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

Abstract

Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI+-HRMS3 analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub ppm concentrations of BD (0.5–1.5 ppm). EB-GII concentrations increased linearly from 1.15±0.23 to 10.11±0.45adducts per 108 nucleotides in HT1080 cells treated with 0.5–10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0 and 1.5 ppm BD were 0.17±0.05, 0.33±0.08, and 0.50±0.04 adducts per 108 nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20±0.12 days) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI+-HRMS3 Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

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

“…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. 19 Although EB-GII was detectable in blood DNA of most human subjects, its concentrations were below the LOQ of the method (0.4 adducts/10 9 nucleotides or 0.2 fmol/150 μg of DNA). N7-guanine adducts such as those induced by BD-derived epoxides are hydrolytically unstable due to the presence of a positive charge on the N7 position of the alkylated base, and are spontaneously released from the DNA backbone over time (Scheme 1).…”

“…Therefore, SPE enriched fractions were subjected to additional enrichment by an offline HPLC method developed in an earlier study. 19 HPLC fractions containing EB-GII and its internal standard were collected, concentrated under vacuum, and reconstituted with 10 μL of water for nanoLC/ESI + -HRMS 3 analysis. HPLC blanks were injected every 10–15 samples to detect any analyte carryover during the offline HPLC clean up procedure.…”

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…Aside from proteins and digestion enzymes, ultrafiltration can also remove the partially depurinated DNA backbone from which adducted bases are selectively released following the aforementioned neutral thermal hydrolysis. 182 Despite being simple and rapid, ultrafiltration is always coupled with further clean-up procedures, such as SPE and off-line HPLC because of its relatively low efficiency and specificity in sample purification.…”

“…Sangaraju et al 182 found that the sensitivity of nLC-nESI-MS/MS was insufficient for the detection of 1,3-butadiene (BD)-DNA adducts in human samples (typical LOD, 0.5 adducts/10 6 nucleosides). To overcome this, the authors developed an nLC-nESI-HRMS 3 method to decrease detection limits to the low fmol to amol range (1–10 per 10 9 nucleosides with 3–76 µg DNA) for their in vivo studies.…”

Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry

“…Sangaraju et al. (, ) employed nanoHPLC‐LIT‐HRMS n for the quantitation of DNA adducts formed with 1,3‐butadiene (BD). BD is a known human carcinogen present in automobile exhaust and cigarette smoke (Lewtas, ).…”