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adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N

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.

Abstract1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A → T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N 6 ,N 6 -(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N 6 ,N 6 -DHB-dA), 1,N 6 -(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -γ-HMHP-dA), and 1,N 6 -(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -α-HMHP-dA) (Seneviratne et al Chem. Res. Toxicol. 2010, 23, 118-133). In the present work, a post-synthetic methodology for preparing DNA oligomers containing stereo-and site-specific N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA adducts was developed. DNA oligomers containing site specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N 6 -(2-hydroxy-3,4-epoxybut-1-yl)adenine adducts, followed by their spontaneous cyclization to 1,N 6 -γ-HMHP-dA lesions. N 6 ,N 6 -DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with 3S,4S or 3R,4R pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when adenine residue was placed opposite 1,N 6 -γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.

Human carcinogen 1,3-butadiene (BD) undergoes metabolic activation to 3,4-epoxy-1-butene (EB), hydroxymethylvinyl ketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD) and 1,2,3,4-diepoxybutane (DEB). Among these, DEB is by far the most genotoxic metabolite and is considered the ultimate carcinogenic species of BD. We have shown previously that BD-exposed laboratory mice form 8- to 10-fold more DEB-DNA adducts than rats exposed at the same conditions, which may be responsible for the enhanced sensitivity of mice to BD-mediated cancer. In the present study, we have identified 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA) as a novel DEB-specific urinary biomarker. Isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was employed to quantify bis-BDMA and three other BD-mercapturic acids, 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxy-but-3-ene (MHBMA, from EB), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA, from HMVK) and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA, from EBD), in urine of confirmed smokers, occupationally exposed workers and BD-exposed laboratory rats. Bis-BDMA was formed in a dose-dependent manner in urine of rats exposed to 0-200 p.p.m. BD by inhalation, although it was a minor metabolite (1%) as compared with DHBMA (47%) and THBMA (37%). In humans, DHBMA was the most abundant BD-mercapturic acid excreted (93%), followed by THBMA (5%) and MHBMA (2%), whereas no bis-BDMA was detected. These results reveal significant differences in metabolism of BD between rats and humans.