ABSTRACT
Sister of P-glycoprotein (SPGP) is the major hepatic bile salt export pump (BSEP). BSEP/SPGP expression varies dramatically among human livers. The potency and hierarchy of bile acids as ligands for the farnesyl/bile acid receptor (FXR/BAR) paralleled their ability to induce BSEP in human hepatocyte cultures. FXR:RXR heterodimers bound to IR1 elements and enhanced bile acid transcriptional activation of the mouse and human BSEP/SPGP promoters. In FXR/BAR nullizygous mice, which have dramatically reduced BSEP/SPGP levels, hepatic CYP3A11 and CYP2B10 were strongly but unexpectedly induced. Notably, the rank order of bile acids as CYP3A4 inducers and activators of pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR) closely paralleled each other but was markedly different from their hierarchy and potency as inducers of BSEP in human hepatocytes. Moreover, the hepatoprotective bile acid ursodeoxycholic acid, which reverses hydrophobic bile acid hepatotoxicity, activates PXR and efficaciously induces CYP3A4 (a bile-metabolizing enzyme) in primary human hepatocytes thus providing one mechanism for its hepatoprotection. Because serum and urinary bile acids increased in FXR/BAR -/- mice, we evaluated hepatic transporters for compensatory changes that might circumvent the profound decrease in BSEP/SPGP. We found weak MRP3 up-regulation. In contrast, MRP4 was substantially increased in the FXR/BAR nullizygous mice and was further elevated by cholic acid. Thus, enhanced hepatocellular concentrations of bile acids, due to the down-regulation of BSEP/SPGP-mediated efflux in FXR nullizygous mice, result in an alternate but apparent compensatory up-regulation of CYP3A, CYP2B, and some ABC transporters that is consistent with activation of PXR/SXR by bile acids.
Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Cell Nucleus/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Animals , Base Sequence , Bile Acids and Salts/metabolism , Cell Line , Cells, Cultured , Cytochrome P-450 CYP3A , Dimerization , Dose-Response Relationship, Drug , Down-Regulation , Genes, Reporter , Hepatocytes/metabolism , Humans , Immunoblotting , Ligands , Liver/metabolism , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Mice, Transgenic , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Multidrug Resistance-Associated Proteins/metabolism , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Proteins/metabolism , Sequence Homology, Nucleic Acid , Transfection , Up-Regulation , Ursodeoxycholic Acid/pharmacologyABSTRACT
Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. Milk thistle is known to contain a number of flavonolignans. We evaluated the effect of silymarin, on the activity of various hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with silymarin (0.1 and 0.25 mM) significantly reduced the activity of CYP3A4 enzyme (by 50 and 100%, respectively) as determined by the formation of 6-beta-hydroxy testosterone and the activity of uridine diphosphoglucuronosyl transferase (UGT1A6/9) (by 65 and 100%, respectively) as measured by the formation of 4-methylumbelliferone glucuronide. Silymarin (0.5 mM) also significantly decreased mitochondrial respiration as determined by MTT reduction in human hepatocytes. These observations point to the potential of silymarin to impair hepatic metabolism of certain coadministered drugs in humans. Indiscriminate use of herbal products may lead to altered pharmacokinetics of certain drugs and may result in increased toxicity of certain drugs.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Glucuronosyltransferase/antagonists & inhibitors , Hepatocytes/drug effects , Mixed Function Oxygenases/antagonists & inhibitors , Plants, Medicinal , Silybum marianum/chemistry , Silymarin/pharmacology , Cells, Cultured , Cytochrome P-450 CYP3A , HumansABSTRACT
Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.
