Heterocyclic aromatic amines: Microbial metabolism and interaction with fruit and vegetable dietary fiber

杂环芳胺(HAA)是一种具有诱变性和潜在致癌性的化合物，可在热处理的食品中发现。在小肠吸收后，HAA被一系列复杂的I相酶反应和II相酶反应代谢激活。在外环氨基的N-羟基化后，N-羟基代谢物可以偶联形成N-乙酰氧基和N-磺酰氧基酯。异解裂解产生高活性的芳基氮离子，可以形成DNA加合物。然而，在小肠中，HAA可以被膳食纤维和细菌吸附，从而逃脱吸收。纤维/细菌结合的HAA到达结肠，并可能被肠道微生物区系代谢，从而被激活或灭活。然而，到目前为止，在体外和体内只鉴定了两种微生物HAA代谢物(PhIP-M1和7-OH-IQ)。初步数据表明，其他HAA如AalphaC也可以被肠道微生物代谢。该项目致力于在体外鉴定由人粪便微生物区系形成的AalphaC、MeIQx、MeIQ、Trp-P-1和Harman/Norharman的未知代谢物。通过LC-MS和核磁共振对代谢产物进行了结构鉴定。在半制备规模上分离主要代谢物，并分别使用Ames试验和Caco-2模型测试它们的诱变潜力和在结肠中的吸收。此外，还研究了木质化果蔬膳食纤维吸附HAA对细菌HAA代谢产物形成的影响。此外，还将分析微生物HAA代谢产物在部分发酵纤维上的吸附情况。通过使用合成的木质素模型膳食纤维，我们先前证明了HAA的吸附取决于木质素的浓度和组成。研究还表明，模型膳食纤维的部分细菌发酵导致了纤维/HAA相互作用的减少。本研究旨在从以下几个方面对木质化果蔬纤维进行分离和结构表征：碳水化合物含量和结构(HPAEC，GC-FID/MS)，木质素含量和结构(DFRC法，2D-核磁共振)。分离出的纤维在体外用人类粪便悬浮液发酵。随后，将部分发酵的纤维用于与HAA代谢物的结合研究。此外，还将HAA与膳食纤维在小肠条件下孵育，制备纤维-HAA-复合体。利用粪便样品进行发酵实验，研究了这些纤维-HAA-复合体形成HAA-代谢物的过程。本申请中提出的研究具有重要意义，因为关于微生物HAA代谢物的形成及其与来自水果和蔬菜的膳食纤维相互作用的数据将有助于重新评估经常被建议的膳食纤维如何降低各种形式癌症风险的机制。

Heterocyclic aromatic amines (HAA) are mutagenic and potentially carcinogenic compounds, which can be found in thermally treated food products. After absorption in the small intestine, HAA are metabolically activated by a complex sequence of phase-I- and phase-II-enzyme reactions. Following N-hydroxylation of the exocyclic amino group, the N-hydroxy metabolites can be conjugated to form N-acetoxy- and N-sulfonyloxy esters. Heterolytic cleavage generates highly reactive aryl nitrenium ions, which can form DNA adducts. However, in the small intestine, HAA can be adsorbed to dietary fiber and bacteria, thus escaping absorption. Fiber/bacteria bound HAA reach the colon and are potentially metabolized by the gut microbiota, thus being activated or inactivated. However, to date, only two microbial HAA metabolites (PhIP-M1 und 7-OH-IQ) were identified both in vitro and in vivo. Preliminary data demonstrated that other HAA such as AalphaC can also be metabolized by the gut microbiota.The proposed project strives to identify unknown metabolites of AalphaC, MeIQx, MeIQ, Trp-P-1 and Harman/Norharman formed by human fecal microbiota in vitro. Structural elucidation of the metabolites is performed by LC-MS and NMR. Dominant metabolites are isolated on a semipreparative scale and tested for their mutagenic potential and absorption in the colon by using the Ames test and a Caco-2-model, respectively. In addition, the impact of the adsorption of HAA to lignified dietary fiber from fruits and vegetables on the formation of bacterial HAA metabolites is studied. Also, the adsorption of microbial HAA metabolites to partially fermented fiber will be analyzed. By using synthetically lignified model dietary fibers we previously demonstrated that HAA adsorption is dependent on both lignin concentration and composition. It was also shown that partial bacterial fermentation of the model dietary fibers led to a reduced fiber/HAA interaction. In this study we aim to isolate and structurally characterize lignified fruit and vegetable fibers with regard to the following characteristics: Carbohydrate contents and structures (HPAEC, GC-FID/MS), lignin contents and structures (DFRC-method, 2D-NMR). The isolated fibers are fermented in vitro with human fecal suspensions. Subsequently, the partially fermented fibers are used in binding studies with HAA metabolites. In addition, fiber-HAA-complexes are prepared by incubating HAA and dietary fibers under small intestine conditions. The formation of HAA-metabolites from these fiber-HAA-complexes is studied in fermentation experiments using fecal samples. The research proposed in this application is significant because the resulting data on the formation of microbial HAA metabolites and their interaction with dietary fiber from fruits and vegetables will contribute to a re-evaluation of an often suggested mechanism how dietary fiber can reduce the risk of various forms of cancer.