Protective effect of quercetin on ER stress caused by calcium dynamics dysregulation in intestinal epithelial cells.

Quercetin, one of the flavonoids present in plants, expresses several physiological functions including antioxidative and anti-inflammatory properties. However, its effect on intestinal epithelia remains to be elucidated. Endoplasmic reticulum (ER) stress has been attracting considerable attention since ER stress triggers such disorders as inflammation and cancer. The effect of quercetin on ER stress was investigated in this present study. Several ER stress inducers (tunicamycin, A23187, thapsigargin and brefeldin A) were added to human colonic LS180 cells or Caco-2 cells with quercetin, and the GRP78 expression as an ER stress marker was determined. The results showed that quercetin suppressed the induction of GRP78 expression by these ER stressors, excepting brefeldin A, at both the mRNA and protein levels. Additionally, XBP-1 mRNA splicing was determined to evaluate the activation of IRE1. The phosphorylation of eIF2alpha and shutdown of protein synthesis were determined to evaluate the activation of PERK. Although quercetin activated IRE1 and PERK when added to LS180 cells alone, it suppressed the activation of IRE1 and PERK induced by A23187 or thapsigargin. The suppressive effect of quercetin on GRP78 mRNA induction was reproduced by PI3K inhibitors (LY294002 and wortmannin), but not by vitamin C and E. LY294002 failed to suppress the GRP78 mRNA induction in combination with quercetin. In conclusion, this study indicates for the first time that quercetin suppressed the ER stress caused by calcium dynamics dysregulation by the inhibition of PI3K. This study helps to clarify the mechanism for quercetin presenting its versatility.

Effect of quercetin on the gene expression profile of the mouse intestine.

BALB/c mice were administered with quercetin or its derivatives (quercetin-3-glucoside, quercetin-4'-glucoside and rutin, 5 or 50 mg/kg BW) for 2 weeks. The mucosa of the small intestine were recovered and subjected to a microarray analysis using Affymetrix Mouse Genome 430 2.0 Array. A cluster analysis shows that only the "low dose group" had a significantly different gene expression profile from the negative control group.

TCDD-induced CYP1A1 expression, an index of dioxin toxicity, is suppressed by flavonoids permeating the human intestinal Caco-2 cell monolayers.

Since the toxicological effects of dioxins are mainly mediated by the aryl hydrocarbon receptor (AhR), an in vitro assessment system for AhR activity was used in this study to search for flavonoids that attenuated dioxin toxicity through the intestinal epithelial monolayer. When AhR transformation in Hepa-1c1c7 cells was examined by southwestern ELISA, nine flavonoids among 34 kinds of flavonoids inhibited the transformation by more than one-half. When each flavonoid with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to dioxin-responsive HepG2 cells, seven flavonoids significantly restrained the TCDD-induced transcriptional activity of the CYP1A1 promoter. Furthermore, those seven flavonoids that had permeated the Caco-2 cell monolayers demonstrated an inhibitory effect on both the AhR transformation and on the transcriptional activity of the CYP1A1 promoter. The expression level of the CYP1A1 mRNA and protein induced by TCDD was suppressed by flavone, galangin, and tangeretin. It is proposed from these results that some flavonoids have the ability to suppress dioxin-induced AhR activity after permeating the human intestinal epithelial cell monolayer.

Assessment system for dioxin absorption in the small intestine and prevention of its absorption by food factors.

It has been reported that 90% of the amount of dioxin in the whole body is absorbed orally with food. However, a concise and simple system to assess dioxin absorption in the small intestine has not yet been established. The present study reports a new in vitro assessment system for this purpose. A stable dioxin-responsive cell line was established by introducing a plasmid that incorporates a xenobiotic-responsive element upstream of the luciferase gene into human hepatic HepG2 genomic DNA. Dioxin was added to the apical side of differentiated human intestinal epithelial Caco-2 cell monolayers that had been cultured on a semipermeable membrane, and the basal medium was recovered after an appropriate incubation time. To the recovered medium was added dioxin-responsive HepG2, and a luciferase assay was performed. The established stable cell line clearly showed dose-and time-dependent response to dioxin. When a food factor such as chlorophyll, which has been reported to increase dioxin excretion in in vivo studies, was added with dioxin, a significant decrease in dioxin permeability to the Caco-2 monolayer was observed. This assessment system would be useful to search for those food factors that could prevent dioxin absorption in the small intestine.

In vitro System for Assessing Dioxin Absorption by Intestinal Epithelial Cells and for Preventing this Absorption by Food Substances.

A system for assessing intestinal dioxin absorption was established by applying a Caco-2 cell monolayer and stable dioxin-responsive cell line. The stable dioxin-responsive cell line was established by introducing a plasmid incorporating the human CYP1A1 promoter into human hepatic HepG2 genomic DNA upstream of the luciferase gene. 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) was added to the apical side of differentiated human intestinal epithelial Caco-2 cell monolayers that had been cultured on a semipermeable membrane. The basal medium was taken after an appropriate incubation time and added to the dioxin-responsive cells, the TCDD content then being analyzed by a luciferase assay. The amount of TCDD in the basal medium increased in a dose- and time-dependent manner, the results being sufficiently sensitive and reproducible. The inhibition of TCDD permeability to the Caco-2 cell monolayer by such food substances as chlorophyll, insoluble corn fiber and tea dregs were observed by this in vitro assessment system. The system will therefore be useful to identify food substances having a preventive effect on the intestinal absorption of dioxins.