in urine of laboratory mice exposed to either filtered air (control) or 1,3-butadiene. Asterisk (**) denotes a statistically significant (p < 0.01) inter-sex difference (unpaired two-tailed t-test).

Figure 4

Correlation of urinary DNA adduct bis-N7G-BD with BD metabolites bis-BDMA (A) and EB-GII (B) in urine of laboratory mouse exposed to 1,3-butadiene. (C) Pearson correlation of urinary DNA adducts bis-N7G-BD and EB-GII with BD metabolites bis-BDMA, MHBMA, and DHBMA in urine of laboratory mouse exposed to 1,3-butadiene.