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.

Background: DNA-protein conjugates can be induced by reactive oxygen species and proteolytically cleaved to the corresponding peptide conjugates. Results: Polymerase bypass past C5-dT peptide conjugates catalyzed by human polymerases and gives rise to base substitutions and deletions. Conclusion: Replication past C5-T peptide conjugates is mutagenic. Significance: This study provides the first evidence for error-prone replication of DPCs cross-linked to pyrimidines in DNA.

Background 1,3-Butadiene (BD) is an important carcinogen in tobacco smoke that undergoes metabolic activation to DNA-reactive epoxides. These species can be detoxified via glutathione conjugation and excreted in urine as the corresponding N-acetylcysteine conjugates. We hypothesize that single nucleotide polymorphisms in BD-metabolizing genes may change the balance of BD bioactivation and detoxification in White, Japanese American, and African American smokers, potentially contributing to ethnic differences in lung cancer risk. Methods We measured the levels of BD metabolites, 1- and 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (MHBMA) and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), in urine samples from a total of 1,072 White, Japanese American, and African American smokers and adjusted these values for body mass index, age, batch, and total nicotine equivalents. We also conducted a genome wide association study to identify genetic determinants of BD metabolism. Results We found that mean urinary MHBMA concentrations differed significantly by ethnicity (p = 4.0 × 10−25). African Americans excreted the highest levels of MHBMA followed by Whites and Japanese Americans. MHBMA levels were affected by GSTT1 gene copy number (p < 0.0001); conditional on GSTT1, no other polymorphisms showed a significant association. Urinary DHBMA levels also differed between ethnic groups (p = 3.3 ×10−4), but were not affected by GSTT1 copy number (p = 0.226). Conclusions GSTT1 gene deletion has a strong effect on urinary MHBMA levels, and therefore BD metabolism, in smokers. Impact Our results show that the order of MHBMA levels among ethnic groups is consistent with their respective lung cancer risk and can be partially explained by GSTT1 genotype.

N 6-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine (N6-HB-dA I) and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N6,N6-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N6 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 N6-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N6-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N6-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N6-HB-dA I and (R,R)-N6,N6-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N6-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N6,N6-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N6-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N6,N6-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 (S)-N6-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N6,N6-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N6-HB-dA I lesions are not miscoding, but exocyclic (R,R)-N6,N6-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.

◥ 2-Phenethyl isothiocyanate (PEITC) is a natural product found as a conjugate in cruciferous vegetables. It has been reported to have preventative properties against lung cancer and to inhibit metabolic activation of tobacco carcinogens. In this study, we evaluated the ability of PEITC to influence the metabolism of the human carcinogen 1,3-butadiene in current smokers in a phase II clinical trial with a crossover design. Urinary mercapturic acids of 1,3-butadiene were quantified at baseline and during PEITC treatment. Seventy-nine smokers were randomly assigned to one of two arms: PEITC followed by placebo or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. Oral ingestion of PEITC increased urinary levels of BDmercapturic acids (MHBMA and DHBMA) by 11.1% and 3.7%, respectively, but these increases were not statistically significant (P ¼ 0.17 and 0.64, respectively). A much stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased urinary levels of MHBMA by 58.7% (P ¼ 0.004) and 90.0% (P ¼ 0.001), respectively, but did not have a significant effect on urinary DHBMA. These results reveal a potentially protective effect of PEITC treatment with respect to the detoxification of 1,3-butadiene in cigarette smokers, specifically in those null for GSTT1, and provide further evidence in support of stronger chemopreventive effects from consumption of dietary isothiocyanates in these individuals.