Fecal Water Genotoxicity Research Articles

A high-fungi diet differentially attenuates the gut mycobiota relative to a high meat diet; consequences for chronic disease risk?

The fungal cell wall facilitates an immune response and may be involved in intestinal immune training (1,2). It is also fermentable by the gut microbiome, thus, consumption of fungi changes gut microbial ecology (3). Yet, the specific effects of consuming fungal foods on the gut mycobiota (i.e., gut fungi) have not been well studied. An interesting case study is mycoprotein, a fungal based protein produced from Fusarium Venenatum (4). We have previously reported that mycoprotein consumption attenuates faecal water genotoxicity, a surrogate marker of colorectal cancer risk, as well as modulates faecal metabolite excretion and gut bacterial composition (5). Here, we aimed to evaluate the impact of consuming a diet high in mycoprotein on gut mycobial ecology, and to explore relationships between mycobial composition and faecal genotoxicity.Here we leverage stool samples from Mycomeat: a randomised crossover-controlled trial, recruiting 20 healthy male adults to adhere to 2-week diets comprising 240 g/day of mycoprotein based foods or red and processed meat, separated by a 4-week washout. Internal transcriber spacer (ITS) sequencing was performed to characterise the mycobiota. Alpha diversity before and after study phases was compared using Wilcoxon tests. Beta diversity was compared by permutational multivariate analysis of variance (PERMANOVA) based on Bay-Curtis dissimilarities. Differences in mycobial taxa within and between study phases were compared using Wilcoxon tests. Changes in mycobiota composition was then regressed against faecal excretion of metabolites using mixed- effects models to understand the impact of myco-ecology on the wider colonic environment. Finally, given the abundance of mycobial genotoxins in nature, we regressed mycobial taxa against faecal water genotoxicity.There were significant shifts in the abundance of several taxa following both diets. Notably, mycoprotein consumption was associated with an increase in the abundance of Malasseziales sp. (P = 0.02) and a reduction in Candida Albicans (P = 0.01). Meat consumption was associated with an increase in Phaeoacremonium Tuscanum (P = 0.01) and Rhodotorula Mucilaginosa (P = 0.008), and reduction in Penicillium Commune (P = 0.02). In addition, Aspergillus Caesiellus was associated with lower faecal genotoxicity (P = 0.04), whereas Penicillium Commune (P = 0.04) and Penicillium Olsonii (P = 0.03) were both associated with higher genotoxicity. Regressing mycobial taxa against faecal metabolites revealed a number of significant associations, including between Penicillium Commune and austdiol, a putative mycotoxin (P < 0.001) as well as 1-methyladenine, a methylated DNA base with cytotoxic properties (P = 0.001).The gut mycobiota is malleable through a fungi rich diet alongside changes within the wider microbiome. Members of the gut mycobiota predicted faecal genotoxicity and faecal excretion of toxins in this model, and there may be value in further exploring the gut mycobiota and the contribution of mycotoxins in gut health and colorectal cancer risk.

Daily berry consumption attenuates β-catenin signalling and genotoxicity in colon carcinoma cells exposed to faecal water from healthy volunteers in a clinical trial

Berries are known to have disease preventive and anticancer properties. We studied whether supplementing a habitual diet rich in red meat with berries affects cancer biomarkers in a clinical trial with 43 healthy volunteers. We exposed Caco-2 and HCA-7 colon adenocarcinoma cells and HaCaT cells, representing non-cancerous epithelial tissue, in 2D and 3D cultures to faecal water extracted from stool samples collected at baseline and at the end of the 4-wk trial. Expression of markers from the Wnt/β-catenin and PI3K/AKT/mTOR pathways, the cell proliferation marker Ki67, and the apoptosis marker cleaved caspase-3 were detected by Western blotting and immunofluorescence staining methods. In addition, genotoxicity of faecal water and the concentration of N-nitroso compounds in the faeces were analysed. The diet supplemented with berries caused downregulation of β-catenin, PS6 as well as Ki67 signalling in the cells exposed to faecal water. Our results suggest that consuming berries as part of a habitual Western-type diet could lead to less cancerous colon metabolism and possibly lower the risk for colorectal cancer by modulating the central signalling pathways in cancer.

The role of diet in genotoxicity of fecal water derived from IBD patients and healthy controls.

Certain dietary factors with anti-inflammatory and/or anti-cancer properties would be a promising preventive strategy for inflammatory bowel disease (IBD) patients against developing colitis-associated colorectal cancer (CAC). In this study, fecal water (FW) was obtained from 80 IBD patients and 20 healthy controls (HCs). The comet assay was applied to determine the DNA damage induced by FW, and the protective potential of FW against hydrogen peroxide (H2O2) induced DNA damage in Caco-2cells. Information on diet was obtained via food frequency questionnaires. The results showed that FW from IBD patients, especially patients with flares, induced higher levels of direct DNA damage in Caco-2cells and showed less protection against H2O2-induced DNA damage, when compared to HCs. The DNA damage induced by FW was positively associated with consumption of processed meat and sugary foods, and nutrient intakes including heme iron and added sugars, whereas negatively correlated to intakes of soy products, and a dietary pattern characterized by high consumption of potatoes, white meat, nuts and seeds, eggs, legumes and soy products. FW from subjects with high coffee consumption protected against H2O2-induced DNA damage. These results can help to develop potential preventive strategies for IBD patients to reduce the CAC risk.

The comet assay for the evaluation of gut content genotoxicity: Use in human studies as an early biomarker of colon cancer risk

This short narrative review describes the use of the comet assay to evaluate the formation of genotoxic compounds in the gut lumen in human studies. The fecal water genotoxicity assay is based on ability of the gut content to induce genotoxicity in a cellular model, employing the aqueous component of the feces (fecal water) as this is supposed to contain most of the reactive species and to convey them to the intestinal epithelium. This non-invasive and low-cost assay has been demonstrated to be associated with colon cancer risk in animal models, and although the final validation against human tumors is lacking, it is widely used as a colo-rectal cancer risk biomarker in human nutritional intervention studies. The contribution given to the field of nutrition and cancer by the FW genotoxicity assay is highlighted, particularly in conjunction with other risk biomarkers, to shed light on the complex relationship among diet, microbiota, individual subject characteristics and the formation of genotoxic compounds in the gut.

Probiotic microorganisms detoxify ochratoxin A in both a chicken liver cell line and chickens.

The administration of probiotics and prebiotics (synbiotics) is a promising method for detoxification of ochratoxin A (OTA) in animals. The aim of this study was to investigate the ability of five probiotic strains of lactic acid bacteria (LAB) and one Saccharomyces cerevisiae yeast strain, from three different synbiotics for poultry, to detoxify OTA. In addition, we also investigated the genotoxicity of faecal water (FW) of chickens after administering OTA and/or synbiotics for 42 days. All tested LAB and yeast strains had the ability to detoxify OTA by significant (P < 0.05) reducing its concentration (by 31.3-47.7% and 31.9%, respectively, after 24 h incubation) and genotoxicity (by 22.6-51.8% and 52.7%, respectively). Synbiotics composed of four and five probiotic strains significantly (P < 0.05) decreased FW genotoxicity of chicks, after exposure to OTA, to the level seen in the control group (21.8% ± 1.7%) and were more effective than synbiotics composed of three probiotic strains (31.5%). These results showed that there was a beneficial effect of the synbiotics on the gastrointestinal tract of animals. Furthermore, synbiotic preparations containing four or five of tested strains can be considered as preventive agents in the contamination of poultry with OTA. © 2019 Society of Chemical Industry.

High phosphorus intake and gut-related parameters \u2013 results of a randomized placebo-controlled human intervention study

BackgroundIn recent years, high phosphate intakes were discussed critically. In the small intestine, a part of the ingested phosphate and calcium precipitates to amorphous calcium phosphate (ACP), which in turn can precipitate other intestinal substances, thus leading to a beneficial modulation of the intestinal environment. Therefore, we analysed faecal samples obtained from a human intervention study regarding gut-related parameters.MethodsSixty-two healthy subjects (men, n = 30; women, n = 32) completed the double-blind, placebo-controlled and parallel designed study (mean age: 29 ± 7 years; mean BMI: 24 ± 3 kg/m2). Supplements were monosodium phosphate and calcium carbonate. During the first 2 weeks, all groups consumed a placebo sherbet powder, and afterwards a sherbet powder for 8 weeks according to the intervention group: P1000/Ca0 (1000 mg/d phosphorus), P1000/Ca500 (1000 mg/d phosphorus and 500 mg/d calcium) and P1000/Ca1000 (1000 mg/d phosphorus and 1000 mg/d calcium). After the placebo period and after 8 weeks of intervention faecal collections took place. We determined in faeces: short-chain fatty acids (SCFA) and fat as well as the composition of the microbiome (subgroup) and cyto- and genotoxicity of faecal water (FW). By questionnaire evaluation we examined tolerability of the used phosphorus supplement.ResultsFaecal fat concentrations did not change significantly due to the interventions. Concentrations of faecal total SCFA and acetate were significantly higher after 8 weeks of P1000/Ca500 supplementation compared to the P1000/Ca0 supplementation. In men, faecal total SCFA and acetate concentrations were significantly higher after 8 weeks in the P1000/Ca1000 group compared to the P1000/Ca0 one. None of the interventions markedly affected cyto- and genotoxic activity of FW. Men of the P1000/Ca1000 intervention had a significantly different gut microbial community compared to the men of the P1000/Ca0 and P1000/Ca500 ones. The genus Clostridium XVIII was significantly more abundant in men of the P1000/Ca1000 intervention group compared to the other groups. Supplementations did not cause increased intestinal distress.ConclusionsThe used high phosphorus diet did not influence cyto- and genotoxicity of FW and the concentrations of faecal fat independent of calcium intake. Our study provides first hints for a potential phosphorus-induced modulation of the gut community and the faecal total SCFA content.Trial registrationThe trial is registered at ClinicalTrials.gov as NCT02095392.

Enhancement of colon carcinogenesis by the combination of indole-3 carbinol and synbiotics in hemin-fed rats

The risk of developing colorectal cancer (CRC) could be associated with red and processed meat intake. Experimental data supports that hemin iron, found abundantly in red meat, promotes CRC in mice and rats, while indole-3 carbinol (I3C) and synbiotics (syn) exert anti-carcinogenic activities in most studies of colon carcinogenesis. This study aimed to investigate the modifying effects of I3C and syn (inulin + Bifidobacterium lactis), given separately or together, on dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed rats. All animals were given four subcutaneous DMH injections and then, two weeks after carcinogen exposure, they began a basal diet containing hemin, hemin + I3C, hemin + syn, or hemin + I3C + syn for 23 weeks. The combination of I3C + syn significantly increased fecal water genotoxicity, tumor volume and invasiveness when compared to the hemin-fed control group. The groups fed I3C or syn alone had a significant reduction in the number of preneoplastic aberrant crypt foci (ACF) lesions compared to the hemin-fed group. Dietary I3C also reduced fecal water genotoxicity. Gene expression analysis of colorectal tumors demonstrated that the combination of dietary I3C + syn increased transcript levels for Raf1 and decreased tumor progression and invasiveness related to the genes Cdh1 and Appl1. This analysis also revealed that the Tnf and Cdh1 genes were significantly up- and down-regulated, respectively, in tumors of rats that received I3C, in comparison with the hemin-fed group. These findings reveal that the joint administration of I3C and syn enhanced the development of colon tumors induced by DMH in hemin-fed rats, while they potentially reduced ACF development when given alone.

Bacterial Composition, Genotoxicity, and Cytotoxicity of Fecal Samples from Individuals Consuming Omnivorous or Vegetarian Diets.

This study analyzes the composition of viable fecal bacteria and gut toxicology biomarkers of 29 healthy volunteers, who followed omnivorous, lacto-ovo-vegetarian, or vegan diets. In particular, the research was focused on the prevalence of some representative viable bacteria from the four dominant phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria) commonly present in human feces, in order to evaluate the relationship between microorganisms selected by the habitual dietary patterns and the potential risk due to fecal water (FW) genotoxicity and cytotoxicity, considered as biomarkers for cancer risk and protective food activity. The relative differences of viable bacteria among dietary groups were generally not statistically significant. However, compared to omnivores, lacto-ovo-vegetarians showed low levels of total anaerobes. Otherwise, vegans showed total anaerobes counts similar to those of omnivores, but with lower number of bifidobacteria and the highest levels of bacteria from the Bacteroides–Prevotella genera. FW genotoxicity of lacto-ovo-vegetarians resulted significantly lower either in relation to that of omnivores and vegans. Lacto-ovo-vegetarians also showed the lowest levels of cytotoxicity, while the highest were found for vegans. These results highlighted that lacto-ovo-vegetarian diet was particularly effective in a favorable modulation of microbial activity, thus contributing to a significant reduction of the genotoxic and cytotoxic risk in the gut.

Impact of β-glucan on the Fecal Water Genotoxicity of Polypectomized Patients

ABSTRACTThe aim of the study was to determine the effect of β-glucan on the cytotoxicity and genotoxicity of polypectomized patient's fecal water (FW). Polypectomized volunteers (n = 69) were randomly assigned to consume bread with or without β-glucan, for 3 months. FW was collected at the beginning (t = 0), the 30th and 90th day and 2 wk after the intervention. Cytotoxicity and genotoxicity were estimated on Caco-2 cells, using trypan blue exclusion test and comet assay, respectively. Gastrointestinal symptoms were recorded and subjects kept a 3-day food diary at baseline and after completion. Trypan blue exclusion test revealed cell survival of approximately 87% after incubation with FW. The FW samples showed 49% genotoxicity at the baseline. Genotoxicity in the intervention group decreased during the trial reaching statistical significance on the 90th day compared to control. An increase was noticed 2 wk after the trial, but it still remained significantly lower compared to control. Group-specific analysis for β-glucan also revealed significant decrease in the genotoxicity on the 90th day compared to baseline. β-glucan ingestion in polypectomized patients significantly decreased the genotoxicity of their FW. Our findings suggest that β-glucan consumption could possibly provide protection against colon cancer development.

Probiotic preparation reduces faecal water genotoxicity and cytotoxicity in chickens fed ochratoxin A contaminated feed (in vivo study).

The aim of the present study was to assess the genotoxicity and cytotoxicity of the faecal water of chickens fed ochratoxin A (OTA) contaminated feed with and without probiotic preparation. The study was performed on 20 healthy female Ross broiler chickens divided into 4 groups: control chickens - fed with non-supplemented feed; PP chickens - fed feed supplemented with the probiotic preparation; OTA chickens - fed feed contaminated with 1 mg per kg of OTA; OTA + PP chickens - fed feed contaminated with 1 mg per kg of OTA and supplemented with the probiotic preparation. Faecal water samples were collected on the 35(th) day of life of chickens from each group. Genotoxicity was measured using the comet assay, and cytotoxicity by means of MTT tests. Mean DNA damage, measured as the percentage of DNA in the tails of the comets, was 8.50 ± 1.10 for chickens fed OTA at 1 mg/kg and 6.41 ± 0.67 in the controls. The supplementation of feed with the probiotic preparation decreased the extent of DNA damage to 4.74 ± 0.78. In the control group of chickens the average cytotoxicity was 38.5 ± 0.5 (in MTT), while in the probiotic preparation group (PP group) it was 31.8 ± 0.7 (in MTT). After supplementation of the feed with the probiotic preparation, the genotoxicity and cytotoxicity were decreased in a statistically significant manner.

Impact of palm date consumption on microbiota growth and large intestinal health: a randomised, controlled, cross-over, human intervention study.

The reported inverse association between the intake of plant-based foods and a reduction in the prevalence of colorectal cancer may be partly mediated by interactions between insoluble fibre and (poly)phenols and the intestinal microbiota. In the present study, we assessed the impact of palm date consumption, rich in both polyphenols and fibre, on the growth of colonic microbiota and markers of colon cancer risk in a randomised, controlled, cross-over human intervention study. A total of twenty-two healthy human volunteers were randomly assigned to either a control group (maltodextrin-dextrose, 37·1 g) or an intervention group (seven dates, approximately 50 g). Each arm was of 21 d duration and was separated by a 14-d washout period in a cross-over manner. Changes in the growth of microbiota were assessed by fluorescence in situ hybridisation analysis, whereas SCFA levels were assessed using HPLC. Further, ammonia concentrations, faecal water genotoxicity and anti-proliferation ability were also assessed using different assays, which included cell work and the Comet assay. Accordingly, dietary intakes, anthropometric measurements and bowel movement assessment were also carried out. Although the consumption of dates did not induce significant changes in the growth of select bacterial groups or SCFA, there were significant increases in bowel movements and stool frequency (P<0·01; n 21) and significant reductions in stool ammonia concentration (P<0·05; n 21) after consumption of dates, relative to baseline. Furthermore, date fruit intake significantly reduced genotoxicity in human faecal water relative to control (P<0·01; n 21). Our data indicate that consumption of date fruit may reduce colon cancer risk without inducing changes in the microbiota.

Su1875 High Levels of Sulfate-Reducing Bacteria Predispose to Protein-Induced Fecal Water Genotoxicity

Su1875 High Levels of Sulfate-Reducing Bacteria Predispose to Protein-Induced Fecal Water Genotoxicity

High dose of prebiotics reduces fecal water cytotoxicity in healthy subjects.

In vitro and animal studies have shown differential colonic fermentation of structurally different prebiotics. We evaluated the impact of two structurally different prebiotics (wheat bran extract (WBE, containing arabinoxylan-oligosaccharides) and oligofructose) on colonic fermentation and markers of bowel health in healthy volunteers. Nineteen healthy subjects completed a double-blind, cross-over randomized controlled trial. Interventions with WBE, oligofructose or placebo for 2 wk (week 1: 15 g/day; week 2: 30 g/day) were separated by 2-wk wash-out periods. At the end of each study period, colonic fermentation was characterized through a metabolomics approach. Fecal water genotoxicity and cytotoxicity were determined using the comet and WST-1 assay, respectively, as parameters of gut health. Cluster analysis revealed differences in effects of WBE and oligofructose on colonic fermentation. WBE, but not oligofructose, reduced fecal p-cresol (p = 0.009) and isovaleric acid concentrations (p = 0.022), markers of protein fermentation. Fecal water cytotoxicity was significantly lower after intake of WBE (p = 0.015). Both WBE- and oligofructose-intake tended to reduce fecal water genotoxicity compared to placebo (WBE: p = 0.060; oligofructose: p = 0.057). Changes in fermentation were not related to changes in fecal water toxicity. Structurally different prebiotics affect colonic fermentation and gut health in a different way.

The influence of Lactobacillus casei DN 114 001 on the activity of faecal enzymes and genotoxicity of faecal water in the presence of heterocyclic aromatic amines

High activity of bacterial enzymes in human colon and genotoxicity of faecal water (FW) are biomarkers of the harmful action of microbiota. The aim of the present study was to assess the activity of β-glucuronidase and β-glucosidase and the genotoxicity of FW in vitro after incubation with 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) or 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and probiotic Lactobacillus casei DN 114 001 (Actimel). Our results indicate, that IQ and PhIP greatly increased the activity of faecal enzymes (it was up to four times higher, as measured by spectrophotometric methods) and the genotoxicity of FW (% DNA in the tail was up to 3.2 times higher, as evaluated by the comet assay on Caco-2 cells) in 15 individuals from three age-dependent groups (breast-fed children, adults aged 30–40 years, elderly aged 75–85 years). Lb. casei DN 114 001 decreased the activity of faecal enzymes and the genotoxicity of FW exposed to PhIP and IQ mostly to control values. The activity of faecal enzymes after incubation with IQ was reduced by 71.8% in the FW of children, 37.5% in adults and 64.2% in elderly (β-glucuronidase); as well as by 59.9% in children and 87.9% in elderly (β-glucosidase). For PhIP the reduction was by 59.0% in the FW of children, 50.0% in adults and 81.2% in elderly (β-glucuronidase) and by 20.2% in children, 20.7% in adults and 84.1% in elderly (β-glucosidase). Lb. casei DN 114 001 also decreased the genotoxicity of FW to the greatest extent in adults after incubation with IQ (by 65.4%) and PhIP (by 69.6%) and it was found to correlate positively with the decrease in faecal enzymes activity. In conclusion, Lb. casei DN 114 001 may exert the protective effects against genotoxic and possibly pro-carcinogenic effects of food processing-derived chemicals present in faecal water.

Fecal water genotoxicity in healthy free-living young Italian people

Dietary habit affects the composition of human feces thus determining intestinal environment and exposure of colon mucosa to risk factors. Fecal water (FW) citotoxicity and genotoxicity were investigated in 33 healthy young Italian people, as well as the relationship between genotoxicity and nutrient intake or microflora composition. Two fecal samples were collected at 2weeks apart and 3-d dietary diary was recorded for each volunteer. Cytotoxicity was measured using the Trypan Blue Dye Exclusion assay and genotoxicity using the Comet Assay (alkaline single-cell electrophoresis). Fecal bifidobacteria, total microbial count and nutrient intakes were also assessed.High intra- and inter-variability in genotoxicity data and in bacteria counts were found. None of the FW samples were citotoxic, but 90% of FW samples were genotoxic. Seventy five percent indicated intermediate and 15% were highly genotoxic. There was a different sex-related distribution. Genotoxicity was positively correlated to the total lipid intake in females and to the bifidobacteria/total bacteria count ratio in male volunteers.These results demonstrate that the majority of FW samples isolated from free-living Italian people show intermediate level of genotoxicity and sustain a relation between this possible non-invasive marker of colorectal cancer risk with both dietary habits and colonic ecosystem.

β-Glucuronidase and β-glucosidase activity and human fecal water genotoxicity in the presence of probiotic lactobacilli and the heterocyclic aromatic amine IQ in vitro

The aim of the study was to assess the genotoxicity of fecal water (FW) and the activity of fecal enzymes (β-glucuronidase and β-glucosidase) after incubation with 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) and probiotic lactobacilli: Lb. casei 0900, Lb. casei 0908, and Lb. paracasei 0919. Our results show that the carcinogen IQ greatly increased FW genotoxicity (up to 16.92±3.03U/mg) and the activity of fecal enzymes (up to even 1.4±0.16U/mg) in 15 individuals (children, adults and elderly). After incubation with IQ, the activity of β-glucuronidase was reduced by Lactobacillus bacteria by 76.0% (Lb. paracasei 0908) in the FW of children, and by 82.0% (Lb. paracasei 0919) in the elderly; while that of β-glucosidase was reduced by 55.0% in children (Lb. casei 0908) and 90.0% (Lb. paracasei 0919) in elderly subjects. Lactobacilli decreased the genotoxicity of FW after incubation with IQ to the greatest extent in adults (by 64.5%). Probiotic lactobacilli, in the presence of IQ, efficiently inhibits activity of fecal enzymes to the level of control. Genotoxicity inhibition depends on the person's age, its individual microbiota and diet.

Effect of AXOS on fecal water cytotoxicity and genotoxicity: a randomized, double‐blind, placebo‐controlled, cross‐over study

This study evaluated the effect of wheat bran derived arabinoxylan‐oligosaccharides (AXOS) on fecal water (FW) cytotoxicity and genotoxicity in healthy subjects.Twenty healthy subjects performed a double‐blind, cross‐over RCT in which they ingested AXOS (10g/d) or placebo (maltodextrine, 10g/d) for 3 weeks. The intervention periods were separated by a 3‐week wash‐out period. At the end of the run‐in, intervention and wash‐out periods the subjects collected feces for 72h. Metabolite profiles were analyzed in fecal samples using GC‐MS. FW cytotoxicity was assessed using the WST‐1 assay and FW genotoxicity using the Comet Assay. Metabolite profiles were correlated to cytotoxicity and genotoxicity using cluster analysis.AXOS did not differentially affect FW cytotoxicity and genotoxicity as compared to placebo. Cluster analysis of metabolite profiles in fecal samples according to both cytotoxicity and genotoxicity revealed a separation between the high toxicity samples and the low toxicity samples. The separation according to cytotoxicity was mainly due to the presence of alcohols and acids in the high toxicity samples, while the separation according to genotoxicity was mainly due to the presence of aldehydes and branched chain fatty acids in the very genotoxic samples.Presence of specific fermentation metabolites was associated with FW cytotoxicity and genotoxicity.Support: Fund for Scientific Research‐Flanders, Belgium (FWO project G.0674.10)

Modulation of protein fermentation does not affect fecal water toxicity: a randomized cross-over study in healthy subjects.

ObjectiveProtein fermentation results in production of metabolites such as ammonia, amines and indolic, phenolic and sulfur-containing compounds. In vitro studies suggest that these metabolites might be toxic. However, human and animal studies do not consistently support these findings. We modified protein fermentation in healthy subjects to assess the effects on colonic metabolism and parameters of gut health, and to identify metabolites associated with toxicity.DesignAfter a 2-week run-in period with normal protein intake (NP), 20 healthy subjects followed an isocaloric high protein (HP) and low protein (LP) diet for 2 weeks in a cross-over design. Protein fermentation was estimated from urinary p-cresol excretion. Fecal metabolite profiles were analyzed using GC-MS and compared using cluster analysis. DGGE was used to analyze microbiota composition. Fecal water genotoxicity and cytotoxicity were determined using the Comet assay and the WST-1-assay, respectively, and were related to the metabolite profiles.ResultsDietary protein intake was significantly higher during the HP diet compared to the NP and LP diet. Urinary p-cresol excretion correlated positively with protein intake. Fecal water cytotoxicity correlated negatively with protein fermentation, while fecal water genotoxicity was not correlated with protein fermentation. Heptanal, 3-methyl-2-butanone, dimethyl disulfide and 2-propenyl ester of acetic acid are associated with genotoxicity and indole, 1-octanol, heptanal, 2,4-dithiapentane, allyl-isothiocyanate, 1-methyl-4-(1-methylethenyl)-benzene, propionic acid, octanoic acid, nonanoic acid and decanoic acid with cytotoxicity.ConclusionThis study does not support a role of protein fermentation in gut toxicity. The identified metabolites can provide new insight into colonic health.Trial RegistrationClinicalTrial.gov NCT01280513

Protective effect of lactofermented red beetroot juice against aberrant crypt foci formation, genotoxicity of fecal water and oxidative stress induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine in rats model

The aim of the study was to investigate the effects of beetroot juice fermented by Lactobacillus brevis 0944 and Lactobacillus paracasei 0920 (FBJ) on carcinogen induction of aberrant crypt foci (ACF) in rat colon. 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was used as carcinogen, which was administrated intragastrically at a dose of 10μg/day, every day of the experiment. Additionally, we investigated the cytotoxicity and genotoxicity of fecal water from experimental animals in the Caco-2 cell line, evaluated by MTT test and the comet assay, respectively, as well as by the count of bacteria adhered to colon epithelium assessed by fluorescence in situ hybridization. Oxidative stress in rats was expressed by measuring serum antioxidant status and the level of malondialdehyde in the kidneys and liver. The experimental rats were divided into four groups based on diet type: basal diet, basal diet supplemented with FBJ, basal diet and PhIP treatment, and basal diet supplemented with FBJ and PhIP treatment. FBJ significantly reduced the number of ACF in PhIP-treated rats (from 59±18 to 26±4). Moreover, the number of extensive aberrations (more than 4 crypts in a focus) decreased from 52±18 to 18±4. Fecal water obtained from rats fed with a PhIP-containing diet induced pronounced cytotoxic and genotoxic effects in Caco-2 cells, but FBJ supplementation of the diet abolished these effects. In groups fed dietary PhP and FBJ the latter was found to increase the antioxidant status of serum from 40% to 66% depending on the fraction. Reduced concentration of malondialdehyde was found only in the kidneys of rats fed with PhIP and FBJ. FBJ present in the diet of rats causes a reduction of MDA in the kidneys from 118.7nmol/g tissue to 100nmol/g tissue. The presence of FBJ in the diet of rats significantly increased the count of bacteria, including Lactobacillus/Enterococcus and Bacteroides–Prevotella group adhered to colonic epithelium. In conclusion, supplementation of the diet with lactofermented beetroot juice may provide protection against precancerous aberrant crypt formation and reduce the cytotoxic and genotoxic effects of fecal water and improve the oxidative status of the organism.

Red meat intake-induced increases in fecal water genotoxicity correlate with pro-carcinogenic gene expression changes in the human colon

Red meat consumption is associated with an increased colorectal cancer (CRC) risk, which may be due to an increased endogenous formation of genotoxic N-nitroso compounds (NOCs). To assess the impact of red meat consumption on potential risk factors of CRC, we investigated the effect of a 7-day dietary red meat intervention in human subjects on endogenous NOC formation and fecal water genotoxicity in relation to genome-wide transcriptomic changes induced in colonic tissue. The intervention showed no effect on fecal NOC excretion but fecal water genotoxicity significantly increased in response to red meat intake. Colonic inflammation caused by inflammatory bowel disease, which has been suggested to stimulate endogenous nitrosation, did not influence fecal NOC excretion or fecal water genotoxicity. Transcriptomic analyses revealed that genes significantly correlating with the increase in fecal water genotoxicity were involved in biological pathways indicative of genotoxic effects, including modifications in DNA damage repair, cell cycle, and apoptosis pathways. Moreover, WNT signaling and nucleosome remodeling pathways were modulated which are implicated in human CRC development. We conclude that the gene expression changes identified in this study corroborate the genotoxic potential of diets high in red meat and point towards a potentially increased CRC risk in humans.