The Role of N1-Inosine Adducts in Butadiene Mutagenesis

性状（由申请方提供）：1，3-丁二烯（BD）是一种已知的啮齿动物和人类致癌物。 然而，负责碱基对突变的DNA加合物尚未确定。 我们推测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定点突变研究中显示N1-HB-dl加合物具有高度致突变性的研究。coli和COS细胞。 没有关于这些加合物在体外或体内的形成、持久性或修复的信息。 因此，在所提出的研究中，我们将合成N1-HB-dl和N1-THB-dl加合物标准品，并开发一种通过LC-ESI-MS对其进行检测和分析的方法。然后将开发的方法应用于来自暴露于1，2-环氧-3-丁烯（EB）或1，2; 3，4-二环氧丁烷（DFB）的人TK 6细胞的DNA样品。 此外，这些方法将用于分析暴露于不同浓度BD、EB或DEB的小鼠和大鼠中的N1-肌苷加合物，以评价体内加合物形成的剂量学。 此外，将分析BD处理10天并在暴露后2小时、1、3或6天处死的小鼠和大鼠的组织，以计算加合物半衰期并了解修复率。 最后，将N1-肌苷加合物的数量与先前分析的小鼠和TK 6细胞中N7-gunaine加合物（N7-HB-gunaine和N7-THB-gunine）和hprt突变的量进行比较。 解决这一假设对于确定BD突变的来源至关重要，并最终提高我们准确评估BD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.