Ethinyl Estradiol-induced Cholestasis Research Articles

Ursodeoxycholic acid and 18β-glycyrrhetinic acid alleviate ethinylestradiol-induced cholestasis via downregulating RORγt and CXCR3 signaling pathway in iNKT cells

Estrogen-induced intrahepatic cholestasis (IHC) is a mild but potentially serious risk and urges for new therapeutic targets and effective treatment. Our previous study demonstrated that RORγt and CXCR3 signaling pathway of invariant natural killer T (iNKT) 17 cells play pathogenic roles in 17α-ethinylestradiol (EE)-induced IHC. Ursodeoxycholic acid (UDCA) and 18β-glycyrrhetinic acid (GA) present a protective effect on IHC partially due to their immunomodulatory properties. Hence in present study, we aim to investigate the effectiveness of UDCA and 18β-GA in vitro and verify the accessibility of the above targets. Biochemical index measurement indicated that UDCA and 18β-GA presented efficacy to alleviate EE-induced cholestatic cytotoxicity. Both UDCA and 18β-GA exhibited suppression on the CXCL9/10-CXCR3 axis, and significantly restrained the expression of RORγt in vitro. In conclusion, our observations provide new therapeutic targets of UDCA and 18β-GA, and 18β-GA as an alternative treatment for EE-induced cholestasis.

Soluble endoglin exacerbates bile acid retention during labetalol treatment in mice with ethinylestradiol-induced cholestasis

Soluble endoglin exacerbates bile acid retention during labetalol treatment in mice with ethinylestradiol-induced cholestasis

Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis.

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10mg/kg s.c., the mice model of ICP) and labetalol (10mg/kg s.c.) for 5days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Protective impact of lycopene on ethinylestradiol-induced cholestasis in rats.

Protection against cholestasis and its consequences are considered an essential issue to improve the quality of a patient's life and reduce the number of death every year from liver diseases. Lycopene, a natural carotenoid, has antioxidant scavenger capacity and inhibits inflammation in many experimental models. The present study aimed to elucidate the potential protective effects of lycopene, in comparison to silymarin, in a rat model of cholestatic liver. Animals were daily injected with 17α-ethinylestradiol (EE; 5mg/kg) for 18 successive days. Silymarin (100mg/kg) and lycopene (10mg/kg) were orally administered once per day through the experimental period. Lycopene significantly decreased the EE-induced rise in the serum levels of total bile acid and total bilirubin as well as the activities of alanine aminotransaminase, aspartate aminotransaminase, alkaline phosphatase, and gamma-glutamyl transaminase. Moreover, lycopene reduced the hepatic levels of thiobarbituric acid reactive substances and tumor necrosis factor-α as well as the hepatic activity of myeloperoxidase that were markedly elevated by EE. Lycopene increased the hepatic levels of total protein and albumin and reduced glutathione. In addition, lycopene improved the hepatic histopathological changes induced by EE. These protective effects of lycopene were comparable to that of silymarin. In conclusion, lycopene was effective in protecting against estrogen-induced cholestatic liver injury through its antioxidant and anti-inflammatory activities. Therefore, lycopene might be a potentially effective drug for protection against cholestasis in susceptible women during pregnancy, administration of oral contraceptives, or postmenopausal replacement therapy.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Protective effect of heme oxygenase induction in ethinylestradiol-induced cholestasis.

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis

Intrahepatic cholestasis of pregnancy is a high-risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis. CoQ, vitamin E and malondialdehyde were measured in plasma and/or tissues by HPLC-UV method whereas serum bile acids by capillary electrophoresis in rats with ethinyl estradiol-induced cholestasis and women with pregnancy cholestasis. CoQ and vitamin E plasma levels were diminished in both rats and women with intrahepatic cholestasis. Furthermore, reduced CoQ was also found in muscle and brain of cholestatic rats but no changes were observed in heart or liver. In addition, a positive correlation between CoQ and ursodeoxycholic/lithocholic acid ratio was found in intrahepatic cholestasis suggesting that increased plasma lithocholic acid may be intimately related to CoQ depletion in blood and tissues. Significant CoQ and vitamin E depletion occur in both animals and humans with intrahepatic cholestasis likely as the result of increased hydrophobic bile acids known to produce significant oxidative stress. Present findings further suggest that antioxidant supplementation complementary to traditional treatment may improve cholestasis outcome.

Protective effect of Jasonia montana against ethinylestradiol-induced cholestasis in rats

Estrogens, and particularly glucuronides such as ethinylestradiol (EE), have been shown to cause cholestasis in animal studies, by reducing bile acid uptake by hepatocytes. The aim of the present article is to investigate anticholestatic activity of the ethanolic extract of the aerial parts of Jasonia montana against liver cholestasis induced by EE in adult female rats in an attempt to understand its mechanism of action, which may pave the way for possible therapeutic applications. Subcutaneous administration of 100 μg/kg b.w. ethinylestradiol to rats induced hepatocellular cholestasis with a significant decrease in serum cholesterol, bile acids and bilirubin levels as well as in hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) activities and hepatic total, protein-bound and non-protein sulfhydryl groups. Also, treatment with EE produced significant increase in serum Pi-glutathione-s-transferase (Pi-GST), gamma glutamyl transpeptidase (γ-GT) and alpha-glutathione-s-transferase (α-GST) activities as well as serum nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) level and hepatic malondialdehyde (MDA) level as compare to control group. Oral administration of the aerial parts of ethanolic extract at a concentration of 150 mg/kg b.w. daily to rats treated with EE for 15 days showed a significant protection against-induced decrease in serum cholesterol, bile acids and bilirubin levels. The treatment also resulted in a significant increase in hepatic SOD, GPx and GR activities as well as hepatic total, protein-bound and non-protein sulfhydryl groups. In addition, the extract could inhibit serum Pi-GST, γ-GT and α-GST activities as well as reduce serum TNF-α, NO and hepatic MDA as compare to ethinylestradiol treated rats. High content of flavonoids and phenolic compounds was found in ethanolic extract, which may be responsible for free radical activity. The results clearly suggest that the aerial parts of J. montana extract may effectively normalize the impaired antioxidant status in ethinylestradiol (EE)-cholestatic model. Thus the extract may have a therapeutic value in drug-induced biliary cholestasis as well as in hormonal therapy.

Nuclear Receptor Ligands: Rational and Effective Therapy for Chronic Cholestatic Liver Disease?

p r f c t p p f c he regulation of genes that are essential to many hepatic metabolic and transport functions are medited in large part by the action of small molecules that unction as nuclear receptor ligands.1 This process has een labeled chemical genomics.2 These ligands bind to heir specific nuclear receptors, activating the receptor hat then binds to specific elements in a gene’s promoter esulting in stimulation or inhibition of gene expresion.3 In the liver many drugs, metabolites, and herbal ompounds exert their biologic properties as ligands for uclear receptors.4,5 Some familiar examples of drugs hat activate nuclear receptors include phenobarbital and t John’s wort. Both drugs induce hepatic drug metablizing enzymes by acting as ligands for the pregnane X eceptor (PXR), which binds to specific response eleents in the promoter of cytochrome P-450 3A CYP3A), a major hepatic microsomal drug–metabolizng enzyme. This induction then indirectly affects the etabolism and systemic effects of a wide variety of other ompounds metabolized by CYP3A. An extract of 4 ifferent plants, Yin Zhi Huang, has been used tradiionally for treating neonatal jaundice in China and is a igand for the constitutive androstane receptor (CAR) hat up-regulates the expression of several different liver ransporters and enzymes involved in the hepatic clearnce of serum bilirubin.5 Indeed, more than 10% of edically useful drugs are known to exert their biologic ehavior by binding to ligand-binding domains on speific nuclear receptors.4 Table 1 shows some of the most mportant nuclear receptors and their physiologic ligands hat determine the hepatic transport and metabolism of variety of xenobiotics and endogenous substrates. In this issue of GASTROENTEROLOGY, Marschall et al6 eport that rifampicin and ursodeoxycholic acid (UDCA), wo well-known drugs used in the treatment of cholesatic liver disease, each stimulate the transcription of a istinct set of genes that together decrease bile acid ptake, enhance bile acid detoxification and excretion, nd stimulate the clearance of bilirubin. Rifampicin inreased the expression of CYP3A4, uridine 5=-glucuonosyl transferase (UGT1A1), and multidrug-resisance–associated protein (MRP2) whereas UDCA timulated the bile salt export pump (BSEP), and mulidrug resistance protein (MDR3), and MRP4. Although hese effects were observed in patients undergoing choecystectomy who otherwise were healthy, these separate ut complementary effects on gene and protein expresion are predicted to have beneficial effects if they ocurred in patients with cholestatic liver disease. These ndings thus suggest that there is therapeutic benefit rom combining drugs such as rifampicin and UDCA hat target nuclear receptors that have coordinated effects n the transcriptional regulation of hepatobiliary excreory mechanisms.7 How does this occur? The hepatic clearance of bile acids and bilirubin can be ivided into 4 phases that include the following: phase 0, epatic uptake; phase I, metabolism (eg, hydroxylation); hase II, detoxification (eg, conjugation); and phase III, xcretion. Nuclear receptors and their ligands that are he major determinants of the functional expression of enes that determine these pathways are shown in Figure , together with enzymes that control bile acid synthesis. In the enterohepatic circulation, the hepatic uptake of onjugated bile acids (phase 0) is mediated predominantly y the sodium taurocholate cotransporting polypeptide TCP (SLC10A1), whereas unconjugated bile acid uptake s facilitated by several organic anion transporting polypepides (OATPs) on the sinusoidal membrane, but predomi-

Ursodeoxycholate reduces ethinylestradiol glucuronidation in the rat: role in prevention of estrogen-induced cholestasis.

Ethinylestradiol (EE) administration (5 mg/kg, s.c., daily for 5 days) to rats leads to cholestasis, and its derivative EE 17beta-glucuronide is a likely mediator of this effect. Coadministration of ursodeoxycholate (UDC) was shown to prevent ethinylestradiol-induced cholestasis. The aim of this study was to evaluate the inhibitory effect of UDC on EE glucuronidation in vivo and in vitro as a potential mechanism to explain UDC protection. UDC treatment (25 mg/kg, i.p., daily for 5 days) decreased the biliary excretion of EE 17beta-glucuronide in bile after administration of a trace dose of [3H]EE and reduced microsomal EE 17beta-glucuronidation activity by 20% and expression of UGT2B1, one of the enzymes involved in EE conjugation, by 30%. Glucuronidation kinetic studies were performed in vitro using normal microsomes and isolated hepatocytes in the presence of tauroursodeoxycholate (TUDC), the major endogenous derivative of UDC in the rat. Kinetic enzymatic studies in microsomes showed a noncompetitive inhibition of EE 17beta-glucuronidation by TUDC, which was unique for this bile salt since other endogenous bile salts such as taurocholate, taurochenodeoxycholate, or taurodeoxycholate did not affect the enzyme activity. Studies in isolated hepatocytes confirmed the inhibitory effect of TUDC on EE glucuronidation and indicated that TUDC can reach the enzyme active site in intact cells. In conclusion, both in vivo and in vitro experiments indicate that UDC decreased the metabolic pathways involved in EE glucuronidation, hence decreasing the formation of the cholestatic derivative EE 17beta-glucuronide.

Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat

Background/Aims: Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na +/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. Methods: Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. Results: All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30–40% of controls, but less pronounced than Ntcp (minus 70–80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40–90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein α (C/EBPα) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. Conclusions: Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.

Effect of Ursodeoxycholic Acid on the Expression of the Hepatocellular Bile Acid Transporters (Ntcp and bsep) in Rats With Estrogen-Induced Cholestasis

Rats with ethinyl estradiol-induced cholestasis have a decreased bile flow and a decreased expression of basolateral and canalicular hepatocyte membrane transporters. The bile acid ursodeoxycholic acid improves bile flow in these animals. The purpose of this study was to examine the effect of ursodeoxycholic acid on the expression of hepatocellular bile acid carriers. Rats received either ethinyl estradiol (5 mg.kg body wt. for 10 days) or ethinyl estradiol associated with ursodeoxycholic acid (1% in the diet). A third group of rats received ursodeoxycholic acid alone. Bile flow, bile acid, and glutathione biliary outputs were measured. Messenger RNA levels and protein expression of Na -dependent taurocholate co-transporting polypeptide, and bile salt export pump were determined in basolateral and canalicular membrane preparations by Northern and Western blot analysis. Ursodeoxycholic acid restored bile flow in ethinyl estradiol-treated rats by increasing bile acid secretion. It did not improve glutathione output nor bile acid-independent flow. Na -dependent taurocholate co-transporting polypeptide mRNA and protein were decreased by ethinyl estradiol and not restored by ursodeoxycholic acid. In contrast, canalicular bile salt export pump protein expression was decreased by ethinyl estradiol and fully restored to control levels by ursodeoxycholic acid. Ursodeoxycholic acid increases bile flow in ethinyl estradiol-treated rats by increasing bile acid secretion. This increase is possibly mediated by a normalization of the expression of the canalicular bile salt export pump.

Ursodeoxycholate (UDC) reduces ethinylestradiol (EE) glucuronidation: role in the prevention of ethinylestradiol-induced cholestasis

Ursodeoxycholate (UDC) reduces ethinylestradiol (EE) glucuronidation: role in the prevention of ethinylestradiol-induced cholestasis

Impaired activity of the bile canalicular organic anion transporter (Mrp2/cmoat) is not the main cause of ethinylestradiol-induced cholestasis in the rat.

To test the hypothesis that impaired activity of the bile canalicular organic anion transporting system mrp2 (cmoat) is a key event in the etiology of 17alpha-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats, EE (5 mg/kg subcutaneously daily) was administered to male normal Wistar (NW) and mrp2-deficient Groningen Yellow/Transport-deficient Wistar (GY/TR-) rats. Elevated plasma bilirubin levels in GY/TR- rats increased upon EE-treatment from 65 +/- 8.4 micromol/L to 183 +/- 22.7 micromol/L within 3 days, whereas bilirubin levels remained unaffected in NW rats. Biliary bilirubin secretion was 1.5-fold increased in NW rats but remained unaltered in GY/TR- rats. Plasma bile salt concentrations remained unchanged in both strains, although hepatic levels of the sinusoidal Na+-taurocholate cotransporting protein (ntcp) were markedly reduced. Biliary secretion of endogenous bile salt was not affected in either strain. A clear reduction of mrp2 levels in liver plasma membranes of NW rats was found after 3 days of treatment. The bile salt-independent fraction of bile flow (BSIF) was reduced from 2.6 to 2.0 microL/min/100 g body weight in NW rats with a concomitant 62% reduction of biliary glutathione secretion. The absence of mrp2 and biliary glutathione in GY/TR- rats did not prevent induction of EE-cholestasis; a similar absolute reduction of BSIF, i.e., from 1.1 to 0.6 microL/min/100 g bodyweight, was found in these animals. EE treatment caused a reduction of the maximal biliary secretory rate (S(RM)) of the mrp2 substrate, dibromosulphthalein (DBSP), from 1,040 to 695 nmol/min/100 g body weight (-38%) in NW rats and from 615 to 327 nmol/min/100 g body weight (-46%) in GY/TR- rats. These results demonstrate that inhibition of mrp2 activity and/or biliary glutathione secretion is not the main cause of EE-induced cholestasis in rats. The data indicate that alternative pathways exist for the biliary secretion of bilirubin and related organic anions that are also affected by EE.

Inhibition of biliary bicarbonate secretion in ethinyl estradiol-induced cholestasis is not associated with impaired activity of the Cl −/HCO 3− exchanger in the rat

Background/Aims: Bicarbonate is a major component of bile salt independent bile flow, which is impaired in ethinyl estradiol (EE)-cholestasis. To examine this subject in EE-cholestasis, we studied: 1) basal and glucagon-stimulated biliary bicarbonate secretion both in vivo and in the isolated perfused rat liver (IPRL); 2) H +/HCO 3 − transport processes in isolated rat hepatocyte couplets. Methods: Rats received (5 mg·kg b.w. −1 for 5 days. Intracellular pH (pH i) was measured (BCECF-AM) using a single-cell microfluorimetric setup. Results: Bile flow was markedly ( p<0.01) decreased in EE-treated rats. Bicarbonate concentration in bile was decreased ( p<0.01) and bicarbonate secretion was 2.5-fold lower in EE-treated animals than in controls, both in bile-fistula rats [19.5±5.1 ( n= 23) vs 54.2±5.7 ( n=20) nmol·min −1·gliver −1; p<0.01] and in the IPRL [11±2 ( n=8) vs 24±3 ( n=8) nmol·min −1·gliver −1; p<0.01]. In control IPRL, a bile/perfusate gradient for bicarbonate is maintained, while it is lost in EE-treated IPRL because of the lower bicarbonate concentration in bile. Glucagon stimulated bile flow and bicarbonate secretion to a similar extent in EE-treated and control IPRL (+25% vs +23%). Resting pH i of EE-treated hepatocyte couplets was higher in comparison with controls in KRB [7.25±0.07 ( n=35) vs 7.20±0.05 ( n=33); p<0.02] but similar in Hepes [7.08±0.07 ( n=24) vs 7.05±0.06 ( n=26)]. Basal activity of the Cl −/HCO 3 − exchanger was similar in EE-treated and control hepatocyte couplets [H + flux= 2.87±1.12 ( n=18) vs 3.01±1.23 mM/min ( n=15)] and was stimulated to a similar extent by glucagon. Na +/HCO 3 − symport activity was increased in EE-treated hepatocyte couplets ( p<0.05) while the Na +/H + exchanger was unchanged. Conclusions: Bicarbonate biliary secretion is markedly impaired during EE-cholestasis in association with a marked decrease of bile salt independent bile flow. However, the Cl −/HCO 3 − exchanger and its hormonal regulation are normal, indicating that the lower bicarbonate excretion in EE-cholestasis is not due to a compromised activity of this anion exchanger. Since the bile/perfusate gradient for bicarbonate is dissipated in EE-treated IPRL, the impaired bicarbonate excretion could be caused by a reflux of biliary bicarbonate via leaky tight junctions.

Ethinyl estradiol cholestasis involves alterations in expression of liver sinusoidal transporters.

The mechanisms involved in ethinyl estradiol-induced cholestasis are controversial. Basal bile flow was reduced by ethinyl estradiol administration, with a half time (t1/2) of 12.5 +/- 0.6 h. In contrast, initial taurocholate uptake was not significantly reduced until 3 days to 59% of control and to 13 and 10% of control at 5 and 7 days, respectively. The t1/2 was 4.3 +/- 0.1 days. These physiological changes were correlated with measurement of protein mass and steady-state mRNA for Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), Na(+)-dependent taurocholate transporter, organic anion transporters, and membrane lipid fluidity. Ethinyl estradiol significantly decreased Na(+)-K(+)-ATPase activity and membrane fluidity. However, neither Na(+)-K(+)-ATPase alpha-subunit nor beta-subunit mass was altered by ethinyl estradiol administration. In contrast, protein content of the Na(+)-dependent taurocholate transporter was significantly reduced to 21% of control (P < 0.001) at 5 days. The Na(+)-dependent taurocholate transporter was identified in sinusoidal membrane fractions as a doublet with a molecular size estimated to be 51 and 56 kDa. Although both bands were reduced with ethinyl estradiol treatment, the 56-kDa band was decreased more rapidly and to a greater extent than the 51-kDa band. The estimated t1/2 of 4.8 +/- 0.6 days for the doublet was similar to that for Na(+)-dependent taurocholate uptake. The organic anion transporter protein mass was similarly reduced with time of ethinyl estradiol administration to 21% of control (P < 0.01) at 5 days. Ethinyl estradiol also rapidly decreased the steady-state mRNA levels of Na(+)-dependent and organic anion transporters to approximately 50% and 15% of control at 5 days, respectively. These studies indicate early generalized abnormalities of the sinusoidal membrane lipid fluidity, Na(+)-K(+)-ATPase activity, and bile acid transport protein content.

Low protein diet protects against ethinyl estradiol-induced cholestasis

Ethinyl estradiol (EE) administration is associated with impairment of bile formation (cholestasis) in both humans and animals. Since low protein diets (LPD) interfere with normal bile secretory function, we examined bile flow, the bile acid (BA) secretory rate and biliary cholesterol (CH) and phospholipid (PH) output in rats fed LPD and given EE (5 mg/kg for body weight 4 days). In another experimental series, we assessed the effects of LPD and EE on the secretory rate maximum (SRm) of cholic acid. Rats fed LPD had significantly lower bile flow and biliary BA secretion rate than animals given an adequate or normal protein diet (NPD). In the NPD group, EE resulted in a cholestatic response. In contrast, bile flow in LPD rats, was not significantly changed, and the BA secretory rate was increased in comparison with corresponding LPD controls not receiving EE. Biliary PH output was enhanced in both the controls and LPD-fed, EE-treated animals. CH secretion was decreased in the NPD and LPD groups as a result of EE administration, indicating a dissociation between bile acid and PH or CH secretion in bile. In rats fed an adequate diet, EE reduced the SRm of cholic acid but in the LPD group, the SRm was not inhibited and even reached higher values than in the untreated controls. The changes in bile flow paralleled those of the BA secretory rate. Estimation of the bile acid dependent (BADF) and independent fractions (BAIF) of bile flow indicated that EE depressed BAIF in the NPD and LPD groups. However, BADF and the choleretic activity of BA secreted were markedly enhanced in the LPD group, which explains, in part, the observation that EE did not induce a cholestatic response in rats fed LPD.

Ursodeoxycholic acid improves ethinyl estradiol-induced cholestasis in the rat.

The effect of oral chronic administration of ursodeoxycholic acid has been examined in rats with cholestasis induced by ethinyl estradiol. Ursodeoxycholic acid at the dose of 25 mg kg-1 per day during 4 days, did not improve the decrease in basal bile flow and bile acid secretion induced by ethinyl estradiol alone. In contrast, when ursodeoxycholic acid was given at the same dose during 10 days, basal bile flow was significantly improved and basal bile acid secretion was restored to control values. When ursodeoxycholic acid was given at the dose of 500 mg kg-1 per day, basal bile flow and bile acid output were not further improved. However, bile flow and bile acid output under taurocholate infusion were restored to control values. Bile of rats treated with ursodeoxycholic acid was enriched with this bile acid. These results show a significant improvement of ethinyl estradiol-induced cholestasis in rats after chronic administration of ursodeoxycholic acid and support the use of this bile acid in intrahepatic cholestasis in man.

Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver.

We investigated the effects of 17 alpha-ethinylestradiol treatment of rats on various transport functions in isolated basolateral and canalicular liver plasma membrane vesicles. Both membrane subfractions were purified to a similar degree from control and cholestatic livers. Although moderate membrane lipid alterations were predominantly observed in basolateral vesicles, no change in basolateral Na+/K(+)-ATPase activity was found. Furthermore, while Na(+)-dependent taurocholate uptake was decreased by approximately 40% in basolateral vesicles, the maximal velocity of ATP-dependent taurocholate transport was decreased by 63% in canalicular membranes. In contrast, only minimal changes or no changes at all were observed for electrogenic taurocholate transport in "cholestatic" canalicular membranes and total microsomes, respectively. However, canalicular vesicles from cholestatic livers also exhibited marked reductions in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione and in Na(+)-dependent uptake of adenosine, while in the same vesicles HCO3-/SO4- exchange and Na+/glycine cotransport activities were markedly stimulated. These data show that in addition to the previously demonstrated sinusoidal transport abnormalities ethinylestradiol-induced cholestasis is also associated with multiple canalicular membrane transport alterations in rat liver. Hence, functional transport alterations at both polar surface domains might ultimately be responsible for the inhibitory effects of estrogens on the organic anion excretory capacity and on bile formation in rat liver.

Zonal changes of hepatobiliary taurocholate transport in intrahepatic cholestasis induced by 17α-ethinyl estradiol: A histoautoradiographic study in rats

The liver has a great reserve capacity for hepatobiliary bile salt transport. This study was performed to elucidate the significance of this capacity in ethinyl estradiol-induced cholestasis by direct visualization of the zonal involvement in taurocholate transport. The acinar distribution of [3H]taurocholate was determined by histoautoradiographical study of cryopreserved liver slices in normal rats and rats treated with ethinyl estradiol for 5 days. Silver grain densities over the different acinar zones were estimated on digitized image analysis. In control animals, histoautoradiographical study performed 4 min after the start of perfusion showed restriction of taurocholate to acinar zone 1. In contrast, in ethinyl estradiol-treated animals, taurocholate was also found in zone 2 and, in smaller concentrations, in zone 3. In control animals, the relative blackenings by silver grains of acinar zones 1, 2 and 3 were 66% +/- 1.2%, 25% +/- 1.6% and 5% +/- 0.6%, respectively. After 5 days of ethinyl estradiol treatment, blackenings were 58% +/- 1.5%, 36% +/- 2.1% and 12% +/- 0.8%, respectively. As early as 15 sec after injection of [3H]taurocholate, the bile canalicular areas of the cell plates and the bile ductules of ethinyl estradiol-treated animals were labeled as intensely as those of control animals. Our results demonstrate ethinyl estradiol-induced recruitment of the acinar zones 2 and 3 for hepatobiliary taurocholate transport. This recruitment may largely compensate for reduction of transport capacity of periportal hepatocytes in early cholestasis.