The Role of N1-Inosine Adducts in Butadiene Mutagenesis

描述（由申请人提供）：1,3-丁二烯（BD）是已知的啮齿动物和人类致癌物。然而，导致碱基对突变的DNA加合物尚未被确定。我们假设N1-（羟基丁烯）-脱氧肌苷（N1- hb - di）和N1-三羟基丁烷-脱氧肌苷（N1- thb -dl）加合物是导致A:T碱基对突变的原因。这一假设是基于在大肠杆菌和COS细胞的定点突变研究中显示N1-HB-dl加合物具有高度诱变性的研究。没有关于这些加合物在体外或体内形成、持续或修复的信息。因此，在本研究中，我们将合成N1-HB-dl和N1-THB-dl加合物标准物，并建立一种LC-ESI-MS检测和分析方法。开发的方法将随后应用于人TK6细胞的DNA样本，暴露于1,2-环氧-3-丁烯（EB）或1,2；3, 4-diepoxybutane(足协)。此外，这些方法将用于分析暴露于不同浓度BD、EB或DEB的小鼠和大鼠体内的n1 -肌苷加合物，以评估加合物形成的剂量学。此外，研究人员将分析小鼠和大鼠的组织，并分别在暴露后2小时、1天、3天或6天处死，以计算加合物的半衰期，并了解修复率。最后，将n1 -肌苷加合物的数量与之前分析的N7-gunaine加合物（N7-HB-gunaine和N7-THB-gunine）的数量以及小鼠和TK6细胞中的hprt突变进行比较。解决这一假设对于确定双相障碍突变的来源至关重要，并将最终提高我们准确评估双相障碍对人类风险的能力。

DESCRIPTION (provided by applicant): 1,3-butadiene (BD) is a known rodent and human carcinogen. However, the DNA adducts responsible for base pair mutations have not been identified. We hypothesize that N1- (hydroxybutene)-deoxyinosine (N1-HB-dI) and N1-trihydroxybutane-deoxyinosine (N1-THB-dl) adducts are responsible of the mutations at A:T base pairs. This hypothesis is based on studies showing that N1-HB-dl adducts are highly mutagenic in site directed mutation studies in E. coli and COS cells. There is no information on the formation, persistence or repair of these adducts in vitro or in vivo. Therefore, in the proposed studies we will synthesize the N1-HB-dl and N1-THB-dl adduct standards and develop a method for their detection and analysis by LC-ESI-MS. The developed methods will then be applied to DNA samples from human TK6 cells, exposed to 1,2-epoxy-3-butene (EB) or 1,2;3,4-diepoxybutane (DFB). Additionally, these methods will be used to analyze N1-inosine adducts in mice and rats exposed to different concentrations of BD, EB or DEB to evaluate the dosimetry of adduct formation in vivo. Further, tissues from mice and rats treated with BD for 10 days and sacrificed 2h, 1, 3 or 6 days post exposure will be analyzed to calculate the adduct half-lives and give insight on the rate of repair. Finally, the number of N1-inosine adducts will be compared with previously analyzed amounts of N7-gunaine adducts (N7-HB-gunaine and N7-THB-gunine) and the hprt mutations in mice and in TK6 cells. Addressing this hypothesis is essential for determining the sources of BD mutations and will ultimately increase our ability to accurately assess the risk of BD to humans.