The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organised inside glands and crypts. Regeneration depends on WNT/β-Catenin pathway activation, but to understand homeostasis and its dysregulation in disease we need to identify the signalling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen- and gastric acid- producing cells. We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief and parietal cells. We localised the source of the growth factors in the tissue of origin. We show that EGF is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with BMP/NOGGIN signals, EGF controls the differentiation of foveolar cells vs. parietal or chief cells. We also show that EGF is likely to underlie alteration of the gastric mucosa in the pre-cancerous condition atrophic gastritis. Use of our recently established mucosoid cultures in combination with analysis of the tissue-of-origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signalling microenvironment in the human gastric glands.

The liver is the central metabolic organ of the body. The plethora of anabolic and catabolic pathways in the liver is tightly regulated by physiological signaling but may become imbalanced as a consequence of malnutrition or exposure to certain chemicals, so-called metabolic endocrine disrupters, or metabolism-disrupting chemicals (MDCs). Among different metabolism-related diseases, obesity and non-alcoholic fatty liver disease (NAFLD) constitute a growing health problem, which has been associated with a western lifestyle combining excessive caloric intake and reduced physical activity. In the past years, awareness of chemical exposure as an underlying cause of metabolic endocrine effects has continuously increased. Within this review, we have collected and summarized evidence that certain environmental MDCs are capable of contributing to metabolic diseases such as liver steatosis and cholestasis by different molecular mechanisms, thereby contributing to the metabolic syndrome. Despite the high relevance of metabolism-related diseases, standardized mechanistic assays for the identification and characterization of MDCs are missing. Therefore, the current state of candidate test systems to identify MDCs is presented, and their possible implementation into a testing strategy for MDCs is discussed.

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),¹ Lichtenstein et al. (2020),² and Knebel et al. (2019).³.

4-Nonylphenol (4-NP), a para-substituted phenolic compound mixture comprising a straight or branched carbon chain, is a widespread ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor with potent activities on estrogen nuclear receptors. Constitutive Androstane Receptor (CAR, NR1I3) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in endocrine disruption mechanism. The nonylphenol mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by NP are enigmatic and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR and its variants using molecular dynamics (MD) simulations, cellular experiments with various CAR constructs or CAR ligand binding domain (LBD) mutants, or in differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type (wt)CAR1 as well as CAR3 variant LBDs in MD simulations. Consistently, branched 4-NP activated CAR3 and wtCAR1 LBD more efficiently than linear 4-NP or tert-nonylphenol (22NP). Furthermore, in HepaRG cells, we observed that all NP significantly up-regulated CYP2B6, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and the human CAR-LBD, as well as its dominant variant CAR3 The work could contribute to the safer use of individual NPs in many areas of industry.

Background: Epigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail. Results: Here, we describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach (i.e., antrum, corpus, fundus) together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1, and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. Conclusions: By extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation already characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics as a starting point for further studies.