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.

Prooxidant Effects of Verbascoside, a Bioactive Compound from Olive Oil Mill Wastewater, onIn VitroDevelopmental Potential of Ovine Prepubertal Oocytes and Bioenergetic/Oxidative Stress Parameters of Fresh and Vitrified Oocytes

“…Probably, other products derived from oxidation activity were present but, at this point of the research, no information about these products is available. These data are in agreement with the results obtained from other polyphenols that were also unstable under the incubation conditions used [71]. Moreover, VB uptake experiments demonstrated that the compound was rapidly incorporated into the OCCs in a time-dependent manner with an accumulation efficiency of about 0.2% after 30 min.…”

“…For these reasons, we performed experiments to test (1) VB stability in our culture conditions and (2) its uptake by the OCCs. Determinations of VB stability in an oocyte-free IVM medium at 38.5°C indicated that VB was unstable under our IVM culture conditions, as previously reported [70], also for other polyphenols such as catechin, chlorogenic acid [60, 71–73]. After 4 hours of incubation, an isomerization process occurred, with the appearance of the positional isomer, isoVB.…”

Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber

“…After membrane filtration (0.45 µm, PVDF, Pall Corporation, Port Washington, New York, USA), 30 µL of the internal standard 3,4,5-trimethoxycinnamic acid (100 mg/L in methanol) was added to an aliquot of 300 µL and the samples were analyzed using HPLC-DAD, as previously described by Bergmann et al [15]. Peak identities were confirmed by HPLC-ESI-MS/ MS, according to [15]. D-(-)-Quinic acid was quantified using stable isotope dilution analysis and HPLC-ESI-MS/ MS, as previously described by Erk et al [27].…”

“…We studied the absorption of apple polyphenols and their intestinal metabolites in physiological concentrations using a monolayer of the colon carcinoma cell line T84 [15]. In this model, absorption from the luminal to the basolateral side was structure dependent.…”

Chlorogenic Acid Decreases Intestinal Permeability and Increases Expression of Intestinal Tight Junction Proteins in Weaned Rats Challenged with LPS

“…Although the information about the effect of phenolic acid on intestinal function is quite limited, some papers disclosed that phenolic acids or flavonoids affected intestinal permeability [15]. Studies in vitro showed that ferulic acid increases zonula occludens-1 (ZO-1) and claudin-4 transcription in T84 colon cells [16]. In addition, Suzuki et al [17] reported that quercetin and myricetin stabilized intestinal barrier function integrity by the maintenance of tight junction protein expression through by inhibiting the PKC-δ isoform in human intestinal Caco-2 cells.…”