Impact of the human intestinal microbiota on the metabolism and toxic properties of the meat contaminant 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Cancer is a major disease burden worldwide that accounts in countries with a Western lifestyle for 20% of the mortality rate. Epidemiological evidence suggests that diet makes a substantial contribution to the burden of human cancer. It is the consumption of meat, and in particular red meat, that has shown the strongest association with human neoplastic disease, particularly tumors of the colon and rectum. Cooking of meat is known to generate a family of promutagenic/procarcinogenic compounds, including the heterocyclic aromatic amine class of chemical compounds. Of the 19 heterocyclic amines identified so far, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is frequently the most mass abundant heterocyclic amine produced during the cooking of beef, pork and chicken. The human intake of PhIP varies with food type and cooking conditions and is estimated to range from nanograms to tens of micrograms per day, depending on individual dietary and cooking preferences. Assessment studies based on rodent tumor data and the abundance of PhIP in the diet have indicated that this heterocyclic amine may be a risk factor in human colon, breast and prostate carcinogenesis; which co-incidentally are the three most common sites of diet-associated cancer in the Western world. As a means of determining the potential health risks associated with heterocyclic amines, several dietary studies have been conducted on the metabolism and disposition of these compounds in humans. So far, most investigations focused on the activation and detoxification of heterocyclic amines by mammalian phase I and II enzymes. In common with other aromatic amines, PhIP is metabolically activated by N-oxidation of the exocyclic amino group, a reaction mediated mainly by the cytochrome P450 isoenzyme CYP1A2. On the other hand, the involvement of the intestinal microbiota in the digestive fate of heterocyclic amines remains poorly investigated. Recent research has however shown that the amount of PhIP metabolites excreted in the urine of humans following ingestion of PhIP in a meat matrix is significantly lower than that of patients administered PhIP in a capsule. This indicates that PhIP provided in capsule form is more bioavailable than PhIP ingested from meat. The non-bioavailable fraction reaches the colon and becomes available for biotransformation by the colonic bacteria. At the start of this research only a few, partly conflicting results from studies with lactobacilli and intestinal microorganisms were available. Indications exist that the intestinal microbiota are essential to the induction of DNA damage by PhIP in HFA rats. Information on the bacterial metabolism of native heterocyclic amines is however scarce and limited to some studies on the quinoline type heterocyclic amines. Therefore, the main objective of this work was to explore the possible role of the human intestinal microbiota in the metabolism and biological activity of PhIP. To do this, an integrated in vitro-in vivo approach was followed, combining fecal incubations, human studies and mammalian cell lines. In the first part of this research, the microbial metabolism of PhIP was investigated. A preliminar explorative study in which PhIP was anaerobically incubated with stools freshly collected from six healthy volunteers demonstrated that PhIP was extensively transformed by the human intestinal bacteria. HPLC analysis revealed that the human fecal microbiota converted PhIP specifically into one major derivative. ESI-MS/MS, HRMS, 1D (1H, 13C, DEPT) and 2D (gCOSY, gTOCSY, gHMBC, gHSQC) NMR and IC analysis elucidated the complete chemical identity of the microbial PhIP metabolite, as 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3,2:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1). To evaluate whether this newly identified microbial PhIP metabolite could be produced by the intestinal bacteria in vivo as well, a human intervention trial was set up. Six human subjects were fed 150 g of cooked chicken containing 0.88-4.7 µg PhIP, and urine and feces collections were obtained during 72 h after the meal. PhIP-M1 and its trideuterated derivate were synthesized and a rapid and accurate solid-phase extraction LC-ESI-MS/MS method for the simultaneous quantification of PhIP and PhIP-M1 in human urine and feces was developed. Of the ingested PhIP dose, volunteers excreted 12-21% as PhIP and 1.2-15% as PhIP-M1 in urine, and 26-42% as PhIP and 0.9-11% as PhIP-M1 in feces. The rate of PhIP-M1 excretion varied among the subjects. Yet, an increase in urinary excretion was observed for successive time increments, whereas for PhIP the majority was excreted in the first 24 h. These findings confirmed that the human intestinal bacteria significantly contribute to the overall metabolism and disposition of PhIP in vivo. After the observation that PhIP could be metabolically converted by the human intestinal bacteria in vitro and in vivo, the next step was to identify and characterize the bacterial species responsible for this process. Two PhIP transforming strains PhIP-M1-a and PhIP-M1-b were isolated from human feces and identified by a combination of microscopy, PCR-DGGE, FAFLPTM and pheS sequence analyses as Enterococcus faecium. Some strains from culture collections belonging to the species Enterococcus durans, Enterococcus avium, Enterococcus faecium and Lactobacillus reuteri were also able to perform this transformation. Glycerol was identified as a fecal matrix constituent required for PhIP conversion. Abiotic synthesis of PhIP-M1 and quantification of the glycerol metabolite 3-hydroxypropopionaldehyde (3-HPA) confirmed that the anaerobic fermentation of glycerol via 3-HPA is the critical bacterial transformation process responsible for the formation of PhIP-M1. Although several lactobacilli, as well as other bacterial species have been shown to use glycerol as an external electron acceptor, we are the first to relate bacterial species of the genus Enterococcus to this anaerobic pathway of glycerol dissimilation. In addition, we have shown that PhIP-M1 production occurs under proteolytic conditions. This was true for mixed fecal microbiota as well as for the Enterococcus faecium PhIP-M1-a transforming strain. The production of PhIP-M1 was shown to be dependent on interindividual differences. A first explorative experiment with six human fecal samples demonstrated this factor. Subsequent fecal incubations with eighteen human microbiota confirmed that individuals could be separated into low, moderate and high PhIP-M1 producers with transformation efficiencies ranging from 1.8 to 96%. Finally, significant differences in intestinal PhIP-M1 production were found to determine differences in urinary and fecal PhIP-M1 excretion in vivo in humans. This indicated that interindividual differences in microbial composition and metabolism may at least be equally important than differential expression and genetic polymorphisms in phase I and II endogenous enzymes, which have been considered so far as the obvious candidates responsible for individual variability in PhIP metabolism, bioavailability and carcinogenicity. In the second part of this doctoral research, the impact of the intestinal microbiota on the biological activity of PhIP was evaluated. Since ligation of the biliary duct has been shown not to alter the genotoxic potential of PhIP, the deconjugation of reactive glucuronides by bacterial β-glucuronidase is most likely not to alter the metabolic fate and bioactivity of PhIP. Therefore, it was very much conceivable that the microbial formation of PhIP-M1 contributed to the final genotoxic and carcinogenic activity of PhIP. Firstly, it was observed that PhIP-M1, as analyzed using the Salmonella typhimurium strains TA98, TA100 and TA102, yielded no significant mutagenic response. Subsequently, it was assessed whether PhIP-M1 could exert any cytotoxic or genotoxic effects towards a human intestinal cell line. PhIP-M1 was shown to induce DNA damage, cell cycle arrest, apoptosis and eventually cell death and growth inhibition towards the epithelial Caco-2 cell line. DNA damage in Caco-2 cells was detected using the Comet assay. This assay is recognized as a sensitive tool widely used for the evaluation of primary DNA damages at the individual cell level, while the bacterial Ames assay only detects mutagenic effects if the DNA damage induced remained after cell division. The conversion of PhIP into PhIP-M1 was therefore considered as a microbial bioactivation. As the genomic and cellular events of CYP1A2-activated PhIP in different in vitro cell systems are not significantly higher than those observed for PhIP-M1 in our test system, the physiological relevance of this newly identified microbial PhIP derivate in PhIP carcinogenicity may not be neglected. Finally, it was investigated whether addition of native chicory inulin could inhibit the extent of microbial PhIP bioactivation. Inulin is generally considered to exert prebiotic effects as it stimulates health-promoting bacteria in the human gut such as bifidobacteria. However, it is also hypothesized that it may exert chemopreventive effects by the indirect suppression of microbial groups such as enterococci that are responsible for the hazardous conversion of carcinogenic compounds such as PhIP. In addition, inulin is known to bring about prebiotic effects at the level of the metabolic activity, resulting in a saccharolytic fermentation pattern and acidic environment. Supplementation of inulin during several weeks to a full-scale SHIME reactor showed significant inhibitory effects towards PhIP bioactivation, in particular in the transverse colon compartment. Interestingly, the strongest decrease in proteolytic end products was also observed in this region of the colon, indicating an indirect relationship with the chemopreventive effects from inulin. As the typical proteolytic conditions in the distal parts of the colon are normally more detrimental to the host in vivo, in particular in the light of the microbial PhIP bioactivation process, these positive modifications in the metabolism and microbial community indicate that inulin is a promising chemopreventive agent

Metabolic fingerprinting to assess the impact of salinity on carotenoid content in developing tomato fruits

As the presence of health-promoting substances has become a significant aspect of tomato fruit appreciation, this study investigated nutrient solution salinity as a tool to enhance carotenoid accumulation in cherry tomato fruit (Solanum lycopersicum L. cv. Juanita). Hereby, a key objective was to uncover the underlying mechanisms of carotenoid metabolism, moving away from typical black box research strategies. To this end, a greenhouse experiment with five salinity treatments (ranging from 2.0 to 5.0 decisiemens (dS) m(-1)) was carried out and a metabolomic fingerprinting approach was applied to obtain valuable insights on the complicated interactions between salinity treatments, environmental conditions, and the plant's genetic background. Hereby, several hundreds of metabolites were attributed a role in the plant's salinity response (at the fruit level), whereby the overall impact turned out to be highly depending on the developmental stage. In addition, 46 of these metabolites embraced a dual significance as they were ascribed a prominent role in carotenoid metabolism as well. Based on the specific mediating actions of the retained metabolites, it could be determined that altered salinity had only marginal potential to enhance carotenoid accumulation in the concerned tomato fruit cultivar. This study invigorates the usefulness of metabolomics in modern agriculture, for instance in modeling tomato fruit quality. Moreover, the metabolome changes that were caused by the different salinity levels may enclose valuable information towards other salinity-related plant processes as well

Plant-based beverages as good sources of free and glycosidic plant sterols

To address the ever-growing group of health-conscious consumers, more and more nutritional and health claims are being used on food products. Nevertheless, only very few food constituents, including plant sterols, have been appointed an approved health claim (European Commission and Food and Drugs Administration). Plant sterols are part of those limited lists of approved compounds for their cholesterol-lowering properties but have been praised for their anti-inflammatory and anti-carcinogenic properties as well. Despite this indisputable reputation, direct quantitative data is still lacking for naturally present (conjugated) plant sterols in beverages. This study aimed to fill this gap by applying a validated extraction and UPLC-MS/MS detection method to a diverse range of everyday plant-based beverages. B-sitosterol--D-glucoside (BSSG) showed to be by far the most abundant sterol in all beverages studied, with concentrations up to 60–90 mg per 100 mL in plant-based milk alternatives and fresh fruit juices. Ergosterol (provitamin D2) could be found in beers (0.8–6.1 g per 100 mL, from the yeast) and occasionally in juices (17–29 g per 100 mL). Overall, the results demonstrated that the concentrations of water-soluble sterol conjugates have been underestimated significantly and that specific plant-based beverages can be good, low-fat sources of these plant sterols