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

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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.