Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo
Version of Record online: 20 MAR 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Molecular Nutrition & Food Research
Special Issue: Cocoa and Human Health
Volume 57, Issue 6, pages 1055–1066, June 2013
How to Cite
Dietz, B. M., Hagos, G. K., Eskra, J. N., Wijewickrama, G. T., Anderson, J. R., Nikolic, D., Guo, J., Wright, B., Chen, S.-N., Pauli, G. F., van Breemen, R. B. and Bolton, J. L. (2013), Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo. Mol. Nutr. Food Res., 57: 1055–1066. doi: 10.1002/mnfr.201200534
- Issue online: 7 JUN 2013
- Version of Record online: 20 MAR 2013
- Manuscript Accepted: 3 JAN 2013
- Manuscript Revised: 21 DEC 2012
- Manuscript Received: 8 AUG 2012
- Office of Dietary Supplements. Grant Number: P50 AT00155
- National Center for Complementary and Alternative Medicine. Grant Number: CA135237
- National Cancer Institute
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Figure S1: Combined negative ion electrospray LC-MS-MS SRM chromatogram of 8-PN, 6-PN, IX, XH, and their respective glucuronides in rat liver homogenates from a rat that was treated with XH subcutaneously (A). LC-MS-MS SRM chromatograms of 8-PN/6-PN SRM transitions (B), and of 8-PN/6-PN glucuronides SRM transitions (C) of the same sample. SRM transitions: XH/IX: m/z 353–119; 8-PN/6-PN: m/z 339–219. XH GlcA/IX GlcA: m/z 529–353; 8-PN GlcA/6-PN GlcA: m/z 515–339.
Figure S2: Negative ion electrospray LC-MS-MS SRM chromatogram of XH GlcAs (A), of 8-PN GlcAs (B) and of 6-PN GlcA (C) after rat microsomal incubations with pure XH (A), 8-PN (B), and 6-PN (C). SRM transitions: XH GlcA/IX GlcA: m/z 529–353 (A); 8-PN GlcA/6-PN GlcA: m/z 515–339 (B and C). To generate glucuronide conjugates of prenylated flavonoids (10 μM), rat liver microsomes (0.5 mg/ml) were preincubated on ice with alamethicin (25 μg/ml) in a 50 mM Tris-HCl buffer, pH 7.5, supplemented with 10 mM magnesium chloride. The reaction was started by adding uridine diphospho glucuronic acid (2 mM) and incubations were carried out for 30 min at 37 °C. The reaction was stopped by adding acetonitrile. After centrifugation, an aliquot of the supernatant was directly analyzed by LC-MS-MS.
Figure S3: Combined negative ion electrospray LC-MS-MS SRM chromatogram of 8-PN, 6-PN, IX, XH, and their respective glucuronides in rat mammary gland homogenate from a rat that was treated with XH subcutaneously (A). LC-MS-MS SRM chromatogram of 8-PN/6-PN glucuronides (B). SRM transitions: XH/IX: m/z 353–119; 8-PN/6-PN: m/z 339–219. XH GlcA/IX GlcA: m/z 529–353; 8-PN GlcA/6-PN GlcA: m/z 515–339.
Figure S4: LC-MS-MS chromatograms of XH and its metabolites in plasma from a rat dosed with XH subcutaneously. SRM was carried out with negative ion electrospray and collision-induced dissociation. The precursor and product ion transition for each measurement are indicated above each chromatogram.
Figure S5: Densiometric analysis of western blots demonstrating Nrf2 nuclear accumulation by hops. HepG2 cells were treated with DMSO (0.2%), positive control BF (1.7 μM), XH (8 μM), and hop extract (20 μg/mL) for 6 hrs and Nrf2 protein was visualized by western blot analysis. Imaging and analysis was performed using FluoroChem software (Cell Biosciences). Each protein band density was normalized to the respective histone 3 band density and was represented as the fold induction of Nrf2 nuclear accumulation in relation to the DMSO control. Three independent experiments were performed to obtain the average ± SD. *: p < 0.05 BF significantly induced Nrf2 nuclear accumulation (ANOVA with Dunnett's post test).
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