Synthesis and characterisation of DNA adducts derived from the bracken toxin ptaquiloside.

蕨菜是一种高度侵入性的植物，在英国和欧洲广泛发现，对牛和其他动物有很好的毒性。蕨菜的毒性来自于存在的Ptaquiloside，一种既有致突变性又有致癌性的降倍半萜类葡萄糖苷。Ptaquiloside可以通过直接接触受污染的地表水或地下水或间接接触受污染的乳制品传播给人类。在微碱性pH下，Ptaquiloside分解形成一种称为蕨菜二烯酮的有效烷基化剂。DNA暴露于蕨菜二烯酮产生N3-腺嘌呤和N7-鸟嘌呤碱基加合物。相比之下，相应的O 6-烷基鸟嘌呤加合物（O 6-（pta）G加合物）尚未在DNA中表征，尽管其形成的生物学后果显著更显著：O 6-烷基鸟嘌呤加合物是前诱变剂和致癌剂，在DNA复制过程中，O 6-烷基鸟嘌呤通常编码胸腺嘧啶的错误掺入，导致GC到AT的转换突变。（特别是O 6-（pta）鸟嘌呤）加合物的DNA暴露后，ptaquiloside/蕨菜二烯酮，我们将提取和纯化的蕨菜。含O 6-（pta）鸟嘌呤加合物的DNA将使用O 6-烷基鸟嘌呤结合蛋白TRIP 1进行表征，并与酶消化含O 6-（pta）鸟嘌呤的DNA后合成的O 6-修饰的脱氧核糖核苷标准品进行比较。不可或缺的这个建议，我们将开发化学合成的天然存在的对映异构体的pterosin B和新的ODNs含有O 6-（pta）鸟嘌呤加合物。将检查蛋白质O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）修复含O 6-（pta）鸟嘌呤的DNA的能力以及该加合物对人DNA聚合酶的底物性质。Ptaquiloside或蕨菜二烯酮与水（分解）或DNA碱基的反应产生蝶呤B及其衍生物。我们的目标是开发用于检测此类含蝶呤B的化合物的方法。我们的建议将导致对接触蕨菜及其成分的性质和人类风险的更一致的理解。

Bracken is a highly invasive plant found extensively in the UK and Europe with a well-documented toxicity in cattle and other animals. Bracken toxicity derives from the presence of ptaquiloside, a norsesquiterpenoid glucoside that is both mutagenic and carcinogenic. Ptaquiloside can be transmitted to humans following direct exposure from contaminated surface or ground water in bracken infested areas, or indirectly for example from contaminated dairy products. At slightly alkaline pH ptaquiloside decomposes to form a potent alkylating agent known as bracken dienone. The exposure of DNA to bracken dienone produces N3-adenine and N7-guanine base adducts. In contrast, the corresponding O6-alkylguanine adducts (O6-(pta)G adducts) have not been characterized in DNA, although the biological consequences of their formation are substantially more significant: O6-alkylguanine adducts are promutagenic and carcinogenic, typically coding for the misincorporation of thymine during DNA replication leading to GC to AT transition mutations.This proposal seeks the first characterization of O6-alkylguanine (specifically O6-(pta)guanine) adducts in DNA following exposure to ptaquiloside/bracken dienone that we will extract and purify from bracken. DNA containing O6-(pta)guanine adducts will be characterised using the O6-alkylguanine-binding protein Atl1 and by comparison with a synthesized O6-modified deoxyribonucleoside standard following enzymatic digestion of O6-(pta)guanine-containing DNA. Integral to this proposal, we will develop chemical syntheses of the naturally occurring enantiomer of pterosin B and novel ODNs containing O6-(pta)guanine adducts. The ability of the protein O6-methylguanine-DNA methyltransferase (MGMT) to repair O6-(pta)guanine-containing DNA and the substrate properties of this adduct for human DNA polymerase enzymes will be examined. The reaction of ptaquiloside or bracken dienone with water (decomposition) or the bases of DNA produces pterosin B and derivatives thereof. We aim to develop methods for the detection of such pterosin B-containing compounds. Our proposal will lead to a much more coherent understanding of the nature and risk to humans of exposure to bracken and its constituents.