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

Polymerase Bypass of <i>N</i>⁶‑Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

<i>N</i>⁶-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine (<i>N</i>⁶-HB-dA I) and <i>N</i>⁶,<i>N</i>⁶-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (<i>N</i>⁶,<i>N</i>⁶-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the <i>N</i>⁶ position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the <i>N</i>⁶-H atom of adenine is required for Watson–Crick hydrogen bonding with thymine, <i>N</i>⁶-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived <i>N</i>⁶-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (<i>S</i>)-<i>N</i>⁶-HB-dA I and (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts were subjected to <i>in vitro</i> translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (<i>S</i>)-<i>N</i>⁶-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (<i>S</i>)-<i>N</i>⁶-HB-dA I. In contrast, hPol β was completely blocked by (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (<i>S</i>)-<i>N</i>⁶-HB-dA I was mostly error-free, replication of DNA containing (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (<i>S</i>)-<i>N</i>⁶-HB-dA I lesions are not miscoding, but that exocyclic (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson–Crick pairs with dT