Haem iron reshapes colonic luminal environment: impact on mucosal homeostasis and microbiome through aldehyde formation.

BACKGROUND: The World Health Organization classified processed and red meat consumption as "carcinogenic" and "probably carcinogenic", respectively, to humans. Haem iron from meat plays a role in the promotion of colorectal cancer in rodent models, in association with enhanced luminal lipoperoxidation and subsequent formation of aldehydes. Here, we investigated the short-term effects of this haem-induced lipoperoxidation on mucosal and luminal gut homeostasis including microbiome in F344 male rats fed with a haem-enriched diet (1.5 µmol/g) 14-21 days. RESULTS: Changes in permeability, inflammation, and genotoxicity observed in the mucosal colonic barrier correlated with luminal haem and lipoperoxidation markers. Trapping of luminal haem-induced aldehydes normalised cellular genotoxicity, permeability, and ROS formation on a colon epithelial cell line. Addition of calcium carbonate (2%) to the haem-enriched diet allowed the luminal haem to be trapped in vivo and counteracted these haem-induced physiological traits. Similar covariations of faecal metabolites and bacterial taxa according to haem-induced lipoperoxidation were identified. CONCLUSIONS: This integrated approach provides an overview of haem-induced modulations of the main actors in the colonic barrier. All alterations were closely linked to haem-induced lipoperoxidation, which is associated with red meat-induced colorectal cancer risk.

Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal Carcinogenesis.

The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRAS (V12) were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRAS(V12) HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC.

Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells.

Consumers are exposed daily to several pesticide residues in food, which can be of potential concern for human health. Based on a previous study dealing with exposure of the French population to pesticide residues via the food, we selected 14 pesticides frequently found in foodstuffs, on the basis of their persistence in the environment or their bioaccumulation in the food chain. In a first step, the objective of this study was to investigate if the 14 selected pesticides were potentially cytotoxic and genotoxic. For this purpose, we used a new and sensitive genotoxicity assay (the γH2AX test, involving phosphorylation of histone H2AX) with four human cell lines (ACHN, SH-SY5Y, LS-174T and HepG2), each originating from a potential target tissue of food contaminants (kidney, nervous system, colon, and liver, respectively). Tebufenpyrad was the only compound identified as genotoxic and the effect was only observed in the SH-SY5Y neuroblastoma cell-line. A time-course study showed that DNA damage appeared early after treatment (1h), suggesting that oxidative stress could be responsible for the induction of γH2AX. In a second step, three other pesticides were studied, i.e. bixafen, fenpyroximate and tolfenpyrad, which - like tebufenpad - also had a methyl-pyrazole structure. All these compounds demonstrated genotoxic activity in SH-SY5Y cells at low concentration (nanomolar range). Complementary experiments demonstrated that the same compounds show genotoxicity in a human T-cell leukemia cell line (Jurkat). Moreover, we observed an increased production of reactive oxygen species in Jurkat cells in the presence of the four methyl-pyrazoles. These results demonstrate that tebufenpyrad, bixafen, fenpyroximat and tolfenpyrad induce DNA damage in human cell lines, very likely by a mode of action that involves oxidative stress. Nonetheless, additional in vivo data are required before a definitive conclusion can be drawn regarding hazard prediction to humans.

Genotoxicity of pesticide mixtures present in the diet of the French population.

Consumers may be simultaneously exposed to several pesticide residues in their diet. A previous study identified the seven most common pesticide mixtures to which the French population was exposed through food consumption in 2006. The aim of this study was to investigate if the seven mixtures are potentially cytotoxic and genotoxic and if so, whether compounds in a same mixture have a combined effect. The cytotoxicity and genotoxicity of the seven mixtures were investigated with a new assay (γ-H2AX) using four human cell lines (ACHN, SH-SY5Y, LS-174T, and HepG2). Mixtures were tested at equimolar concentrations and also at concentrations reflecting their actual proportion in the diet. Irrespective of the cell line tested, parallel cytotoxicity of the seven mixtures was observed. Only one mixture was genotoxic for the HepG2 cells at concentrations = 3 µM in equimolar proportion and at 30 µM in actual proportion. Caspase 3/7 activity, the comet assay, and reactive oxygen species production were also investigated using the same mixture and HepG2 cells. Our results suggest that pesticide metabolites from the mixture generated by HepG2 cells were responsible for the observed damage to DNA. Among the five compounds in the genotoxic mixture, only fludioxonil and cyprodinil were genotoxic for HepG2 cells alone at concentrations = 4 and 20 µM, respectively. Our data suggest a combined genotoxic effect of the mixture at low concentrations with a significantly higher effect of the mixture of pesticides than would be expected from the response to the individual compounds. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.

Genotoxic activities of the food contaminant 5-hydroxymethylfurfural using different in vitro bioassays.

5-Hydroxymethylfurfural (5-HMF) is known as an indicator of quality deterioration in a wide range of foods. 5-HMF is formed as an intermediate in the Maillard reaction and has been identified in a wide variety of heat-processed foods. In recent years, the presence of 5-HMF in foods has raised toxicological concerns: data have shown cytotoxic, genotoxic and tumoral effects but further studies suggest that 5-HMF does not pose a serious health risk. However the subject is still a matter of debate. We investigated the genotoxicity of the food-borne contaminant 5-HMF using the Ames test, the micronucleus (MN) and the single-cell gel electrophoresis (SCGE) assays in the human metabolically active HepG2 cell line. Cytotoxic effect of 5-HMF was first assessed using Alamar Blue as a sensitive sub-lethal assay. 5-HMF did not induce any genic mutation in bacteria whatever the concentration in the Ames test. Furthermore, it does not induce clastogenic or aneugenic effects in the HepG2 cells. In contrast, 5-HMF induced HepG2 DNA damage at concentrations from 7.87 to 25 mM in the comet assay suggesting a weak genotoxic effect of 5-HMF in the HepG2 cells probably repaired.