taurochenodeoxycholic-acid and estradiol-3-glucuronide

Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response.. To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M.. The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (μmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17β-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level.. The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT.. SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Topics: Adenosine Triphosphatases; Baculoviridae; Biomarkers, Pharmacological; Drug Resistance, Multiple; Estradiol; Genetic Vectors; Glucuronides; Glutathione; Humans; Leukotriene C4; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Polymorphism, Single Nucleotide; Taurochenodeoxycholic Acid

Chlorpromazine at a concentration of 250 microM and estradiol-17 beta-D-glucuronide at 17.5 microM on infusion led to a sharp reduction in bile flow by the in vitro perfused rat liver. This was accompanied by fragmentation and a loss of canalicular microvilli, dilatation of canaliculi, and thickening of pericanalicular ectoplasm. Less prominent were the smooth endoplasmic reticulum dilatation, lysosomal lamination, and the appearance of amorphous bile in hepatocyte cytoplasm. The bile flow and electron microscopy appearance were restored to normal by infusion of tauroursodeoxycholate in a concentration of 5 mumols/min for the estradiol-17 beta-D-glucuronide-induced cholestasis and 1.5 mumol/min for the chlorpromazine-induced cholestasis. Changes in ultrastructure paralleled changes in bile flow. These observations demonstrate the feasibility of electron microscopy studies on the perfused liver, and the rapidity with which cholestatic changes appear.

Topics: Animals; Bile; Chlorpromazine; Cholestasis; Estradiol; In Vitro Techniques; Kinetics; Liver; Microscopy, Electron; Perfusion; Rats; Taurochenodeoxycholic Acid; Time Factors