1. Volume 44, Issue 6, Pages 1150-1157 (June 2006)
Potential role of trans-inhibition of the bile salt export pump by progesterone metabolites in the etiopathogenesis of intrahepatic cholestasis of pregnancy 
Marta Vallejo, Oscar Briz, Maria A. Serrano, Maria J. Monte, Jose J.G. Marin 
Journal of Hepatology 
Volume 44, Issue 6, Pages (June 2006)
DOI: /j.jhep
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Effect of several steroids on bile flow (A) and bile acid output (B). The indicated compound (3μmol) was added to recirculating media (150ml) used to in situ perfuse the rat liver. Values are means±SD (n≧4). *, P<0.05 as compared with control by the Bonferroni method of multiple range testing. E217βG, estradiol 17β-d-glucuronide; PM4, 5α-pregnan-3α-ol-20-one; PM4-S, 5α-pregnan-3α-ol-20-one sulfate.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Steroid bile secretion by isolated in situ perfused rat livers. (A) Mass spectrum of 5α-pregnan-3α-ol-20-one (PM4). (B) Bile concentration and bile output of progesterone, estradiol 17β-d-glucuronide (E217βG), PM4 and PM4 sulfate (PM4-S) after administration of one of these compounds (3μmol) to the recirculating media (150ml) used to in situ perfuse the rat liver. Values are means±SD (n≧4). *, P<0.05 as compared with basal period (0–20min) by paired t-test.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Effect on bile flow, bile acid output and PM4 and PM4-S concentrations in bile. 5α-pregnan-3α-ol-20-one (PM4) sulfate (PM4-S) was added (3μmol) to the recirculating media (150ml) used to in situ perfuse the rat liver during continuous infusion of sodium taurocholate (TC, 0,25μmol/min). Values are means±SD (n≥4). *, P<0.05 by comparing, by paired t-test, samples collected just before and after drug bolus administration.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Effect of temperature on uptake of cholic acid (CA) or its methyl ester (ME) derivative by X. laevis oocytes. Oocytes expressing human CES1 were incubated in medium A (100mM choline chloride, 2mM KCl, 1mM CaCl2, 1mM MgCl2 and 10mM Hepes, Tris pH 7.0) containing 50μM [14C]-CA or [14C]-CA-ME. The inset shows the result of a representative thin layer chromatography carried out to check the purity of the product obtained by chemical synthesize, in this case CA-ME (lane 2), as compared with unlabeled standards of CA-ME (lane 1) and CA (lane 3). Values are means±SD.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Loading, cleavage and efflux of cholic acid (CA) by X. laevis oocytes. (A) Comparison of the proportions between cleaved and non-cleaved radiolabeled cholic acid methyl ester ([14C]-CA-ME) in oocytes expressing human CES1 or not. Oocytes were incubated with [14C]-CA-ME for the indicated times before the cell content was extracted, separated by thin layer chromatography (TLC) and the amount of [14C]-CA-ME and [14C]-CA was measured. (B) Time-course of radiolabeled bile acid intracellular content. Oocytes expressing rat BSEP, rat BSEP and CES1 or none of them were first incubated with 50μM [14C]-CA-ME at 15°C for 60min. To determine efflux, oocytes were then transferred to bile acid-free medium and incubated at 25°C for 120min. (C) BSEP-mediated efflux was calculated as the difference in the radioactivity content of oocytes injected with TE buffer alone or containing the cRNA of human CES1 and rat BSEP. Values are means±SD. †, P<0.05 by comparing -CES1 versus +CES1 (in A); *, P<0.05 as compared with the control by the Bonferroni method of multiple range testing (in B).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Inhibition of cholic acid (CA) efflux from X. laevis oocytes. Efflux (at 25°C for 1h) from oocytes expressing human CES1 with (A) or without (B) rat BSEP that were previously loaded with 50μM radiolabeled cholic acid methyl ester ([14C]-CA-ME) at 15°C for 1h was measured in the presence of 10μM cyclosporin A (CsA) or 50μM of either estradiol 17β-d-glucuronide (E217βG), progesterone, 5β-pregnan-3α,20α-diol (PM3), 5α-pregnan-3α-ol-20-one (PM4), 5α-pregnan-3β-ol-20-one (PM5), PM4 sulfate (PM4-S), PM5 sulfate (PM5-S), or rifampicin. Values are means±SD. *, P<0.05 as compared with oocytes incubated in the absence of inhibitors (control) by the Bonferroni method of multiple range testing.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

8. Fig. 7
Dixon plots of trans-inhibition of BSEP-dependent cholic acid (CA) efflux from X. laevis oocytes. Oocytes expressing human CES1 and rat BSEP were previously loaded with 5 or 50μM [14C]-CA-ME at 15°C for 1h. CA efflux was measured in the presence of estradiol 17β-d-glucuronide (E217βG) (A) or 5α-pregnan-3α-ol-20-one sulfate (PM4-S) (B) (at 25°C for 15min. Values are means±SD.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

9. Fig. 8
Effect of the expression of rat BSEP and trans-inhibition of its function on drug-induced toxicity in X. laevis oocytes. Oocytes expressing human CES1 alone or together with rat BSEP were previously incubated (at 18°C for 6h) with a non-specific toxic agent (10μM potassium cyanide, KCN), two natural bile acids (100μM CA or CDCA), or their methyl esters (100μM CA-ME or CDCA-ME) in the absence or presence of 50μM estradiol 17β-d-glucuronide (E217βG), which was used as a BSEP trans-inhibitor. Toxic effect was measured by the reduction in the ability of the cells to biotransform tetrazolium salts into formazan. *, P<0.001 on comparing oocytes expressing and not expressing rat BSEP under similar incubation conditions by the paired t-test. Values are means±SD from four different experiments carried out in quadruplicate. Groups of 5 oocytes were incubated together to obtain each measurement.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions