Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.

OBJECTIVE: IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. DESIGN: We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. RESULTS: CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). CONCLUSIONS: SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Dense genotyping of immune-related loci identifies HLA variants associated with increased risk of collagenous colitis.

OBJECTIVE: Collagenous colitis (CC) is a major cause of chronic non-bloody diarrhoea, particularly in the elderly female population. The aetiology of CC is unknown, and still poor is the understanding of its pathogenesis. This possibly involves dysregulated inflammation and immune-mediated reactions in genetically predisposed individuals, but the contribution of genetic factors to CC is underinvestigated. We systematically tested immune-related genes known to impact the risk of several autoimmune diseases for their potential CC-predisposing role. DESIGN: Three independent cohorts of histologically confirmed CC cases (N=314) and controls (N=4299) from Sweden and Germany were included in a 2-step association analysis. Immunochip and targeted single nucleotide polymorphism (SNP) genotype data were produced, respectively, for discovery and replication purposes. Classical human leucocyte antigen (HLA) variants at 2-digit and 4-digit resolution were obtained via imputation from single marker genotypes. SNPs and HLA variants passing quality control filters were tested for association with CC with logistic regression adjusting for age, sex and country of origin. RESULTS: Forty-two markers gave rise to genome-wide significant association signals, all contained within the HLA region on chromosome 6 (best p=4.2×10-10 for SNP rs4143332). Among the HLA variants, most pronounced risk effects were observed for 8.1 haplotype alleles including DQ2.5, which was targeted and confirmed in the replication data set (p=2.3×10-11; OR=2.06; 95% CI (1.67 to 2.55) in the combined analysis). CONCLUSIONS: HLA genotype associates with CC, thus implicating HLA-related immune mechanisms in its pathogenesis.

Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts.

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11,326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31×10(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.

Evaluation of the cyclooxygenase inhibiting effects of six major cannabinoids isolated from Cannabis sativa.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in the development of colon cancer, which makes this subject interesting to study further. In the present work, the six cannabinoids tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa, were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids inhibited cyclooxygenase enzyme activity with IC50 values ranging from 1.7·10⁻³ to 2.0·10⁻4 M.

Guidance for substantiating the evidence for beneficial effects of probiotics: impact of probiotics on digestive system metabolism.

Probiotic bacteria have been studied for their potential impact on the metabolism of dietary components in the small intestine lumen including lactose digestion, metabolism of lipids such as cholesterol, and oxalate metabolism. In the large intestine, they contribute to the metabolism of otherwise indigestible dietary carbohydrates (e.g., prebiotics) and have a favorable effect on colonic protein and ammonia metabolism, although their effect on the digestive fate of phytochemicals and xenobiotics is still uncertain. Probiotics also influence metabolism in the host tissues, in particular the gastrointestinal mucosa and the liver. Underlying mechanisms include supply of additional enzymatic activities in the gut lumen and alterations of the composition or metabolic pattern of the gut resident microbiota. For future studies, selection of probiotic strains should include assessment of their metabolic activities, and the outcome of the intervention studies should also take into account the composition of the probiotic matrix and the background diet of the target population. New technologies such as metabolomics hold great promise for assessment of probiotics functionality.

Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research.

Probiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. There is a growing interest in probiotics within the scientific community, with consumers, and in the food industry. The interactions between the gut and intestinal microbiota and between resident and transient microbiota define a new arena in physiology, an understanding of which would shed light on the "cross-talk" between humans and microbes. The different beneficial effects of specific probiotic strains may be translated into different health claims. However, there is a need for comprehensive and harmonized guidelines on the assessment of the characteristics and efficacy of probiotics and of foods containing them. An international expert group of ILSI has evaluated the published evidence of the functionality of different probiotics in 4 areas of (human) application: 1) metabolism, 2) chronic intestinal inflammatory and functional disorders, 3) infections, and 4) allergy. Based on the existing evidence, concrete examples of demonstration of benefits and gaps are listed, and guidelines and recommendations are defined that should help design the next generation of probiotic studies.

Genetic mapping of Mom5, a novel modifier of Apc(Min)-induced intestinal tumorigenesis.

The initial purpose of this study was to assess the role of estrogen receptor beta (ERbeta) in intestinal tumorigenesis by examining the effects of an ERbeta knockout (ERbeta(-/-)) on Apc(Min) mice. In order to accomplish this goal on a uniform genetic background, we were required to backcross the ERbeta knockout from the 129P2 genetic background to the B6 genetic background for 10 generations. Midway through this process, we performed a test cross in which mice from the N(5) backcross generation of the ERbeta knockout strain were intercrossed with Apc(Min/+) mice to obtain Apc(Min/+) ERbeta(+/+), Apc(Min/+) ERbeta(+/-) and Apc(Min/+) ERbeta(-/-) mice. Intestinal tumorigenesis in the N(5)F(2) mice was evaluated at 14 weeks of age. The analysis of the impact of ERbeta in the N(5) cross was complicated by segregating 129P2-derived alleles that affected tumor number and were unlinked to ERbeta. Genetic linkage analysis of this cross permitted the localization of a single genetic modifier of tumor number in Apc(Min/+) mice. This locus, Modifier of Min 5 (Mom5), maps to proximal mouse chromosome 5; the 129P2 allele of this locus is associated with a 50% reduction in mean intestinal tumor number. Through in silico analysis and confirmatory sequencing, we have identified the Rad50-interacting protein-1 gene as a strong candidate for Mom5.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in Apc(Min/+) mice.

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

Gut flora, Toll-like receptors and nuclear receptors: a tripartite communication that tunes innate immunity in large intestine.

Separating the large intestine from gut flora is a robust layer of epithelial cells. This barrier is armed with an array of recognizing receptors that collectively set the host innate response. Here, we use nuclear receptors (NRs) and Toll-like receptors (TLRs), suggested to act as second messengers in the communication between microorganisms and epithelial cells, as probes to assess the impact of gut flora on innate immunity in germ-free (GF) mice. Using quantitative real-time polymerase chain reaction analyses, we show that 37/49 NRs are expressed in colonic cells of GF mice. Of these, 5 can be modulated by resident flora: LXRalpha, RORgamma and CAR show reduced expression and Nur77 and GCNF display elevated expression in conventionally raised mice compared with GF. Moreover, increased expression levels of TLR-2 and TLR-5 are observed in specific pathogen-free (SPF) mice compared with GF mice, and CAR expression is connected to the TLR-2 signalling pathway. Infections of GF or SPF mice with Yersinia pseudotuberculosis, show that GF intestinal epithelial cells fail to respond, except for CAR, which is downregulated. In contrast, SPF epithelial cells show a downregulation of all the NRs except CAR, which appears to be unaffected. Our findings indicate that gut flora contributes to the development of an intact barrier function.

N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway.

The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5 mM), whereas it was necessary to use high doses of NPIP (45 mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.

Antiapoptotic effects of dietary antioxidants towards N-nitrosopiperidine and N-nitrosodibutylamine-induced apoptosis in HL-60 and HepG2 cells.

The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced apoptosis in human leukemia (HL-60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. None of the vitamin C (5-50 microm), DADS and DPDS (1-5 microm) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL-60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'-dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N-nitrosamine was reduced to control levels by vitamin C (5-50 microm) in a dose-dependent manner. However, DADS (5 microm) increased ROS levels induced by NPIP and NDBA in HL-60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 microm) in both HL-60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP- and NDBA-induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP- and NDBA-induced apoptosis in HL-60 and HepG2 cells.

Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients.

BACKGROUND: Animal studies suggest that prebiotics and probiotics exert protective effects against tumor development in the colon, but human data supporting this suggestion are weak. OBJECTIVE: The objective was to verify whether the prebiotic concept (selective interaction with colonic flora of nondigested carbohydrates) as induced by a synbiotic preparation-oligofructose-enriched inulin (SYN1) + Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (BB12)-is able to reduce the risk of colon cancer in humans. DESIGN: The 12-wk randomized, double-blind, placebo-controlled trial of a synbiotic food composed of the prebiotic SYN1 and probiotics LGG and BB12 was conducted in 37 colon cancer patients and 43 polypectomized patients. Fecal and blood samples were obtained before, during, and after the intervention, and colorectal biopsy samples were obtained before and after the intervention. The effect of synbiotic consumption on a battery of intermediate bio-markers for colon cancer was examined. RESULTS: Synbiotic intervention resulted in significant changes in fecal flora: Bifidobacterium and Lactobacillus increased and Clostridium perfringens decreased. The intervention significantly reduced colorectal proliferation and the capacity of fecal water to induce necrosis in colonic cells and improve epithelial barrier function in polypectomized patients. Genotoxicity assays of colonic biopsy samples indicated a decreased exposure to genotoxins in polypectomized patients at the end of the intervention period. Synbiotic consumption prevented an increased secretion of interleukin 2 by peripheral blood mononuclear cells in the polypectomized patients and increased the production of interferon gamma in the cancer patients. CONCLUSIONS: Several colorectal cancer biomarkers can be altered favorably by synbiotic intervention.

Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. None of the vitamin C concentrations tested (1-10 microM) in presence or absence of formamidopyrimidine-DNA glycosylase (Fpg enzyme) caused DNA damage per se. HepG2 cells simultaneously treated with vitamin C and N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner. At concentrations of 1-5 microM vitamin C, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 microM vitamin C led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of vitamin C (10 microM) was higher towards NDBA-induced oxidative DNA damage. One feasible mechanism by which vitamin C exerted its protective effect is that may interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates. Vitamin C (10 microM) strongly reduced the coumarin hydroxylase (82%) activity. However, the p-nitrophenol hydroxylase and the ethoxyresorufine O-deethylation activities were slightly and weakly reduced (32-19%), respectively.

Bidirectional communication between the Aryl hydrocarbon Receptor (AhR) and the microbiome tunes host metabolism.

The ligand-induced transcription factor, aryl hydrocarbon receptor (AhR) is known for its capacity to tune adaptive immunity and xenobiotic metabolism-biological properties subject to regulation by the indigenous microbiome. The objective of this study was to probe the postulated microbiome-AhR crosstalk and whether such an axis could influence metabolic homeostasis of the host. Utilising a systems-biology approach combining in-depth 1H-NMR-based metabonomics (plasma, liver and skeletal muscle) with microbiome profiling (small intestine, colon and faeces) of AhR knockout (AhR-/-) and wild-type (AhR+/+) mice, we assessed AhR function in host metabolism. Microbiome metabolites such as short-chain fatty acids were found to regulate AhR and its target genes in liver and intestine. The AhR signalling pathway, in turn, was able to influence microbiome composition in the small intestine as evident from microbiota profiling of the AhR+/+ and AhR-/- mice fed with diet enriched with a specific AhR ligand or diet depleted of any known AhR ligands. The AhR-/- mice also displayed increased levels of corticosterol and alanine in serum. In addition, activation of gluconeogenic genes in the AhR-/- mice was indicative of on-going metabolic stress. Reduced levels of ketone bodies and reduced expression of genes involved in fatty acid metabolism in the liver further underscored this observation. Interestingly, exposing AhR-/- mice to a high-fat diet showed resilience to glucose intolerance. Our data suggest the existence of a bidirectional AhR-microbiome axis, which influences host metabolic pathways.

Human fecal water content of phenolics: the extent of colonic exposure to aromatic compounds.

Phenolic compounds are not completely absorbed in the small intestine and so enter the colon, where they might exert physiological effects. To identify phenolics that are present in normal human colon, fecal water was prepared from 5 free-living volunteers with no dietary restrictions and analyzed by gas chromatography-mass spectrometry. Daily measurements were also performed on a single individual to examine the variation more closely. Levels of polyphenols were variable between individuals. Naringenin and quercetin had mean concentrations of 1.20 and 0.63 microM. All other flavonoids examined were present < or =0.17 microM. Simple phenolic and other aromatic acids were present at much higher concentrations. The major components were phenylacetic acid, 479 microM; 3-phenylpropionic acid, 166 microM; 3-(4-hydroxy)-phenylpropionic acid, 68 microM; 3,4-dihydroxycinnamic acid, 52 microM; benzoic acid, 51 microM; 3-hydroxyphenylacetic acid, 46 microM; and 4-hydroxyphenylacetic acid, 19 microM. Other phenolic acids ranged from 0.04 to 7 microM. Decreased dietary phenolic intake caused a decrease in polyphenol and monophenolic acid concentration in fecal water 24 h later. This study is the first to measure the range of aromatic compounds in human fecal water and demonstrates that phenolic acid concentrations are high. The biological effects of phenolics may play an important role in colon function.

Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not?

Foods and beverages rich in phenolic compounds, especially flavonoids, have often been associated with decreased risk of developing several diseases. However, it remains unclear whether this protective effect is attributable to the phenols or to other agents in the diet. Alleged health-promoting effects of flavonoids are usually attributed to their powerful antioxidant activities, but evidence for in vivo antioxidant effects of flavonoids is confusing and equivocal. This may be because maximal plasma concentrations, even after extensive flavonoid intake, may be low (insufficient to exert significant systemic antioxidant effects) and because flavonoid metabolites tend to have decreased antioxidant activity. Reports of substantial increases in plasma total antioxidant activity after flavonoid intake must be interpreted with caution; findings may be attributable to changes in urate concentrations. However, phenols might exert direct effects within the gastrointestinal tract, because of the high concentrations present. These effects could include binding of prooxidant iron, scavenging of reactive nitrogen, chlorine, and oxygen species, and perhaps inhibition of cyclooxygenases and lipoxygenases. Our measurements of flavonoids and other phenols in human fecal water are consistent with this concept. We argue that tocopherols and tocotrienols may also exert direct beneficial effects in the gastrointestinal tract and that their return to the gastrointestinal tract by the liver through the bile may be physiologically advantageous.