Assessment of Bioavailability after In Vitro Digestion and First Pass Metabolism of Bioactive Peptides from Collagen Hydrolysates.

Collagen hydrolysates (CHs) are composed of bioactive peptides (BAPs), which possess health enhancing properties. There is a knowledge gap regarding the bioavailability of these BAPs that involves intestinal transport and hepatic first pass effects. A simulated gastrointestinal model was used to generate digesta from two CHs (CH-GL and CH-OPT), which were applied to a novel transwell co-culture of human intestinal epithelium cell line-6 (HIEC-6) and hepatic (HepG2) cells to simulate in vivo conditions of absorption and first pass metabolism. Peptide transport, hepatic first pass effects, and bioavailability were determined by measuring BAPs (Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, Gly-Pro-Hyp) using an innovative capillary electrophoresis method. All peptides were transported across the intestinal cell layer to varying degrees with both CHs; however, Gly-Pro-Hyp was transported only with CH-GL, but not CH-OPT. Notable hepatic production was observed for Ala-Hyp with both CH treatments, and for Pro-Hyp and Gly-Pro with CH-GL only. All peptides were bioavailable (>10%), except for Gly-Pro-Hyp after CH-OPT. Overall, a high degree of transport and hepatic first pass effects on CH-derived BAPs were observed. Further research is needed to explore the hepatic mechanisms related to the production of BAPs and the bifunctional effects of the bioavailable BAPs noted in this study.

Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect.

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.

The inhibitory effect and mechanism of theaflavins on fluoride transport and uptake in HIEC-6 cell model.

Fluoride (F-) is widely present in nature, while long-term excessive F- intake can lead to fluorosis. Theaflavins are an important bioactive ingredient of black and dark tea, and black and dark tea water extracts showed a significantly lower F- bioavailability than NaF solutions in previous studies. In this study, the effect and mechanism of four theaflavins (theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate) on F- bioavailability were investigated using normal human small intestinal epithelial cells (HIEC-6) as a model. The results showed that theaflavins could inhibit the absorptive (apical - basolateral) transport of F- while promote its secretory (basolateral - apical) transport in HIEC-6 cell monolayers in a time- and concentration-dependent (5-100 µg/mL) manner, and significantly reduce the cellular F- uptake. Moreover, the HIEC-6 cells treated with theaflavins showed a reduction in cell membrane fluidity and cell surface microvilli. Transcriptome, qRT-PCR and Western blot analysis revealed that theaflavin-3-gallate (TF3G) addition could significantly enhance the mRNA and protein expression levels of tight junction-related genes in HIEC-6 cells, such as claudin-1, occludin and zonula occludens-1 (ZO-1). Overall, theaflavins may reduce F- absorptive transport by regulating tight junction-related proteins, and decreasing intracellular F- accumulation by affecting the cell membrane structure and properties in HIEC-6 cells.

Primary Cilium Identifies a Quiescent Cell Population in the Human Intestinal Crypt.

Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt-villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics.

Investigation of the inflammatory and oxidative stress-inducing effects of deoxynivalenol and T-2 toxin exposure in non-tumorigenic human intestinal cell model.

Fungi in the Fusarium genus produce trichothecene mycotoxins including deoxynivalenol (DON) and T-2 toxin which may elicit their damaging effects on the gastrointestinal tract following the consumption of contaminated cereal-based foods. The aim of our study was to evaluate the effects of these commonly occurring fusarotoxins alone and in combination using the human, non-cancerous intestinal epithelial cell line HIEC-6. Based on our experimental data, 24 h after treatment with fusarotoxins, hydrogen peroxide levels, intracellular oxidative stress and the amounts of inflammatory interleukins IL-6 and IL-8 significantly increased. Cell membrane localization of the tight junction protein claudin-1 decreased, whereas distribution of occludin remained unchanged. Taken together, the HIEC-6 cell line appears to be a suitable experimental model for monitoring the combined effects of mycotoxins at the cellular level including changes in the redox states of cells.

New Data on Cylindrospermopsin Toxicity.

Cylindrospermopsin (CYN) is a widely spread cyanotoxin that can occur in fresh water and food. This research aims to investigate CYN toxicity by studying the effects of drinking 0.25 nM of CYN-contaminated water from a natural source, and of the direct application of moderate concentrations of CYN on different animal targets. The chosen structures and activities are rat mitochondria inner membrane permeability, mitochondrial ATP synthase (ATPase) and rat liver diamine oxidase (DAO) activities (EC 1.4.3.22.), the force of the contraction of an excised frog heart preparation with functional innervation, and the viability of a human intestinal epithelial cell line (HIEC-6). The oral exposure to CYN decreased the reverse (hydrolase) activity of rat liver ATPase whereas its short-term, in vitro application was without significant effect on this organelle, DAO activity, heart contractions, and their neuronal regulation. The application of CYN reduced HIEC-6 cells' viability dose dependently. It was concluded that CYN is moderately toxic for the human intestinal epithelial cells, where the regeneration of the epithelial layer can be suppressed by CYN. This result suggests that CYN may provoke pathological changes in the human gastrointestinal tract.