Western diet influences on microbiome and carcinogenesis.

The intestinal microbiota composition and associated bioactivities are sensitive to various modifier cues such as stress, inflammation, age, life-style and nutrition, which in turn are associated with susceptibility to developing cancer. Among these modifiers, diet has been shown to influence both microbiota composition as well as being an important source of microbial-derived compounds impacting the immunological, neurological and hormonal systems. Thus, it is necessary to take a holistic view when considering effect of diet on health and diseases. In this review, we focus on the interplay between western diet, the microbiota and cancer development by dissecting key components of the diet and leveraging data from human interventions and pre-clinical studies to better understand this relationship. We highlight key progress as well as stressing limitations in this field of research.

Comparing Transgenic Production to Supplementation of ω-3 PUFA Reveals Distinct But Overlapping Mechanisms Underlying Protection Against Metabolic and Hepatic Disorders.

We compared endogenous ω-3 PUFA production to supplementation for improving obesity-related metabolic dysfunction. Fat-1 transgenic mice, who endogenously convert exogenous ω-6 to ω-3 PUFA, and wild-type littermates were fed a high-fat diet and a daily dose of either ω-3 or ω-6 PUFA-rich oil for 12 wk. The endogenous ω-3 PUFA production improved glucose intolerance and insulin resistance but not hepatic steatosis. Conversely, ω-3 PUFA supplementation fully prevented hepatic steatosis but failed to improve insulin resistance. Both models increased hepatic levels of ω-3 PUFA-containing 2-monoacylglycerol and N-acylethanolamine congeners, and reduced levels of ω-6 PUFA-derived endocannabinoids with ω-3 PUFA supplementation being more efficacious. Reduced hepatic lipid accumulation associated with the endocannabinoidome metabolites EPEA and DHEA, which was causally demonstrated by lower lipid accumulation in oleic acid-treated hepatic cells treated with these metabolites. While both models induced a significant fecal enrichment of the beneficial Allobaculum genus, mice supplemented with ω-3 PUFA displayed additional changes in the gut microbiota functions with a significant reduction of fecal levels of the proinflammatory molecules lipopolysaccharide and flagellin. Multiple-factor analysis identify that the metabolic improvements induced by ω-3 PUFAs were accompanied by a reduced production of the proinflammatory cytokine TNFα, and that ω-3 PUFA supplementation had a stronger effect on improving the hepatic fatty acid profile than endogenous ω-3 PUFA. While endogenous ω-3 PUFA production preferably improves glucose tolerance and insulin resistance, ω-3 PUFA intake appears to be required to elicit selective changes in hepatic endocannabinoidome signaling that are essential to alleviate high-fat diet-induced hepatic steatosis.

Gut Microbiota as a Novel Tool to Dissect the Complex Structures of Black Tea Polymers.

Thearubigins, polymers of tea catechins, account for more than 20% of the black tea polyphenols and have been reported to be the active components in black tea. However, the chemical structures and underlying mechanisms regarding how the thearubigins, being poorly bioavailable, generate in vivo health benefits are still largely unknown. Using germ-free and specific pathogen-free husbandry conditions combined with LC/MS-based nontargeted and targeted metabolomic analyses, we investigated the role of intestinal bacteria in thearubigin metabolism. Theaflavins and theasinensins were identified as the major microbial metabolites of thearubigins, suggesting that these molecules are the building units for the complex thearubigins. To further confirm this, thearubigin depolymerization was done using menthofuran in an acidic condition. Menthofuran-conjugated theaflavins, theasinensins, and catechins as well as their free forms were detected as the major degradation products of thearubigins. This indicated that theaflavins and theasinensins could be further polymerized through B-type proanthocyanidin linkages. Furthermore, four microbial degradation products were able to be detected in urine samples, suggesting that they can be absorbed into the circulatory system. Using the combination of microbial degradation, metabolomics, and chemical degradation, our results demonstrate that thearubigins are the complex polymers of theaflavins, theasinensins, and catechins and can be metabolized by gut microbiota to their corresponding bioactive and bioavailable smaller molecular metabolites.

Black Tea Theaflavin Detoxifies Metabolic Toxins in the Intestinal Tract of Mice.

SCOPE: This study is to determine the in vivo efficacy of black tea theaflavin (TF) to detoxify two metabolic toxins, ammonia and methylglyoxal (MGO), in mice METHODS AND RESULTS: Under in vitro conditions, TF is able to react with ammonia, MGO, and hydrogen peroxide to produce its aminated, MGO conjugated, and oxidized products, respectively. In TF-treated mice, the aminated TF, the MGO conjugates of TF and aminated TF, and the oxidized TF are searched using LC-MS/MS. The results provide the first in vivo evidence that the unabsorbed TF is able to trap ammonia to form the aminated TF; furthermore, both TF and the aminated TF have the capacity to trap MGO to generate the corresponding mono-MGO conjugates. Moreover, TF is oxidized to dehydrotheaflavin, which underwent further amination in the gut. By exposing TF to germ-free (GF) mice and conventionalized mice (GF mice colonized with specific-pathogen-free microbiota), the gut microbiota is demonstrated to facilitate the amination and MGO conjugation of TF. CONCLUSION: TF has the capacity to remove the endogenous metabolic toxins through oxidation, amination, and MGO conjugation in the intestinal tract, which can potentially explain why TF still generates in vivo efficacy while showing a poor systematic bioavailability.

Dietary iron variably modulates assembly of the intestinal microbiota in colitis-resistant and colitis-susceptible mice.

Iron deficiency, a common comorbidity of gastrointestinal inflammatory disorders such as inflammatory bowel diseases (IBD), is often treated with oral iron supplementation. However, the safety of oral iron supplementation remains controversial because of its association with exacerbated disease activity in a subset of IBD patients. Because iron modulates bacterial growth and function, one possible mechanism by which iron may exacerbate inflammation in susceptible hosts is by modulating the intestinal microbiota. We, therefore, investigated the impact of dietary iron on the intestinal microbiota, utilizing the conventionalization of germ-free mice as a model of a microbial community in compositional flux to recapitulate the instability of the IBD-associated intestinal microbiota. Our findings demonstrate that altering intestinal iron availability during community assembly modulated the microbiota in non-inflamed wild type (WT) and colitis-susceptible interleukin-10-deficient (Il10-/-) mice. Depletion of luminal iron availability promoted luminal compositional changes associated with dysbiotic states irrespective of host genotype, including an expansion of Enterobacteriaceae such as Escherichia coli. Mechanistic in vitro growth competitions confirmed that high-affinity iron acquisition systems in E. coli enhance its abundance over other bacteria in iron-restricted conditions, thereby enabling pathobiont iron scavenging during dietary iron restriction. In contrast, distinct luminal community assembly was observed with dietary iron supplementation in WT versus Il10-/- mice, suggesting that the effects of increased iron on the microbiota differ with host inflammation status. Taken together, shifts in dietary iron intake during community assembly modulate the ecological structure of the intestinal microbiota and is dependent on host genotype and inflammation status.

Microbiota facilitates the formation of the aminated metabolite of green tea polyphenol (-)-epigallocatechin-3-gallate which trap deleterious reactive endogenous metabolites.

The in vivo mechanism of tea polyphenol-mediated prevention of many chronic diseases is still largely unknown. Studies have shown that accumulation of toxic reactive cellular metabolites, such as ammonia and reactive carbonyl species (RCS), is one of the causing factors to the development of many chronic diseases. In this study, we investigated the in vivo interaction between (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in tea leaves, and ammonia and RCS. We found that EGCG could be oxidized to EGCG quinone in mice, and then rapidly react with ammonia to generate the aminated EGCG metabolite, 4'-NH2-EGCG. Both EGCG and its aminated metabolite could further scavenge RCS, such as methylglyoxal (MGO), malondialdehyde (MDA), and trans-4-hydroxy-2-nonenal (4-HNE), to produce the RCS conjugates of EGCG and the aminated EGCG. Both the aminated and the RCS conjugated metabolites of EGCG were detected in human after drinking four cups of green tea per day. By comparing the levels of the aminated and the RCS conjugated metabolites in EGCG exposed germ-free (GF) mice and specific-pathogen-free (SPF) mice, we demonstrated that gut microbiota facilitate the formation of the aminated metabolite of EGCG, the RCS conjugates of EGCG, and the RCS conjugates of the aminated EGCG. By comparing the trapping capacities of EGCG and its aminated metabolite under aerobic and anaerobic conditions, we found that oxygen is not essential for the trapping of reactive species by EGCG and 4'-NH2-EGCG suggesting that EGCG and its aminated metabolite could scavenge RCS in the GI track and in the circulation system. Altogether, this study provides in vivo evidences that EGCG has the capacity to scavenge toxic reactive metabolic wastes. This finding opens a new window to understand the underlying mechanisms by which drinking tea could prevent the development of chronic diseases.

A Rapid Screenable Assay for Compounds That Protect Against Intestinal Injury in Zebrafish Larva.

This chapter describes a method to assay compounds modulating NSAID-induced intestinal injury in zebrafish larvae. The assay employs the NSAID glafenine, which causes intestinal epithelial cell damage and death by inducing organelle stress responses (endoplasmic reticulum and mitochondrial) and blocking the unfolded protein response pathway. This epithelial damage includes sloughing of intestinal cells into the lumen and out the cloaca of the zebrafish larvae. Exposing larvae to acridine orange highlights this injury when visualized under fluorescence microscope; injured fish develop intensely red-staining intestines, as well as a "tube" or cord of red color extending through the intestine and out the cloaca. Using this rapid visually screenable method, various candidate compounds were successfully tested for their ability to prevent glafenine-induced intestinal injury. Because this assay involves examination of larval zebrafish intestinal pathology, we have also included our protocol for preparation and analysis of zebrafish histology. The protocol includes numerous steps to generate high-quality zebrafish histology slides, as well as protocols to establish accurate anatomic localization of any given tissue cross-section-processes that are made technically difficult by the small size of zebrafish larvae.

The Role of Curcumin in Modulating Colonic Microbiota During Colitis and Colon Cancer Prevention.

BACKGROUND: Intestinal microbiota influences the progression of colitis-associated colorectal cancer. With diet being a key determinant of the gut microbial ecology, dietary interventions are an attractive avenue for the prevention of colitis-associated colorectal cancer. Curcumin is the most active constituent of the ground rhizome of the Curcuma longa plant, which has been demonstrated to have anti-inflammatory, antioxidative, and antiproliferative properties. METHODS: Il10 mice on 129/SvEv background were used as a model of colitis-associated colorectal cancer. Starting at 10 weeks of age, wild-type or Il10 mice received 6 weekly intraperitoneal injections of azoxymethane (AOM) or phosphate-buffered saline (PBS) and were started on either a control or a curcumin-supplemented diet. Stools were collected every 4 weeks for microbial community analysis. Mice were killed at 30 weeks of age. RESULTS: Curcumin-supplemented diet increased survival, decreased colon weight/length ratio, and, at 0.5%, entirely eliminated tumor burden. Although colonic histology indicated improvement with curcumin, no effects of mucosal immune responses have been observed in PBS/Il10 mice and limited effects were seen in AOM/Il10 mice. In wild-type and in Il10 mice, curcumin increased bacterial richness, prevented age-related decrease in alpha diversity, increased the relative abundance of Lactobacillales, and decreased Coriobacterales order. Taxonomic profile of AOM/Il10 mice receiving curcumin was more similar to those of wild-type mice than those fed control diet. CONCLUSIONS: In AOM/Il10 model, curcumin reduced or eliminated colonic tumor burden with limited effects on mucosal immune responses. The beneficial effect of curcumin on tumorigenesis was associated with the maintenance of a more diverse colonic microbial ecology.

Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute intestinal inflammation.

Oxymatrine is a traditional Chinese herbal product that exhibits anti-inflammatory effects in models of heart, brain and liver injury. We investigated the impact of oxymatrine in an acute model of intestinal injury and inflammation. Oxymatrine significantly decreased LPS-induced NF-κB-driven luciferase activity, correlating with diminished induction of Cxcl2, Tnfα and Il6 mRNA expression in rat IEC-6 and murine BMDC. Although oxymatrine decreased LPS-induced p65 nuclear translocation and binding to the Cxcl2 gene promoter, this effect was independent of IκBα degradation/phosphorylation. DSS-induced weight loss and histological damage were ameliorated in oxymatrine-treated C57BL/6-WT-mice. While this effect correlated with reduced colonic Il6 and Il1ß mRNA accumulation, global NF-κB activity as measured in NF-κB(EGFP) mice was unaffected. Our data demonstrate that oxymatrine reduces LPS-induced NF-κB nuclear translocation and activity independently of IκBα status, prevents intestinal inflammation through blockade of inflammatory signaling and ameliorates overall intestinal inflammation in vivo.

The microbiota is essential for the generation of black tea theaflavins-derived metabolites.

BACKGROUND: Theaflavins including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3'-gallate (TF3'G), and theaflavin-3,3'-digallate (TFDG), are the most important bioactive polyphenols in black tea. Because of their poor systemic bioavailability, it is still unclear how these compounds can exert their biological functions. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans. METHODS AND FINDINGS: In the present study, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3'G, and gallic acid as the major fecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG- gavaged GF mice. The microbial bioconversion of TFDG, TF3G, and TF3'G was also investigated in vitro using fecal slurries collected from three healthy human subjects. Our results indicate that TFDG is metabolized to TF, TF3G, TF3'G, gallic acid, and pyrogallol by human microbiota. Moreover, both TF3G and TF3'G are metabolized to TF, gallic acid, and pyrogallol by human microbiota. Importantly, we observed interindividual differences on the metabolism rate of gallic acid to pyrogallol among the three human subjects. In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG. CONCLUSIONS: The microbiota is important for the metabolism of theaflavins in both mice and humans. The in vivo functional impact of microbiota-generated theaflavins-derived metabolites is worthwhile of further study.

Think small: zebrafish as a model system of human pathology.

Although human pathologies have mostly been modeled using higher mammal systems such as mice, the lower vertebrate zebrafish has gained tremendous attention as a model system. The advantages of zebrafish over classical vertebrate models are multifactorial and include high genetic and organ system homology to humans, high fecundity, external fertilization, ease of genetic manipulation, and transparency through early adulthood that enables powerful imaging modalities. This paper focuses on four areas of human pathology that were developed and/or advanced significantly in zebrafish in the last decade. These areas are (1) wound healing/restitution, (2) gastrointestinal diseases, (3) microbe-host interactions, and (4) genetic diseases and drug screens. Important biological processes and pathologies explored include wound-healing responses, pancreatic cancer, inflammatory bowel diseases, nonalcoholic fatty liver disease, and mycobacterium infection. The utility of zebrafish in screening for novel genes important in various pathologies such as polycystic kidney disease is also discussed.

Structural identification of mouse fecal metabolites of theaflavin 3,3'-digallate using liquid chromatography tandem mass spectrometry.

Black tea consumption has been associated with many health benefits including the prevention of cancer and heart disease. Theaflavins are the major bioactive polyphenols present in black tea. Unfortunately, limited information is available on their biotransformation. In the present study, we investigated the metabolic fate of theaflavin 3,3'-digallate (TFDG), one of the most abundant and bioactive theaflavins, in mouse fecal samples using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MS(n) (n=1-3) spectra. Four metabolites theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, and gallic acid were identified as the major mouse fecal metabolites of TFDG. Glucuronidated and sulfated, instead of methylated metabolites of theaflavin 3-gallate, theaflavin 3'-gallate, and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. Our results indicate that TFDG can be degraded in mice. Further studies on the formation of those metabolites in TFDG-treated mice in germ-free conditions are warranted. To our knowledge, this is the first report on the biotransformation of TFDG in mice.

Alleviating cancer drug toxicity by inhibiting a bacterial enzyme.

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial ß-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial ß-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial ß-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.

Tomato lycopene extract prevents lipopolysaccharide-induced NF-kappaB signaling but worsens dextran sulfate sodium-induced colitis in NF-kappaBEGFP mice.

BACKGROUND: The impact of tomato lycopene extract (TLE) on intestinal inflammation is currently unknown. We investigated the effect of TLE on lipopolysaccharide (LPS)-induced innate signaling and experimental colitis. METHODOLOGY/PRINCIPAL FINDINGS: Mice were fed a diet containing 0.5 and 2% TLE or isoflavone free control (AIN-76). The therapeutic efficacy of TLE diet was assessed using dextran sulfate sodium (DSS) exposed mice and IL-10(-/-);NF-kappaB(EGFP) mice, representing an acute and spontaneous chronic colitis model respectively. A mini-endoscope was used to determine the extent of macroscopic mucosal lesions. Murine splenocytes and intestinal epithelial cells were used to determine the in vitro impact of TLE on LPS-induced NF-kappaB signaling. In vitro, TLE blocked LPS-induced IkappaBalpha degradation, RelA translocation, NF-kappaB transcriptional activity and MIP-2 mRNA accumulation in IEC-18 cells. Moreover, LPS-induced IL-12p40 gene expression was dose-dependently inhibited in TLE-treated splenocytes. Interestingly, DSS-induced acute colitis worsened in TLE-fed NF-kappaB(EGFP) mice compared to control diet as measured by weight loss, colonoscopic analysis and histological scores. In contrast, TLE-fed IL-10(-/-);NF-kappaB(EGFP) mice displayed decreased colonic EGFP expression compared to control diet. IL-6, TNFalpha, and MCP-1 mRNA expression were increased in the colon of TLE-fed, DSS-exposed NF-kappaB(EGFP) mice compared to the control diet. Additionally, caspase-3 activation and TUNEL positive cells were enhanced in TLE diet-fed, DSS-exposed mice as compared to DSS control mice. CONCLUSIONS/ SIGNIFICANCE: These results indicate that TLE prevents LPS-induced proinflammatory gene expression by blocking of NF-kappaB signaling, but aggravates DSS-induced colitis by enhancing epithelial cell apoptosis.