Lipid-Peroxidation Induced Cyclic Adducts in Hepatocarcinogenesis

描述（由申请方提供）：在啮齿动物和人体组织中检测到脂质过氧化反应产生的丙烯醛（Acr-dG）和反式-4-羟基-2-壬烯醛（HNE-dG）的环状1，N2-丙氧脱氧鸟苷加合物为内源性DNA损伤。然而，它们在致癌作用中的作用仍不清楚。最近在体外培养的人类细胞和含有人类p53基因的DNA片段中的研究表明，Acr和HNE可以以序列选择性的方式优先结合到p53基因的鸟嘌呤上，并且结合模式类似于在某些人类癌症中观察到的p53突变热点，这表明Acr-dG和HNE-dG在致癌作用中可能的作用。然而，尚未进行体内研究以直接检查在相关动物肿瘤发生模型中肿瘤发生的组织中的环状加合物。这些研究很重要，因为它们提供了强有力的证据，直接将加合物与靶组织中的突变和肿瘤形成联系起来。在本申请中，我们建议使用遗传缺陷的Long Evans Cinnamon（LEC）大鼠和转基因XPA缺陷（XPA-/-）小鼠，这两种小鼠在没有致癌剂治疗的情况下都非常容易发生自发性肝癌，以研究Acr-dG和HNE-dG作为内源性DNA损伤在致癌作用中的作用。LEC大鼠由于铜的异常积累而遭受肝脏中脂质过氧化作用的增强，而XPA（-/-）小鼠缺乏核苷酸切除修复（NER），这对于去除Acr-和HNE-dG加合物是重要的。我们的假设是，与野生型Long Evans（LE）大鼠和XPA（+/+）小鼠相比，这些动物的环状加合物的形成将增加，这些加合物将在肝脏DNA中积累，这些加合物的形成增加是肝癌发生机制的基础。我们将进行以下目的来检验这一假设。目的一：在LEC大鼠和XPA（-/-）小鼠及其正常品系中进行肿瘤生物测定，研究其一生中肝DNA中环状加合物与肝癌发生不同阶段的关系;在目标2中，我们将绘制LEC大鼠和XPA（-/-）肝DNA p53基因中Acr和HNE加合物结合位点的图谱。在肿瘤生物测定过程中对小鼠的p53突变谱进行分析，并将它们的结合位点与生物测定结束时收获的肝肿瘤的p53突变谱进行比较。在目标3中，我们将研究抗氧化剂对肝DNA中环状加合物的形成和这些动物中自发性肝癌的发展的影响。从这些研究中产生的数据将有助于我们了解内源性环Acr-dG和HNE-dG加合物的作用，从脂质过氧化在肝癌的发生。

DESCRIPTION (provided by applicant): Cyclic 1,N2-propanodeoxyguanosine adducts of acrolein (Acr-dG) and trans-4-hydroxy-2-nonenal (HNE-dG) derived from lipid peroxidation have been detected as endogenous DNA lesions in rodent and human tissues. However, their roles in carcinogenesis are still not clearly understood. Recent in vitro studies in cultured human cells and in DNA fragments containing the human p53 gene have shown that Acr and HNE can bind to the p53 gene preferentially at guanines in a sequence-selective manner and that the binding patterns are similar to p53 mutation hotspots observed in certain human cancers, suggesting the possible roles of Acr-dG and HNE-dG in carcinogenesis. However, no in vivo studies have been conducted to directly examine cyclic adducts in tissues where tumors develop in a relevant animal tumorigenesis model. These studies are important because they provide strong evidence directly linking adducts with mutations and tumor formation in the target tissue. In this application, we propose to use genetically defected Long Evans Cinnamon (LEC) rats and transgenic XPA-deficient (XPA-/-) mice, both of which are highly prone to spontaneous liver cancer without carcinogen treatment, to investigate the roles of Acr-dG and HNE-dG as endogenous DNA lesions in carcinogenesis. LEC rats are inflicted with heightened lipid peroxidation in the liver as a result of abnormal copper accumulation, whereas XPA(-/-) mice are deficient in nucleotide excision repair (NER) which is important for removing Acr- and HNE-dG adducts. Our hypothesis is that the formation of the cyclic adducts will be increased and these adducts will accumulate in the liver DNA of these animals compared with the wild-type Long Evans (LE) rats and XPA(+/+) mice and that the increased formation of these adducts underlies the mechanisms for liver carcinogenesis. We will carry out the following aims to examine this hypothesis. In Aim 1, we will conduct tumor bioassays in LEC rats and XPA(-/-) mice and their normal strains and study throughout their lives the relationships of cyclic adducts in the liver DNA with liver carcinogenesis at different stages; In Aim 2, we will map Acr and HNE adduct binding sites in the p53 gene of the liver DNA from LEC rats and XPA (-/-) mice during the tumor bioassays and compare their binding sites with the p53 mutational spectra of the liver tumors harvested at the termination of bioassays. In Aim 3, we will investigate the effects of antioxidants on the formation of cyclic adducts in the liver DNA and on the development of spontaneous liver cancers in these animals. The data generated from these studies will help us to understand the roles of endogenous cyclic Acr-dG and HNE-dG adducts from lipid peroxidation in hepatocarcinogenesis.