Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites (M1-3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4'-O-sulfate, and trans-resveratrol-3-O-4'-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K(i) of 21.3 +/- 8.73 microM and a V(max)/K(m) of 1.63 +/- 0.41 microLmin(-1)mg(-1) protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V(max)/K(m) values for M3 than for M2 (2.23 +/- 0.14 and 0.04 +/- 0.01 microLmin(-1)mg(-1)). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.

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Epithelium of the gallbladder and biliary tract is exposed to high concentrations of potentially harmful exogenous and endogenous compounds excreted into primary bile. As the ATP-dependent efflux pump ABCG2 can prevent cellular accumulation of anticancer drugs, estrogen sulfate, xenobiotics, porphyrins, and sterols, its expression in the biliary tract might mediate protection by hindering their penetration. We therefore investigated the expression and subcellular distribution of ABCG2 in normal and malignant human gallbladder. After demonstrating ABCG2 expression in gallbladder epithelium by RT-PCR and Western blotting, we analyzed the subcellular localization of ABCG2 by indirect immunofluorescence in gallbladder adenocarcinoma specimens, and compared it to that in cholelithiasis, and normal gallbladder samples (n ¼ 54). In control, cholelithiasis, and well-differentiated tumor samples (grade 1, T1-3), ABCG2 is present at the luminal membrane of epithelial cells, which was proven by colocalization of apical-bound TRITC-labeled lectin (wheat germ agglutinin). In poorly differentiated gallbladder adenocarcinomas, intracellular ABCG2, in addition to luminal ABCG2 immunoreactivity, was found in 13/21 carcinoma samples (grade 2 and 3, T2-4, Po0.01). In 3/11 of grade 3 tumors, ABCG2 was present in the cytoplasmatic compartment only (Po0.01). In proliferating bile ducts of cholangiocarcinomas, ABCG2 showed an analogous staining pattern with presence in cytosolic compartments. However, the apical marker enzyme neutral endopeptidase remained on the membrane in all samples. To study whether phosphatidylinositol 3-kinase (PI3K) signaling might be necessary for ABCG2 membrane insertion, we treated freshly isolated human gallbladder epithelial cells with the PI3K inhibitor wortmannin. As assessed by indirect immunofluorescence, this maneuver redistributes ABCG2 to intracellular compartments. In conclusion, our data suggest a protective role for ABCG2 in well-differentiated gallbladder epithelial cells. Cytoplasmatic accumulation of ABCG2 in poorly differentiated carcinomas might coincide with malfunctioning of PI3K-signaling pathways during tumor progression. Laboratory Investigation (2004) 84, 1024-1036, advance online publication, 7 May 2004; doi:10.1038/labinvest.3700127Keywords: ABCG2; gallbladder epithelium; gallbladder carcinoma; cholelithiasis; phosphatidylinositol 3-kinase; BCRP; neutral endopeptidase Cholelithiasis, and as a complication, acute cholecystitis, are among the most common gastrointestinal diseases in industrialized countries, while carcinomas of the gallbladder are quite rare. Although cholelithiasis is considered an important risk factor for gallbladder carcinoma, only 1-2% of patients who have operations for gallstones are diagnosed with gallbladder cancer at the time of surgery. The epidemiological characteristics of cholelithiasis and gallbladder cancer are closely related. Risk factors associated with the formation of cholesterol gallstones including estrogen exposure, nutritional factors, obesity...

: Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT‐PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine‐N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT‐PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin‐embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5‐hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1‐mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.