Coffee Induces Expression of Glucuronosyltransferases by the Aryl Hydrocarbon Receptor and Nrf2 in Liver and Stomach

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1 GASTROENTEROLOGY 2010;139: 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, and CHRISTIAN P. STRASSBURG Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany See editorial on page BACKGROUND & AIMS: Coffee is one of the most widely consumed beverages worldwide. Epidemiologic data indicate that coffee consumption protects against the progression of chronic liver disease and development of hepatocellular carcinoma and diabetes, but the mechanisms are not clear. UDP glucuronosyltransferases (UGT1A) are proteins with indirect antioxidant, cytoprotective, and genoprotective capabilities; we examined UGT1A regulation in response to coffee in cultured cells and mice. METHODS: HepG2 and CaCo2 cells were incubated with regular, metalor paper-filtered, decaffeinated, or instant coffee; green or black tea; cocoa; or metabolic products of caffeine. The effects of UGT1A regulation were investigated with reporter gene assays, immunoblot, TaqMan polymerase chain reaction, mutagenesis, and short interfering (si)rna analyses. We also studied the effects of coffee in humanized transgenic mice that express human UGT1A. RESULTS: Incubation of cells with coffee induced transcription of UGT1A1 (5.4-fold), UGT1A3 (5.2-fold), UGT1A4 (4.8-fold), UGT1A7 (6.2-fold), UGT1A8 (5.2-fold), UGT1A9 (3.5-fold), and UGT1A10 (6.1-fold). Induction was independent of caffeine, methylxanthines, or the diterpenes cafestol and kahweol. Mutagenesis and short interfering RNA knockdown studies showed that UGT1A is regulated by the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid-related factor 2 (Nrf2) by cis-acting antioxidant and xenobiotic response elements (ARE/XRE). In transgenic UGT1A mice, administration of coffee resulted in a 10- and 14-fold induction of UGT1A transcription in liver and stomach, respectively. CONCLUSIONS: UGT1A genes are induced in vitro and in vivo by coffee, independent of caffeine content, cafestol, or kahweol. Coffee up-regulates glucuronidation by AhR signaling and Nrf2 binding to the ARE/XRE. Glucuronidation could mediate the protective and antioxidant effects of coffee. Keywords: Liver Cancer; Glucuronidation; Coffee. Together with green tea, coffee is one of the most widely consumed beverages worldwide. The value of coffee beans as a commodity is only second to crude oil. Inhabitants of Western countries consume as much as 3 cups of coffee daily. 1 Coffee represents a readily available and much sought after caffeine delivery system because of the desired stimulatory effects on its consumers. However, apart from caffeine, coffee contains a plethora of complex organic compounds. 2 Coffee represents a rich source of phenols, polyphenols, flavanoids, and nonflavanoids, a number of which have been associated with antioxidant properties. A high proportion of chlorogenic acid and coffee bean flavanoids survive typical roasting temperatures of up to 230 C, but roasting also leads to mutagenic polyaromatic hydrocarbons. 2,3 Nevertheless, epidemiologic and study data suggest that coffee consumption is associated with a decreased risk of a number of diseases. In 1986, Arnesen et al 4 observed lower glutamyltransferase activities in coffee drinkers in the Tromso Heart study. This finding has been replicated in subsequent studies, 5 including an analysis of the third National Health and Nutrition Examination Survey (NHANES III) in that showed an inverse correlation of coffee intake and alanine aminotransferase activities. Coffee consumption has been associated with reduced risks of hepatocellular carcinoma (HCC), 7 liver cirrhosis, 8,9 and disease progression in chronic hepatitis C, 10 as well as with protective effects in Parkinson s disease 11 and type 2 diabetes, 12 and a controversial protection against colorectal cancer. 13 To date, the responsible protective mechanisms of coffee remain to be fully elucidated. It has been suggested that caffeine may be responsible, 6 possibly by impaired transforming growth factor signaling. 14 However, the role of caffeine alone was questioned by findings in other studies. 5,15,16 Coffee diterpenes (cafestol and kahweol) have been suggested to induce glutathione-s-transferases (GSTs) preventing benzo( )pyrene genotoxicity, 17 and nuclear factor erythroid-related factor 2 (Nrf2) mediated antioxidant action. 18 Abbreviations used in this paper: ARE, antioxidant response element; HCC, hepatocellular carcinoma; Nrf2, nuclear factor erythroid-related factor 2; PCR, polymerase chain reaction; sirna, short interfering RNA; tbhq, tert-butylhydroquinone; TCDD, 2,3,7,8-tetrachlordibenzo-p-dioxin; UGT, UDP-glucuronosyltransferase; XRE, xenobiotic response element by the AGA Institute /$36.00 doi: /j.gastro

2 1700 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 On the basis of these data, we hypothesized that detoxification systems such as the UDP-glucuronosyltransferases (UGTs) may be regulated by coffee and thereby contribute toward cytoprotection and disease susceptibility. UGTs catalyze the formation of glucuronides from a broad array of potentially cytotoxic or genotoxic compounds, 19 which include human carcinogens and reactive oxygen species In addition, genetic UGT variants with reduced catalytic activity have been identified as risk factors for HCC and other cancers. 19,23 25 Regulation of UGTs by the xenobiotics present in roasted coffee would, therefore, represent a physiologically plausible mechanism of cytoprotection. Material and Methods Cell Culture Experiments Hepatoma (HepG2), colon carcinoma (CaCo2), and esophagus carcinoma (KYSE70) cells were grown in RPMI 1640 (HepG2 and KYSE70) or Dulbecco s modified Eagle s medium with nonessential amino acids (CaCo2) supplemented with 10% fetal bovine serum. Standardized Preparation of Coffee, Cocoa, and Tea Stock solutions were prepared to represent the concentration and preparation mode of commonly used beverages: regular, decaffeinated, filtered, boiled, and instant coffee. For preparation of coffee and decaffeinated coffee, 150 ml of water (Aqua Irrigation Solution; DeltaSelect, Dreieich, Germany) was boiled in a beaker and cooled for 10 seconds. Six grams of ground coffee powder (Jacobs Krönung/Jacobs Krönung decaffeinated; Kraft Foods, Bremen, Germany) was added, incubated for 1 minute, and subsequently filtered through a paper coffee filter (Mellita, Minden, Germany). The undiluted filtrate was used for mice treatment, and diluted (12% coffee 88% medium) filtrate was used for cell culture treatment. Two grams of Jacobs Krönung instant coffee (Kraft Foods) was solubilized in 150 ml of boiled water. For boiled coffee, 150 ml of water was boiled, cooled for 10 seconds, and 6gofJacobs Krönung coffee powder was added and incubated for 1 minute. The mixture was filtered through a metal sieve. Six grams of cocoa (Krüger, Bergisch Gladbach, Germany) was diluted in 150 ml of boiled water. For green tea, 150 ml of water were boiled and cooled for 2 minutes and 1 tea bag (green tea Meßmer, Seevetal, Germany) was incubated for 3 minutes. For preparation of black tea, 1 tea bag (black tea; Thiele Tee, Emden, Germany) was incubated in 150 ml of boiled water for 3 minutes. To show concentration dependency 1%, 4%, 8%, and 12% coffee stock solutions were used for luciferase experiments (data not shown). Induction was highest with 12%, which was used subsequently. As a control for filtered coffee 150 ml of water was boiled, cooled for 10 seconds, and paper filtered. For all other beverages water served as a negative control. Caffeine and theophylline concentrations were determined by enzyme-linked immunoabsorbent assay Table 1. Chemical Composition of the Tested Beverages Caffeine (mg/cup, 150 ml) Theophylline (mg/cup, 150 ml) Coffee Decaffeinated coffee Instant coffee Boiled coffee Cocoa Green tea Black tea Data are mean SD. (Neogen, Lexington, KY; Table 1). The antioxidative capacities indicating polyphenol content were determined by an OxiSelect Oxygen Radical Antioxidant Capacity Activity Assay (Cell BioLabs, San Diego, CA). Methylxanthines and Coffee Lipids Cafestol and Kahweol One cup of coffee following the above protocol contains approximately 100 mg of caffeine, which are metabolized to 70 mg of paraxanthine, 9 mg of theobromine, and 9 mg of theophylline. 26 Accordingly, these methylxanthine amounts (Sigma-Aldrich, Traufkirchen, Germany) were used per 150 ml of medium. Cafestol and kahweol (Sigma-Aldrich) were solubilized in dimethyl sulfoxide and diluted in medium to concentrations of 5, 30, and 56 mol/l. RNA Isolation and Reverse Transcription Polymerase Chain Reaction HepG2, CaCo2, and KYSE70 cells were treated with coffee, tea, or cocoa solutions for 24 hours. Total RNA was isolated with TRIzol (Invitrogen, Karlsruhe, Germany). To isolate RNA from mouse organs, 100 mg of frozen tissue was homogenized in TRIzol. RNA (5 g) was used for cdna synthesis with the use of oligo(dt)- primed Superscript III reverse transcriptase (Invitrogen). Quantitative Real-Time Polymerase Chain Reaction By TaqMan, 0.2 g of cdna were analyzed (ABI Prism 7000 sequence detection system; Applied Biosystems, Foster City, CA) with the use of UGT1A isoformspecific primers and probes (Supplementary Table 1). Ct-values were normalized against -actin. Generation of Luciferase Reporter Gene Constructs A 1000-base pair (bp) DNA fragment of the UGT1A1 5=-upstream region was amplified by polymerase chain reaction (PCR) from 2 healthy blood donors exhibiting the A(TA) 6 TAA or the A(TA) 7 TAA (UGT1A1*28) variant, respectively. The UGT1A3 258-bp DNA fragment and the UGT1A4 513-bp fragment were cloned from genomic DNA. 27,28 UGT1A7 5= upstream DNA fragments

3 November 2010 UGT1A REGULATION BY COFFEE 1701 were amplified by PCR from 2 healthy blood donors exhibiting the 57G or the 57T variant, respectively. 29 A 500-bp (UGT1A8, UGT1A10) and a 530-bp (UGT1A9) DNA fragment of each UGT1A 5= upstream sequence was amplified by PCR from a healthy blood donor (all primers are shown in Supplementary Table 2). The PCR fragments were directionally inserted (XhoI and NheI) into pgl3 basic (Promega, Mannheim, Germany). Mutagenesis of putative xenobiotic response element (XRE) and antioxidant response element (ARE) binding sites was performed by primer extension with the use of primers specified in Supplementary Table 3. All inserts were sequenced in full. Luciferase Assays Dual luciferase assays (Dual-Reporter Assay; Promega) with HepG2, CaCo2, or KYSE70 cells were performed as previously described. 27 One day after transfection, cells were treated with coffee, cocoa, or tea for 48 hours. All experiments were performed in triplicate in at least 3 10 independent experiments. Results were analyzed with the use of Microsoft Excel software (Microsoft, Redman, WA) and show fold induction of luciferase activity. Error bars represent standard deviations. Statistical analysis was performed with the Student s t test. Differences were considered significant when P values were below.05. Transfection of Short Hairpin RNA For short interfering RNA (sirna) experiments, 100 pmol of sirna (MWG Biotech, Ebersberg, Germany) against Nrf2 (AAGAGUAUGAGCUGGAAAAACTT), AhR (AAGCGGCAUAGAGACCGACUUTT), or nonsilencing control (UAAUGUAUUGGAACGCAUATT) was transfected in 1 ml of OPTI-MEM (Invitrogen) into KYSE70 cells seeded into 12-well plates with the use of lipofectamine 2000 (Invitrogen). The final concentration of sirna was 100 nmol/l. Reporter gene constructs were transfected 6 hours later with the use of lipofectin, and cells were treated with coffee, cocoa, or tea on the next day for a further 48 hours. Knockdown efficiency of sirnas was determined by Western blot analysis (described below). For complete UGT1A knockdown KYSE70, CaCo2, and HepG2 cells were treated with 100 nmol/l sirna (Applied Biosystems) against UGT1A exon 2 (GGAUCAAUGGUCUCAGAAAtt). Cells were counted 24 and 48 hours after treatment. Hydrogen Peroxide Production Hydrogen peroxide concentrations in supernatants of treated cells were determined with the use of the H 2 O 2 detection kit (Assay Designs, Ann Arbor, MI). Western Blot Analysis KYSE70 cells were treated with coffee (12% of stock solution), decaffeinated coffee (12% of stock solution), 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD; 5 nmol/l), or tert-butylhydroquinone (tbhq; 100 m). Total cell lysate (20 g) was boiled for 10 minutes in Laemmli sample buffer (2% sodium dodecyl sulfate, 62.5 mmol/l Tris-HCl, ph 6.8, 10% glycerol, and 0.001% bromphenol blue) and separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis before electrotransfer onto a nitrocellulose membrane. Incubation with primary antibodies (anti-ahr, sc- 8087, anti-nrf2, sc-722, anti-ugt1a1, and sc-27415; Santa Cruz Biotechnology Inc, Santa Cruz, CA) was carried out in 10% dry milk. After incubation with secondary antibodies (Millipore, Schwalbach, Germany), protein was visualized by chemiluminescence (Pierce, Rockford, IL) on X-ray film. Staining with -actin antibody (sc-69879) was used as loading control. UGT1A protein expression in mice tissue was performed as described before 30 (antibodies: UGT1A1: sc-27415, Santa Cruz Biotechnology Inc; UGT1A3: H M02, Abnova, Walnut, CA; UGT1A4: AV46829, Sigma-Aldrich; UGT1A6: , BD Biosciences, San Jose, CA). Humanized UGT1A Transgenic Mice To study in vivo UGT1A regulation by coffee, a humanized transgenic mouse model was used. Recently, a UGT1A transgenic mouse model containing the complete human UGT1A locus was described. 31 In this study a transgenic mouse model was used that contained the human first exons of UGT1A isoforms UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, and UGT1A9 and the respective promoter regions that were based on BAC clone RP11-943B10 (imagenes GmbH, Berlin, Germany). DNA was sequenced by capillary electrophoresis in an ABI Prism 300 automated sequencer (Applied Biosystems) to exclude polymorphisms. In the 7 founder animals the human transgene was confirmed by DNA analysis. A mouse line carrying 6 gene copies was selected (determined by real-time quantitative PCR) with 10 ng of purified genomic DNA in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with the use of qpcr MasterMix (Euro Gentec, Cologne, Germany). Fluorescent in situ hybridization confirmed transgene insertion into a single region of the genome. Six male and 6 female 8-week-old transgenic UGT1A mice were treated with undiluted coffee (for preparation see above) as their drinking water for 3 days. Each control pool contained 4 mice given normal drinking water. The animal treatment was approved by the Animal Ethics Committee of the state Lower Saxony, Germany. Results UGT1A Gene Regulation by Coffee Significant induction of luciferase activity by regular as well as decaffeinated coffee was observed. Regular coffee induced UGT1A1 (5.4-fold), UGT1A3 (5.2-fold), UGT1A4 (4.8-fold), UGT1A7 (6.2-fold), UGT1A8 (5.2-fold), UGT1A9 (3.5-fold), and UGT1A10 (6.1-fold) in KYSE70

4 1702 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 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. cells (Figure 1A). In HepG2 and CaCo2 cells induced luciferase activity was observed for UGT1A1, UGT1A7, and UGT1A10 reporter gene constructs with regular coffee, decaffeinated coffee, and instant coffee (Figure 1B and C). This indicates that UGT1A induction did not require caffeine. In HepG2, CaCo2, and KYSE70 cells treated with regular and decaffeinated coffee, an increase of UGT1A1 protein was observed, which did not depend on caffeine (Figure 1D). Coffee treatment did not lead to significant hydrogen peroxide generation (Figure 1E), nor did coffee exhibit the highest antioxidant activity (Figure 1F) compared with the other beverages used.

5 November 2010 UGT1A REGULATION BY COFFEE 1703 Regulation of UGT1A1, UGT1A7, and UGT1A10 by Different Coffee Preparations, Cocoa, Green Tea, and Black Tea Induction experiments with regular filtered coffee, decaffeinated filter coffee, instant coffee, boiled coffee, cocoa, green tea, and black tea (KYSE70 cells) were performed. The UGT1A bp wild-type reporter gene construct was induced by coffee, decaffeinated coffee, and instant coffee to a comparable extent (ca 5.5-fold; Figure 2A). UGT1A1 induction was weaker with boiled coffee (4.1-fold) and green 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 mol/l).

6 1704 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 tea (4.4-fold) and insignificant with cocoa and black tea. A construct containing UGT1A1*28 [Gilbert s syndrome; A(TA) 6 TAA A(TA) 7 TAA] did not significantly reduce induction compared with wild-type UGT1A1. However, there was a trend toward reduction for coffee (5.4-fold 4.2- fold), decaffeinated coffee (5.4-fold 4.6-fold), and green tea (4.4-fold 3.5-fold). The UGT1A7 wild-type construct was significantly induced by coffee (6.2-fold), decaffeinated coffee (5.9-fold), instant coffee (6.8-fold), boiled coffee (9-fold), and green tea (4.8-fold; Figure 2B). Cocoa and black tea failed to significantly induce UGT1A7. A construct containing the UGT1A7*12 polymorphism ( 57 T G) led to significant reduction of coffee (3.3-fold), decaffeinated coffee (2.7-fold), and instant coffee-mediated induction (3.5- fold) relative to wild type. To assess the effect of UGT1A expression on cell proliferation, sirna knockdown of UGT1A1 exon 2 was performed, which completely abrogates all UGT1A transcripts. In KYSE70, HepG2, and CaCo2 cells this had no effect on cell proliferation, indicating that modulation of UGT1A expression does not affect cell proliferation rates (data not shown). In the absence of known promoter variants of UGT1A10 only the wild-type sequence was studied. There was significant up-regulation by coffee (6.1-fold), decaffeinated coffee (6-fold), instant coffee (9.8-fold), boiled coffee (9.5- fold), cocoa (11.2-fold), green tea (5.7-fold), and black tea (3-fold; Figure 2C). In combination, UGT1A induction depends on the beverage preparation but not on caffeine. Black tea showed only weak inducibility; green tea showed a moderate induction of UGT1A1, UGT1A7, and UGT1A10. Inducibility Is Independent of Methylxanthines, Cafestol, and Kahweol To clarify the role of caffeine or its metabolites, KYSE70, HepG2, and Caco2 cells were treated with caffeine (100 mg/150 ml), paraxanthine (70 mg/150 ml), theobromine (9 mg/150 ml), and theophylline (9 mg/150 ml). 26 In KYSE70 cells, luciferase activity with UGT1A1 and UGT1A10 was not significantly induced (Figure 2D), and UGT1A7 induction by paraxanthine (2-fold) was low. Induction experiments were then expanded to cafestol and kahweol present in non paper-filtered coffee. Because in Figure 2B and C induction was higher with boiled (metalfiltered) coffee, this may be an effect of cafestol and kahweol. However, there was no significant up-regulation of UGT1A7 and UGT1A10 by 30 mol/l cafestol and kahweol (3.4/3.7-fold) compared with the control (basic) vector (Figure 2E). Treatment with 5 mol/l cafestol and kahweol even led to reduced UGT1A7 and UGT1A10 expression, which was not observed for UGT1A1 but is unlikely to affect overall UGT1A inducibility. In combination, induction of UGT1A1, UGT1A7 and UGT1A10 by coffee is not mediated by caffeine, its dimethylxanthine metabolites, or the coffee lipids cafestol and kahweol. Identification of Responsible DNA Binding Motifs in the UGT1A1-, UGT1A7-, and UGT1A10-5= Upstream Regions Previous data suggest an effect of coffee on Nrf2 signaling. In addition, xenobiotics generated by roasting are likely to activate AhR signaling. This may proceed by ARE and XRE DNA binding motifs; thus, the 5= upstream sequence of UGT1A1, UGT1A7, and UGT1A10 was analyzed (Figure 3A). All identified putative XRE and ARE DNA binding motifs were mutagenized individually in the UGT1A bp construct to examine their function. Mutagenesis of XRE-102, XRE-586, XRE-706, or ARE-96 led to a significant reduction of regular coffee and decaffeinated coffee mediated induction of UGT1A1 (Figure 3B). XRE- 586 and XRE-706 are therefore responsible for the higher inducibility of the UGT1A bp construct. In the UGT1A7 promoter, 1 XRE and 2 ARE binding sites were identified (Figure 3A). Mutagenesis of XRE-101 or ARE-143 significantly reduced induction (Figure 3C). However, mutagenesis of ARE-187 led to a lower decrease of UGT1A7 inducibility, which was not significant with the use of decaffeinated coffee. In the UGT1A10 promoter, 4 XRE binding sites and 1 ARE binding site were identified (Figure 3A). Mutagenesis of XRE-176 and XRE-256 did not affect UGT1A10 induction (Figure 3D). However, when XRE-101, XRE- 136, or ARE-149 was mutagenized, inducibility was significantly reduced. In summary, mutagenesis indicates XRE- and ARE-mediated regulation of UGT1A transcription in response to coffee. sirna Mediated Knockdown of AhR and Nrf2 Affects Inducibility of UGT1A1, UGT1A7, and UGT1A10 by Coffee and Decaffeinated Coffee A direct involvement of AhR and Nrf2 was examined by sirna experiments. After treatment with the AhR ligand TCDD or the Nrf2 activator tbhq (for 24 and 48 hours), AhR and Nrf2 protein amounts were increased in KYSE70 cells (Figure 4A), which was reduced or absent in the presence of AhR/Nrf2 sirna treatment (Western blots). AhR as well as Nrf2 sirna abolished regular and decaffeinated coffee mediated induction of UGT1A1, UGT1A7, and UGT1A10 (Figure 4B D). These data suggest that both AhR and Nrf2 are involved in UGT1A induction by coffee. UGT1A mrna Induction by Coffee in HepG2, CaCo2, and KYSE70 Cells UGT1A mrna level induction was examined by TaqMan PCR. In HepG2, CaCo2, and KYSE70 cells treated with either regular or decaffeinated coffee UGT1A1 mrna was most strongly induced (HepG2,

7 November 2010 UGT1A REGULATION BY COFFEE 1705 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. 18-fold; CaCo2, 29-fold; KYSE70, 20-fold; Figure 5). In HepG2 cells inducibility was additionally observed for UGT1A3 (11.8-fold), UGT1A4 (5.9-fold), and UGT1A6 (5.8-fold; Figure 5A) RNAs. Again, decaffeinated coffee showed no differences in inducibility. UGT1A8 mrna was not detectable in HepG2 cells. In CaCo2 cells, inducibility was detected for UGT1A3 (7.7-fold), UGT1A4 (10-fold), UGT1A6 (8.7-fold), UGT1A7 (8.8-fold), and UGT1A8 (8.1-fold) mrnas (Figure 5B). In KYSE70 cells, UGT1A3 (7-fold), UGT1A4 (7.2-fold), UGT1A6 (5.8-fold), UGT1A7 (4-fold), UGT1A8 (4.1-fold), and UGT1A10 (6.6-fold) mrnas were induced (Figure 5C). The KYSE70 cells were additionally treated with AhR, Nrf2, and control sirnas to show the dependence of coffee-mediated inducibility on AhR and Nrf2. SiRNA treatment completely abolished UGT1A induction (Figure 5C). As additional control experiments we examined whether coffee (and not only synthetic inducers as shown in Figure 4) directly increases AhR and Nrf2 protein levels. HepG2, CaCo2, and KYSE70 cell lysates after 48 hours of treatment with coffee showed up-regulation in all 3 cell lines (Figure 5D and E). In combination, coffee regulates human UGT1A genes by AhR and Nrf2 signaling at the transcriptional level. In Vivo Regulation of UGT1A mrna Transcription by Coffee In humanized transgenic UGT1A mice orally exposed to coffee for 3 days UGT1A induction in different hepato-gastrointestinal organs was detectable by Taq- Man PCR. Figure 6A F shows examples of UGT1A mrna as well as protein induction by coffee. In the liver, UGT1A1 expression is highly up-regulated (10-fold upregulation in female liver) in comparison to controls (3 pools of untreated UGT1A transgenic mice; Figure 6A). UGT1A3 and UGT1A6 mrnas are up-regulated only in the colon of male mice (Figure 6B and C). UGT1A mrna induction was highest in the stomach, in which UGT1A1 (11-fold), UGT1A4, and UGT1A6 (14-fold) were up-regulated in male and female humanized UGT1A transgenic mice (Figure 6D F). These data provide direct evidence of significant coffee-mediated in vivo regulation of human UGT1A expression.

8 1706 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 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. Discussion Coffee consumption has been associated with protection against inflammation, fibrosis, and neoplastic disease, spanning conditions, which include liver fibrosis, chronic hepatitis C, Parkinson s disease, and HCC. An attractive mechanistic hypothesis is the induction of cytoprotective and genoprotective antioxidant mechanisms. 32,33 As a roasted plant-derived product, coffee contains a plethora of potentially active ingredients that are potential inducers for oxidative and phase II metabolism. 27,34 The present study performed in vitro and in vivo provides direct evidence that coffee is a potent inducer of the human UGT1A genes. Glucuronidation exerts protective effects by detoxifying and eliminating oxidative intermediates and reactive oxygen species generated by phase I metabolism. Our study shows a surprisingly substantial induction of the entire family of UGT1A genes in reporter gene experiments, at the transcriptional level and at the protein level. This contrasts with earlier findings that failed to identify coffee-mediated UGT induction. 35 In that study, a lower coffee concentration used in vitro with CaCo2 cells did not induce UGT1A1 and UGT1A6 mrnas. In our study a higher concentration of 12% (extracted from 6 g of coffee powder with 150 ml of water) was used that up-regulated mrna not only in CaCo2, HepG2, and KYSE70 cells but also in vivo in mice, possibly indicating that the experimental outline of Okamura et al 35 was not capable of detecting UGT1A inducibility. Because of the variability of common coffee preparations, we developed standard preparations, including metal- and paper-filtered coffee, decaffeinated and instant coffee, as well as green and black tea and cocoa, to re-create everyday practices of beverage preparation. 1 Paper-filtered coffee is less rich in cafestol and kahweol. Decaffeinated coffee lacks caffeine, and tea was included because it contains caffeine but is not processed by roasting. The different preparations analyzed in this study led to various degrees

9 November 2010 UGT1A REGULATION BY COFFEE 1707 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. of UGT1A induction (Figure 2A C). However, apart from black tea, all used preparations were observed to significantly induce UGT1A gene transcription, indicating that compounds other than caffeine and coffee diterpenes are likely to be responsible for UGT1A up-regulation. We were able to exclude caffeine and methylxanthines, as well as the coffee diterpenes cafestol and kahweol as main responsible inducers of UGT1A transcription (Figure 2D and E). These findings are of interest because they expand other hypotheses. Caffeine increases intracellular cyclic adenosine monophosphate and has been shown to inhibit the synthesis of connective tissue growth factor by inducing proteasomal degradation of Sma- and Madrelated proteins and thereby disrupting transforming growth factor signaling. 36 The cytochrome P450 (CYP) 1A2-generated primary caffeine metabolite paraxanthine is capable of exerting this effect 14 but was found to be

10 1708 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 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.

11 November 2010 UGT1A REGULATION BY COFFEE 1709 inactive toward inducing UGT1A transcription. Despite epidemiologic evidence that associated caffeine with reduced aminotransferase activities, 6 some studies suggest that protective effects of coffee may not be related to caffeine alone, 5,15,16 which is supported by this study. In a recent study, caffeine consumption was linked to reduced liver fibrosis. 8 However, caffeine sources other than coffee showed only a modest effect, 8 suggesting that there may be additional mechanisms related to other coffee-derived constituents. Other well-studied ingredients in coffee are the diterpenes cafestol and kahweol. Both have been suggested to be anticarcinogenic by inhibiting aflatoxin B DNA binding, 37 promoting glutathione-s-transferase induction and protection against aromatic hydrocarbon genotoxicity, 17,38 and by protecting against tetrahydrocarbon hepatotoxicity in mice. 39 However, cafestol and kahweol were not observed to play a role in the transcriptional regulation of UGT1A expression. Cafestol and kahweol were previously shown in mice to regulate drug metabolizing enzymes by way of the Nrf2 pathway. 40 We, therefore, demonstrated by mutagenesis of ARE and XRE DNA binding motifs and sirna knockdown that UGT1A regulation by coffee proceeds by cis-acting Nrf2/ARE in addition to AhR/XRE elements (Figures 3 5). This cafestol and kahweol independent transcriptional regulation was coordinated such that sirna knockdown of either Nrf2 or AhR was capable of completely abolishing coffee-mediated inducibility. Nrf2-mediated regulation in response to coffee is in agreement with the findings in another study 18 that additionally postulated an inhibitory effect of coffee on CYP activity. This inhibitory effect may in fact be related to the simultaneous activation of UGT1A genes as a means of antioxidant action. Our data therefore suggest that Nrf2- and AhR-dependent activation of glucuronidation proceeds independent of cafestol and kahweol, as well as caffeine or its methylxanthine metabolites. Glucuronidation may therefore represent a protective mechanism activated by coffee and coordinately controlled by Nrf2/ARE and AhR/XRE signaling. Genetic variants of UGT1A genes that alter catalytic activity are present in whites at frequencies between 1% and 40%. 19 Case control studies have discussed these variants as risk factors for cancer development, including HCC (reviewed in Strassburg et al 19 ). In particular UGT1A7, which catalyzes the detoxification of hydroxylated benzo( )pyrenes 30 has been linked to HCC. The induction of UGT1A7 and other UGT1As by coffee would thus represent a plausible explanation for cancer protection. In this study 2 variants were studied: UGT1A1*28 (Gilbert s syndrome, reviewed in Strassburg 41 ) and UGT1A7* Both are characterized by 70% reduced gene transcription compared with wild-type (UGT1A1*1 and UGT1A7*1) sequence. Our analysis showed that only in UGT1A7*12 (Figure 2B) was inducibility significantly impaired, which strengthens the hypothesis of UGT1A7 polymorphisms as cancer risk factors. 19 This does not appear to be an effect of altered cell proliferation by the modulation of UGT1A transcription because a complete knockdown of all UGT1A transcripts did not alter cell proliferation in different cell lines (data not shown). To show that oral intake of coffee was indeed able to regulate UGT1A expression in the digestive tract that establishes direct contact to coffee but also in the liver, which would require induction through the blood stream, we treated humanized transgenic UGT1A mice with coffee. In vivo induction of human UGT1A gene transcription was observed in the gastrointestinal tract as well as in the liver, indicating that coffee can regulate glucuronidation in various organs on direct and indirect contact (Figure 6). In conclusion, this study adds a new mechanistic hypothesis for the protective action of coffee mediated by UGT1A gene activation capable of conferring indirect antioxidant activity, cytoprotection, and genoprotection. UGT1A regulation is affected by coffee consumption, which is coordinately mediated by the Nrf2/ARE and the AhR/XRE signaling pathways. It does not depend on caffeine or coffee diterpenes. Although the responsible compound (or compounds) for this regulation has not yet been identified, our study indicates that pharmacologic intervention with caffeine-free coffee extracts may represent an attractive future strategy of chemoprotection in risk groups for HCC, chronic hepatitis, or tumor development. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Cadden IS, Partovi N, Yoshida EM. Review article: possible beneficial effects of coffee on liver disease and function. Aliment Pharmacol Ther 2007;26: Crozier A, Jaganath IB, Clifford MN. Dietary phenolics: chemistry, bioavailability and effects on health. Nat Prod Rep 2009;26: Nehlig A, Debry G. Potential genotoxic, mutagenic and antimutagenic effects of coffee: a review. Mutat Res 1994;317: Arnesen E, Huseby NE, Brenn T, et al. 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Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein. Toxicol Appl Pharmacol 2008;226: Strassburg CP. Pharmacogenetics of Gilbert s syndrome. Pharmacogenomics 2008;9: Received December 30, Accepted June 10, Reprint requests Address requests for reprints to: Christian P. Strassburg, MD, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, Germany. strassburg.christian@mh-hannover.de; fax: (49) Conflicts of interest The authors disclose no conflicts. Funding This work was supported by the Deutsche Forschungsgemeinschaft SFB/TRR77 project A4 (to C.P.S.).

13 November 2010 UGT1A REGULATION BY COFFEE 1710.e1 Supplementary Table 1. Primers and Probes for TaqMan PCR Gene UGT1Aall UGT1A1 UGT1A3 UGT1A4 UGT1A5 UGT1A6 UGT1A7 UGT1A8 UGT1A9 UGT1A10 Actin human Actin mouse Primer and probes Forward: GCTATGGCAATTGCTGATGCTTT Reverse: CGATGGTCGGGTTCCAGTGTA Probe: FAM-AAAATCCCTCAGACAGTCCT-MGB Forward: GAATCAACTGCCTTCACCAAAAT Reverse: AGAGAAAACCACAATTCCATGTTCT Probe: FAM-CTATCCCAGGAATTTGAA-MGB Forward: CAGAAGTATGGCAATGTTGAACAATA Reverse: GCCTCATTATGTAGTAGCTCCACACA Probe: FAM-TCTTTGGTCTATCATAGGTC-MGB Forward: TTTTTCTGCCCCTTATGCAAGT Reverse: ACAGCCACACGGATGCATAG Probe: FAM-TCAGAGAGAGGTGTCAGTGGTGGATCTTGT- TAMRA Forward: CCATTTCATGGACCCAGGAC Reverse: AGAAGATGTTCTGTTTCAAAGAACGA Probe: FAM-AATTTGATCGCCTTTTGCTGGGTCACA- TAMRA Forward: CTTCATTGGAGGTATCAACTGTAAGAA Reverse: AAGAGAAAACCACAATTCCATGTTC Probe: FAM-AGGAAAGACTTGTCTCAGGAATTTGAAGCC- TAMRA Forward: GAGGATCAGGACCGGGAGTT Reverse: GAAAATGCACTTCGCAATGGT Probe: VIC-TGGTTTTTGCCGATGCT-MGB Forward: GGAGGATCTGGACCGGGAA Reverse: TGGATGAACTCAGAAATAGAGAAAACAA Probe: FAM-TGGATTTCGCCGATGCTCAATGG-TAMRA Forward: AAACCCGTGATGCCCAAC Reverse: GGCTTCAAATTCCATAGGCAAC Probe: FAM-TGATCTTCATTGGTGGTATCAACTGCCATC- TAMRA Forward: ACCTCGTACACTCTGGAAGATCAGA Reverse: GAACTCATTAATAGAGAAAATATACTTTGTGCC Probe: FAM-AATTCATGGTTTTCGCCCATGCTCA-TAMRA Forward: TGCCGACAGGATGCAGAAG Reverse: GCCGATCCACACGGAGTACT Probe: FAM-AGATCAAGATCATTGCTCCTCCTGAGCGC- TAMRA Forward: ACGGCCAGGTCATCACTATTG Reverse: CAAGAAGGAAGGCTGGAAAAG Probe: FAM-CAACGAGCGGTTCCGATGCCC-MGB Supplementary Table 2. Primers for Cloning UGT1A- Luciferase Constructs Primer 1A Nhe fwd 1A1 pre ATG Xho rev 1A3 258 Nhe fwd 1A3 pre ATG Xho rev 1A4 513 bp Nhe fwd 1A4 pre ATG Xho rev 1A7 530 NheI fwd 1A7 rev XhoI 1A8 500 Nhe fwd 1A8 Xho rev 1A9 530 Nhe F 1A9 pre ATG Xho R 1A Nhe F 1A10 pre ATG Xho R Sequence GGT TGG CTA GCG CTG AGC CCT GAG TGG CTG AGG TTT AAC TCG AGG GCG CCT TTG CTC CTG CCA G TCT AGC TAG CAC TTG GAT GTT CCC CAG AG GTG GCT CGA GCT CAG CAG AAG ACA CG TTT AAG CTA GCC CTG AAC ACT CTC TGT TT TTT AAC TCG AGC TCA GCA GAA GCC ACC G TTT AAG CTA GCT CCC AGC TAC TGA GGC TGA GGC AGG AAA TTC TCG AGC AGC AGA GAA CTT CAG CCC AGA GCC GCC AGC TAG CGC TGG GAA GTC GGT GCT AAG G TTT AAC TCG AGA GAG AAC TGC AGC CCG AGC C AAA TTG CTA GCA ATA TGT ATG CAT TGC AGA G AAA TTC TCG AGC AGC AGA GAA CTG CAG CTG GAG ATG CTA GCG AGC CCC AGT TTC TTG CCA GTT G TTT AAC TCG AGA GAG AAC TGC AGC CCG AGC C

14 1710.e2 KALTHOFF ET AL GASTROENTEROLOGY Vol. 139, No. 5 Supplementary Table 3. Primers for Site-Directed Mutagenesis of Binding Elements Primer 1A1 XRE 102 mut fwd 1A1 XRE 102 mut rev 1A1 XRE 586 mut fwd 1A1 XRE 586 mut rev 1A1 XRE 706 mut fwd 1A1 XRE 706 mut rev 1A1 ARE 96 mut fwd 1A1 ARE 96 mut rev 1A7 XRE 100 mut fwd 1A7 XRE 100 mut rev 1A7 ARE 143 mut fwd 1A7 ARE 146 mut rev 1A7 ARE 187 mut fwd 1A7 ARE 187 mut rev 1A10 XRE 101 mut fwd 1A10 XRE 101 mut rev 1A10 XRE 136 mut fwd 1A10 XRE 136 mut rev 1A10 XRE 176 mut fwd 1A10 XRE 176 mut rev 1A10 XRE 256 mut fwd 1A10 XRE 256 mut rev 1A10 ARE 149 mut fwd 1A10 ARE 149 mut rev Sequence GCT TTT TAT AGT AAT TAA ACA CAG TC GAC TGT GTT TAA TTA CTA TAA AAA GC GGC TCA CCT CAT GGC AAT TAC TCG TGT GG CCA CAC GAG TAA TTG CCA TGA GGT GAG CC CTC TAC CCC AGA ATT ACC CCC ACC CC GGG GTG GGG GTA ATT CTG GGG TAG G TAT AGT CAC GTG AAA TTT AAA AAC ATT AAC GTT AAT GTT TTT AAA TTT CAC GTG ACT ATA ATG AAT AAG TAA TTT GCT TCT TTT GAG GGC GCC CTC AAA AGA AGC AAA TTA CTT ATT CAT TAT GAG TAA AAA ATT TAA AGT GAA TGT GA TCA CAT TCA CTT TAA ATT TTT TAC TCA TA CAT ATA AGC AAA ATT TAA AGC AAA GGC TA TAG CCT TTG CTT TAA ATT TTG CTT ATA TG GGATAAATAAAATTCCTCTATTGGGGTC GACCCCAATAGAGGAATTTTATTTATCC ATCATTGGCAGTGAAAATTATTTTTT AAAAAATAATTTTCACTGCCAATGAT CAGCAAATGATACAAATTTGTTATCGTTC GAACGATAACAAATTTGTATCATTTGCTG CTAAACTCACTTGCAATATACTCTCCCTC GAGGGAGAGTATATTGCAAGTGAGTTTAG TATGAGTAAAAAATTTAAAGTGAGTGTGA TCACACTCACTTTAAATTTTTTACTCATA