title: The Ussing type chamber model to study the intestinal transport and modulation of specific tight-junction genes using a colonic cell line.
abstract: Polyphenols in apples, such as various hydroxycinnamic acids and flavonoids, have positive health effects that strongly depend on their bioavailability. In order to show that the Ussing-type chamber is a useful model to study metabolism, transport, and tightness of cell monolayers in one experimental setup, monolayers of the T84 colon carcinoma cell line mounted in Ussing-type chambers were incubated in the presence of physiological concentrations of various hydroxycinnamic acids (including ferulic, isoferulic, cinnamic, and hydrocinnamic acids) and flavonoids for 4 h. Concentrations of each tested polyphenol in the apical chamber, basolateral chamber, and those associated with the cells were then determined using HPLC with DAD (HPLC-DAD). The transport studies showed that the amounts of the tested polyphenols that passed from the apical to the basolateral side of the T84 monolayers depended on their polarity. Metabolites, such as glucuronides and sulfates of ferulic acid, were also detected at measurable levels by HPLC-ESI-MS/MS in the model system, but only when they were supplied at supra-physiological concentrations (>100 microM). In addition, the transepithelial resistance (TER) of T84 monolayers was measured before and after the addition of polyphenols, with and without short-term exposure to apical sodium caprate (C10), a tight junction (TJ) modulator. Exposure to C10 induced a decrease in TER that was reversible by incubation with polyphenols. However, no increase in paracellular permeability of tested polyphenols was observed after apical C10 exposure, so C10 did not promote fluxes of hydroxycinnamic acids across the monolayers. Further, real-time PCR analysis of the T84 colon cell line showed that ferulic and isoferulic acids induced significant increases in expression of the TJ components zonula occludens-1 (ZO-1) and claudin-4 transcription, but reductions in occludin expression. In contrast, caffeic and p-coumaric acids had no significant effects on the transcription of either ZO-1 or occludin. Our results provide confirmation that T84 cells could be used as model system to simulate the intestinal mucosa, and that polyphenols are able to increase the TER of C10-treated and -untreated T84 monolayers.
authors.affiliation: Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Hannah
authors.initials: H
authors.lastname: Bergmann
authors.forename: Dorothee
authors.initials: D
authors.lastname: Rogoll
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Scheppach
authors.forename: Ralph
authors.initials: R
authors.lastname: Melcher
authors.forename: Elke
authors.initials: E
authors.lastname: Richling
mesh_terms: D045744:Cell Line, Tumor; D002934:Cinnamates / Q000737:chemistry / Q000494:pharmacology*; D062506:Claudin-4; D003652:Decanoic Acids / Q000494:pharmacology; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D005638:Fruit / Q000737:chemistry; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D027845:Malus / Q000737:chemistry; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D062793:Occludin; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D059808:Polyphenols; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
chemical_list: C568157:CLDN4 protein, human; D002934:Cinnamates; D062506:Claudin-4; D003652:Decanoic Acids; D005419:Flavonoids; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010636:Phenols; D010750:Phosphoproteins; D059808:Polyphenols; D012333:RNA, Messenger; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C031071:decanoic acid