Dataset Information

Modelling the role of microbial p-cresol in colorectal genotoxicity.

ABSTRACT: Background: A greater understanding of mechanisms explaining the interactions between diet and the gut microbiota in colorectal cancer is desirable. Genotoxic microbial metabolites present in the colon may be implicated in carcinogenesis and potentially influenced by diet. Aims: We hypothesised that microbial p-cresol is a colonic genotoxin and set out to model potential exposures in the colon and the effects of these exposures on colonic cells. Methods: Batch culture fermentations with human faecal inoculate were used to determine the synthesis of p-cresol and other metabolites in response to various substrates. The fermentation supernatants were evaluated for genotoxicity and the independent effects of p-cresol on colonic cells were studied in vitro. Results: In batch culture fermentation, supplementary protein increased the synthesis of phenols, indoles and p-cresol, whereas supplementary fructoligosaccharide (FOS) increased the synthesis of short chain fatty acids. The p-cresol was the greatest predictor of genotoxicity against colonocytes in the fermentation supernatants. Spiking fermentation supernatants with exogenous p-cresol further increased DNA damage, and independently p-cresol induced DNA damage in a dose-dependent manner against HT29 and Caco-2 cells and influenced cell cycle kinetics. Conclusions: In the colon p-cresol may reach physiologically significant concentrations which contribute to genotoxic exposures in the intestinal lumen, p-cresol production may be attenuated by substrate, and therefore diet, making it a potential modifiable biomarker of genotoxicity in the colon.

SUBMITTER: Al Hinai EA

PROVIDER: S-EPMC6546321 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Modelling the role of microbial p-cresol in colorectal genotoxicity.

Al Hinai Eiman Abdulla EA Kullamethee Piyarach P Rowland Ian R IR Swann Jonathan J Walton Gemma E GE Commane Daniel M DM

Gut microbes 20181025 3

Background: A greater understanding of mechanisms explaining the interactions between diet and the gut microbiota in colorectal cancer is desirable. Genotoxic microbial metabolites present in the colon may be implicated in carcinogenesis and potentially influenced by diet. Aims: We hypothesised that microbial p-cresol is a colonic genotoxin and set out to model potential exposures in the colon and the effects of these exposures on colonic cells. Methods: Batch culture fermen ...[more]

PMID: 30359553

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Project description:Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630?erm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

Project description:The toxicological effects of p-cresol have primarily been attributed to its metabolism products; however, very little human data are available in the key organ (i.e., liver) responsible for the generation of these metabolites. Experiments were conducted in HepaRG cells utilizing the following markers of cellular toxicity: 2'-7'-dichlorofluorescein (DCF; oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; cellular necrosis) release. Concentrations of p-cresol, p-cresol sulfate, and p-cresol glucuronide were determined using validated assays. p-Cresol exposure resulted in concentration- and time-dependent changes in DCF (EC50 = 0.64 ± 0.37 mM at 24 h of exposure) formation, GSH (EC50 = 1.00 ± 0.07 mM) concentration, and LDH (EC50 = 0.85 ± 0.14 mM) release at toxicologically relevant conditions. p-Cresol was also relatively more toxic than 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, and hippuric acid on all markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were less toxic compared to p-cresol at equal-molar conditions. Moreover, p-cresol glucuronide was the predominant metabolite generated in situ from p-cresol exposure. Selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e., 0.75 mM l-borneol, 75 µM amentoflavone, or 100 µM diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure (by 28.3 ± 5.3%, 30.0 ± 8.2% or 27.3 ± 6.8%, respectively, compared to p-cresol treatment). These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely to be associated with the manifestation of its toxic effects in HepaRG cells.

Dataset Information

Modelling the role of microbial p-cresol in colorectal genotoxicity.

Publications

Modelling the role of microbial p-cresol in colorectal genotoxicity.

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