Kotapati, Srikanth2014-12-222014-12-222013-10https://hdl.handle.net/11299/168258University of Minnesota Ph.D. dissertation. October 2013. Major: Medicinal Chemistry. Advisor: Dr. Natalia Y. Tretyakova. 1 computer file (PDF); xx, 285 pages.Cigarette smoking is a known risk factor for the development of lung cancer: approximately 1 out of 5 heavy smokers will develop the disease. However, there are significant differences in risk of lung cancer among smokers from different ethnic/racial groups. African American and Native Hawaiian smokers are at a higher risk of lung cancer than European American, Japanese American or Latin American smokers. Cigarette smoke has more than 70 known carcinogens. Following metabolic activation to electrophilic species, these carcinogens can form covalent DNA adducts, which are capable of inducing heritable mutations ultimately resulting in lung cancer. It has been hypothesized that the observed ethnic/racial differences in lung cancer risk in smokers are due to different frequencies of specific polymorphisms in drug metabolizing genes, leading to a different degree of carcinogen bioactivation to DNA-reactive intermediates. 1,3-Butadiene (BD) is among the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated primarily by CYP2E1 to form 3,4-epoxy-1-butene (EB), hydroxymethyl vinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, HMVK, EBD, and DEB have been shown to modify DNA bases to form promutagenic adducts. Alternatively, EB, EBD, and DEB can undergo detoxification via epoxide hydrolysis (the main pathway in humans) or glutathione conjugation and further metabolic conversion into urinary mercapturic acids, 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetyl cysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-<italic>bis</italic>-(N-acetylcysteinyl)butane-2,3-diol (<italic>bis</italic>-BDMA), respectively. The research presented in this thesis focuses on revealing any ethnic/racial differences in metabolism of BD in smokers and examining the ability of BD-DNA adducts to cause mutations. In the first part of the thesis, we have identified two novel metabolites of BD which have not been previously detected <italic>in vivo</italic>: 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-<italic>bis</italic>-(N-acetylcysteinyl)butane-2,3-diol (<italic>bis</italic>-BDMA). To enable their detection in smokers, sensitive and specific HPLC-ESI<super>-</super>-MS/MS methods were developed for both metabolites in human urine. We observed significant amounts of THBMA in samples from smokers, non-smokers and occupationally exposed workers. In contrast, <italic>bis</italic>-BDMA amounts in urine of smokers and occupationally exposed workers were below the method's limit of detection, although it was found in urine of F344 rats exposed to 62.5 or 200 ppm BD. Additionally we found significant interspecies differences in BD metabolism between laboratory rats and humans. DHBMA accounted for only 47% of BD urinary mercapturic acids in rats while the corresponding percentage in humans is 93%. We further developed a high throughput HPLC-ESI<super>-</super>-MS/MS method for the quantification of MHBMA and DHBMA in humans and applied this method to quantify urinary BD-mercapturic acids metabolites in workers from a BD and styrene butadiene rubber (SBR) manufacturing facility and smokers belonging to different ethnic groups in two separate multi-ethnic cohort studies. Workers occupationally exposed to BD excreted significantly more BD-mercapturic acids than administrative workers at the same plant. In a small multi-ethnic study of smokers belonging to European American, Native Hawaiian and Japanese American (N = 200 per group), mean urinary MHBMA and MHBMA/DHBMA+MHBMA metabolic ratio were highest in European American and lowest in Japanese American smokers. Similar results were obtained in the larger study (N = 450 per group) composed of European American and African American smokers. Urine of European American smokers contained higher concentrations of MHBMA than that of African Americans. Genome-wide association study (GWAS) analysis conducted for the larger multi-ethnic group has revealed significant associations between single-nucleotide polymorphisms (SNPs) in chromosome 22 (22564172bp - 22735492 bp, nearby genes GSTT1, GSTT2, DDT and MIF) and urinary BD-mercapturic acid levels in smokers, providing the first evidence for genetic and ethnic/racial differences in metabolism of BD.The second part of my thesis work has focused on evaluating the mutagenic ability of three recently discovered BD-dA lesions: <italic>N</italic><super>6</super>-(2-hydroxy-3-buten-1-yl)-adenine (<italic>N</italic><super>6</super>-HB-dA), <italic>N</italic><super>6</super>,<italic>N</italic><super>6</super>-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (<italic>N</italic><super>6</super>,<italic>N</italic><super>6</super>-DHB-dA), and 1,<italic>N</italic><super>6</super>-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,<italic>N</italic><super>6</super>-&gamma-HMHP-dA). <italic>In vitro</italic> translesion synthesis experiments were performed on synthetic oligonucleotides containing each of the three lesions at a site-specific position by gel electrophoresis and HPLC-MS/MS. We found that human translesion synthesis (TLS) polymerases hPols &eta, &kappa, &iota and human polymerase &beta were able to bypass (<italic>S</italic>)-<italic>N</italic><super>6</super>-HB-dA in an error-free manner because of the conserved Watson-Crick base pairing with dT. However, replication past both (<italic>R</italic>,<italic>R</italic>)-<italic>N</italic><super>6</super>,<italic>N</italic><super>6</super>-DHB-dA and (<italic>R</italic>,<italic>S</italic>)-1,<italic>N</italic><super>6</super>-&gamma-HMHP-dA lesions by TLS polymerases hPols &eta and &kappa was highly error-prone, resulting in A&rarrT, A&rarrC mutations and frameshift deletions. This is the first study that identifies (<italic>R</italic>,<italic>R</italic>)-<italic>N</italic><super>6</super>,<italic>N</italic><super>6</super>-DHB-dA and (<italic>R</italic>,<italic>S</italic>)-1,<italic>N</italic><super>6</super>-&gamma-HMHP-dA as BD-DNA adducts potentially responsible for the induction of A*rarrT mutations by BD.en1,3-butadieneEthnic differencesHPLCMass spectrometryMetabolismTranslesion synthesisMedicinal chemistryThesis or Dissertation