Association between polyphenol metabolism and polymorphisms in intestinal drug metabolising enzymes and its relation to health benefits

流行病学研究表明，经常饮用红茶和绿色茶与降低一系列病理条件的风险有关，包括心血管疾病和选定的癌症。体外研究表明，流行病学研究中观察到的影响部分是由茶中存在的儿茶素及其聚合物引起的。然而，这些研究的局限性在于，它们大多没有考虑到多酚的生物利用度非常低，血浆峰浓度在亚微摩尔范围内。迄今为止，仍不清楚多酚的哪些分子形式可以介导这些作用，以及潜在的作用机制是什么。多酚的生物利用度差阿利亚是由外源性物质代谢酶的广泛生物转化引起的。已经显示了儿茶素通过肠II相代谢酶如儿茶酚-O-甲基-转移酶（COMT）和UDP-葡糖醛酸基转移酶（UGT）的代谢。此外，体外数据表明，选择的多酚可以诱导许多I相代谢酶的活性，如细胞色素CYP 450 1A 1。反过来，I相和II相酶的激活可能会改变其他多酚化合物的生物利用度。此外，这些酶的多态性已被认为影响多酚的代谢率和绝对生物利用度。总的来说，人们对多酚在吸收过程中如何以及在多大程度上生物转化以及这一过程如何与其潜在的健康有益作用相关缺乏了解。因此，拟议项目的总体目标是利用组织培养和人为干预研究来发现绿色和红茶多酚对I、II和III相酶的影响。该项目的目标是阐明产生了哪些不同的多酚代谢物，以及遗传变异如何解释人类研究中观察到的一些受试者之间的差异。最后，该项目旨在确定多酚代谢物是否能够减少线粒体DNA损伤。主要目的是确定茶多酚在组织培养中的代谢谱，I，II和III相酶参与，以及I，II和III相代谢的遗传差异。一个重要的早期里程碑将是使用适当的肠和肝代谢体外模型鉴定主要的茶多酚代谢物。为了实现这一目标，我们将利用已建立的模型，如肠微粒体，HepG 2细胞和肝切片。将使用最先进的HPLC-MS/MS方法测定代谢特征。代谢谱分析的结果将用于鉴定所涉及的主要异生物质代谢酶。此外，计划确定组织培养物和口腔粘膜细胞中参与茶多酚代谢的关键I、II和III相酶的基因和蛋白质表达。定量PCR以及微阵列分析将突出代谢途径以及茶叶施用后差异调节的关键I、II和III期酶。接下来的步骤将是鉴定人类颊粘膜细胞中关键I、II和III期酶的常见单核苷酸多态性。科学挑战将是将那些频率>5%的多态性与人类的代谢谱联系起来。将进行人为干预研究以解决这些问题。该项目将是揭示茶多酚潜在健康有益作用的一个重要里程碑，它将更多地揭示I、II和III相酶产生的代谢物，它们在血浆中循环的浓度，以及在多大程度上观察到的人类受试者之间的差异与异生物质代谢酶的遗传变异有关。

Epidemiological studies suggest that regular consumption of black and green tea is associated with a reduction in risk of a range of pathological conditions including cardiovascular diseases and selected cancers. In vitro studies suggest that the effects seen in epidemiological studies are partially caused by catechins and polymers thereof present in tea. However, limitations in these studies are that they mostly do not take into account the very low bioavailability of polyphenols with peak plasma concentration in the sub-micromolar range. To date it is still unclear which molecular forms of polyphenols could mediate these effects, and what the potential mechanisms of action are. The poor bioavailability of polyphenols is caused inter alia by extensive biotransformation by xenobiotic metabolising enzymes. Metabolism of catechins by intestinal phase II metabolising enzymes such as catechol-O-methyl-transferase (COMT) and UDP-glucuronosyltransferases (UGT's) has been shown. In addition, in vitro data suggest that selected polyphenols can induce activity of a number of phase I metabolising enzymes such as cytochroms CYP450 1A1. In turn, activation of phase I and phase II enzymes could change bioavailability of other polyphenolic compounds. Furthermore, polymorphism in those enzymes has been suggested to influence metabolic rate and absolute bioavailability of polyphenols. Overall, there is a lack of understanding how and to which extent polyphenols are biotransformed during absorption and how this process is related to their potential health beneficial effects. The overall aim of the proposed project is therefore to discover the effects of green and black tea polyphenols on phase I, II and III enzymes using both tissue culture and a human intervention study. The goal of the project is to elucidate which different polyphenol metabolites are produced and how genetic variation might explain some of the observed between-subject differences seen in human studies. Finally, the project aims at identifying whether polyphenol metabolites are able to reduce mitochondrial DNA damage. The main objectives are to identify metabolic profiles of tea polyphenols in tissue cultures, phase I, II and III enzymes involved, and genetic differences in phase I, II and III metabolism. An important early milestone will be the identification of major tea polyphenol metabolites using appropriate in vitro models for intestinal and liver metabolism. To achieve this we will make use of established models such as intestinal microsomes, HepG2 cells and liver slices. Metabolic profiles will be determined using state-of-the-art HPLC-MS/MS methods. Results from metabolic profiling will be used to identify the major xenobiotic metabolising enzymes involved. In addition, it is planned to determine gene and protein expression of key phase I, II and III enzymes involved in metabolism of tea polyphenols in tissue cultures and buccal mucosal cells. Quantitative PCR as well as microarray analysis will highlight metabolic pathways as well as key phase I, II and III enzymes differentially regulated after tea administration. The subsequent step will be identification of common single nucleotide polymorphisms in key phase I, II and III enzymes in human buccal mucosal cells. The scientific challenge will be to link those polymorphism with a frequency >5% to metabolic profiles in humans. A human intervention study will be performed to address these questions. This project will be a major milestone in unravelling the potential health beneficial effects of tea polyphenol by shedding more light on which metabolites are produced by phase I, II and III enzymes, at which concentrations they circulate in plasma and to what extent the large observed between-subject variation in humans is linked to genetic variations in xenobiotic metabolising enzymes.