Volume 139, Issue 5, Pages 1699-1710.e2 (November 2010) Coffee Induces Expression of Glucuronosyltransferases by the Aryl Hydrocarbon Receptor and Nrf2 in Liver and Stomach  Sandra Kalthoff, Ursula Ehmer, Nicole Freiberg, Michael P. Manns, Christian P. Strassburg  Gastroenterology  Volume 139, Issue 5, Pages 1699-1710.e2 (November 2010) DOI: 10.1053/j.gastro.2010.06.048 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A) Induction of different UGT1A reporter gene constructs by coffee in luciferase assays (KYSE70 cells). Different UGT1A–5′-upstream regions were significantly induced by regular and decaffeinated coffee in comparison to empty vector control (basic). UGT1A1, UGT1A7, and UGT1A10 coffee induction in HepG2 (B) and CaCo2 (C) cells. (D) Western blot of UGT1A1 protein induction by regular and decaffeinated coffee in HepG2, CaCo2, and KYSE70 cells. Human liver microsomes (HLMs) were used as positive control. (E) Hydrogen peroxide concentration in supernatants of treated (48 hours) KYSE70 cells. (F) Antioxidative capacity of tested beverages determined by enzyme-linked immunoabsorbent assay. Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Induction of UGT1A1 (A), UGT1A7 (B), and UGT1A10 reporter gene constructs (C) in KYSE70 cells. Luciferase activity with the UGT1A7 −57G (UGT1A7*12) and UGT1A1 A(TA)7(TAA) (UGT1A1*28) variants was compared with wild type. Significant differences are relative to the basic vector. In single nucleotide polymorphism constructs significance was compared with wild type. (D) Luciferase activity of the UGT1A1, UGT1A7, and UGT1A10 reporter gene constructs in KYSE70 cells after treatment with caffeine (100 mg/150 mL) and its primary dimethylxanthine metabolites paraxanthine (70 mg/150 mL), theobromine (9 mg/150 mL), and theophylline (9 mg/150 mL). Similar results for Caco2 and HepG2 cells are not shown. (E) Treatment of KYSE70 cells with the coffee lipids cafestol and kahweol (C+K) (each 5, 30, or 56 μmol/L). Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Identification of putative DNA binding motifs (A) and their mutagenesis in the UGT1A1 (B), UGT1A7 (C), and UGT1A10 (D) genes. Both XRE and ARE elements participate in the transcriptional regulation of UGT1A genes in response to coffee. Significance was determined in comparison to wild-type (nonmutagenized) constructs. Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 (A) Western blot confirming knockdown of AhR (top) and Nrf2 (bottom) by specific siRNAs at 24 and 48 hours in KYSE70 cells induced with either TCDD or tBHQ 6 hours after knockdown. siRNA-mediated knockdown of AhR and Nrf2 (KYSE70 cells) abolished both regular and decaffeinated coffee–mediated luciferase induction of UGT1A1 (B), UGT1A7 (C), and UGT1A10 (D). Significance was determined in relation to constructs treated with control siRNA. Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 TaqMan PCR quantification of UGT1A mRNA induction by regular and decaffeinated coffee in HepG2 (A), CaCo2 (B), and KYSE70 cells (C). Significance was determined in relation to control-treated cells. In all cell lines UGT1A1 mRNA induction was highest. (C) KYSE70 cells were additionally treated with AhR, Nrf2, and control siRNAs, abolishing inducibility of all UGT1A genes. Significance was determined in relation to cells treated with control siRNA. Increase of AhR (D) and Nrf2 (E) protein in lysates of coffee-treated HepG2, CaCo2, and KYSE70 cells detected by Western blot. Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Induction of UGT1A mRNA (TaqMan PCR relative to actin) and protein (Western blot) in humanized UGT1A transgenic mice after 3 days of oral coffee intake. (A–F) Examples of UGT1A expression in different organs of the hepato-gastrointestinal tract are shown. Significance levels were determined between treated and untreated animals. Gastroenterology 2010 139, 1699-1710.e2DOI: (10.1053/j.gastro.2010.06.048) Copyright © 2010 AGA Institute Terms and Conditions