Urinary N7-(1-hydroxy-3-buten-2-yl) guanine adducts in humans: temporal stability and association with smoking

Abstract

1,3-Butadiene (BD) is a known human carcinogen found in cigarette smoke, automobile exhaust, and urban air. Workers occupationally exposed to BD in the workplace have an increased incidence of leukemia and lymphoma. BD undergoes cytochrome P450-mediated metabolic activation to 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB) and 1,2-dihydroxy-3,4-epoxybutane (EBD), which form covalent adducts with DNA. We have previously reported a quantitative nanoLC/ESI+-HRMS3 method for urinary N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts as a mechanism-based biomarker of BD exposure. In the present study, the method was updated to include high throughput 96-well solid phase extraction (SPE) and employed to establish urinary EB-GII biomarker stability and association with smoking. Urinary EB-GII levels were measured bimonthly for 1 year in 19 smokers to determine whether single adduct measurement provides reliable levels of EB-GII in an individual smoker. In addition, association of EB-GII with smoking was studied in 17 individuals participating in a smoking cessation program. EB-GII levels decreased 34% upon smoking cessation, indicating that it is associated with smoking status, but may also originate from sources other than exposure to cigarette smoke.

Figure 1.

Formation of EB-GII adducts from 1,3-butadiene.

Figure 2.

Sample preparation procedure for high-throughput nanoLC/ESI⁺-HRMS³ analysis of EB-GII in urine.

Figure 3.

NanoLC/ESI⁺HRMS³ method validation: correlation between the added and observed amounts of EB-GII spiked into 30 µl water.

Figure 4.

NanoLC/ESI⁺HRMS³ method validation: correlation between the spiked and observed amounts of EB-GII spiked into 200 µl of synthetic urine.

Figure 5.

Representative nanoLC/ESI⁺HRMS³ detection of EB-GII in urine of a smoker. Top and bottom panels show extracted ion chromatogram MS³ spectra from EB-GII and ¹⁵N₅-EB-GII (internal standard), respectively.

Figure 6.

Urinary EB-GII levels (pg/ml urine) in subjects participating in a smoking cessation program. EB-GII levels decrease significantly from baseline to Day 3. Further decreases are not statistically significant.