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

Abstract

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 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). The new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0 – 200 ppm 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 BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1–2.2 ppm BD (1.25 ± 0.51pg/mg creatinine) as compared to administrative controls exposed to < 0.01 ppm BD (0.22 ± 0.08 and pg/mg creatinine) (p = 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 creatinine, p = 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. Comparison of EB-GII adduct levels in urine to urinary mercapturic acids of BD (MHBMA, DHBMA) and genetic polymorphisms were also attempted in the genotyped smoker ethnic groups cohort.

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

“…Our previous experience with developing methods to quantify urinary DNA adducts showed that SPE alone does not result in sufficient sample purity for nanoLC/MS analyses [ 36 ]. Therefore, following SPE, samples were further purified using an offline HPLC method [ 39 ].…”

“…Earlier studies have described urinary DNA adducts induced by endogenous biomolecules such as estrogens [ 55 ], lipid peroxidation products [ 56 ] as well as by reactive exogenous metabolites such as styrene [ 57 ] and polyaromatic hydrocarbons [ 58 ]. Previous studies employed BD-derived urinary adduct, EB-GII, as a useful biomarker of exposure to BD and metabolic activation [ 34 , 35 , 36 , 59 ]. However, EB-GII is formed from BD monoepoxide (EB) and thus does not reflect the formation of the most genotoxic metabolite of 1,3-butadiene (i.e., DEB).…”

“…To test the stability of bis-N7G-BD in mouse urine, a freeze–thaw stability assay was performed as previously described for 1,3-butadiene urinary DNA adducts [ 36 ]. Urine from 1,3-butadiene exposed mouse was frozen (−20 °C) for 24 h and thawed at room temperature for 2 h (1 freeze–thaw cycle).…”

“…By contrast, DNA adducts are mechanistically relevant, cancer-related markers; DNA is the ultimate biological target of genotoxic carcinogens. We have previously reported analytical methods for sensitive detection of many butadiene-DNA adducts in genomic DNA, [ 24 , 25 , 36 , 37 , 38 ] however, large epidemiological studies do not make DNA available. In the present work, we have developed a quantitative nanoLC/NSI + -HRMS methodology for bis -N7G-BD DNA adducts in urine.…”

1,3-Butadiene: a ubiquitous environmental mutagen and its associations with diseases

“…The DNA adducts released are eventually excreted in urine and thus can also be used as urinary biomarkers. Although many BD-derived DNA adducts have been identified [ 7 ], so far only two DNA adducts, N 7-[1-​(hydroxymethyl)​-​2-​propen-​1-​yl]guanine (EB-GII) and 1,4- bis (gua-7-yl)-2,3-butanediol ( bis -N7G-BD), have been used as urinary biomarkers [ 130 , 131 ].…”