Differential effects of 17a-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat Nynke R. Koopen,* Sabine M. Post,† Henk Wolters,* Rick Havinga,* Frans Stellaard,* Renze Boverhof,* Folkert Kuipers,1,* and Hans M. G. Princen† Groningen Institute for Drug Studies (GIDS),* Center for Liver, Digestive and Metabolic Diseases, Academic Hospital Groningen, and Gaubius Laboratory TNO-Prevention and Health,† Leiden, The Netherlands Abstract Effects of 17a-ethinylestradiol (EE) on the neu- salt-independent fraction of bile flow (BSIF) (1, 2). The tral and acidic biosynthetic pathways of bile salt (BS) syn- mechanisms underlying the decrease in bile flow are not thesis were evaluated in rats with an intact enterohepatic cir- yet clear (3). In addition to reduced BSIF, several authors culation and in rats with long-term bile diversion to induce have reported that EE may also affect bile salt-dependent BS synthesis. For this purpose, bile salt pool composition, Downloaded from bile formation (BSDF) (2, 4, 5). This effect has been at- synthesis of individual BS in vivo, hepatic activities, and ex- pression levels of cholesterol 7a-hydroxylase (CYP7A), and tributed to reduced hepatic BS synthesis (4–6) and to im- sterol 27-hydroxylase (CYP27), as well as of other enzymes paired activities of hepatic transport systems involved in involved in BS synthesis, were analyzed in rats treated with vectorial transport from blood to bile (7–9). With respect EE (5 mg/kg, 3 days) or its vehicle. BS pool size was de- to the first, reduced biliary BS secretion and altered bil- creased by 27% but total BS synthesis was not affected by iary BS composition in EE-treated animals have been www.jlr.org EE in intact rats. Synthesis of cholate was reduced by 68% reported (4, 5, 10). In particular, the contribution of in EE-treated rats, while that of chenodeoxycholate was in- chenodeoxycholate and b-muricholate to the BS pool ap- D22 b creased by 60%. The recently identified -isomer of - pears to increase at the expense of cholate. In a previous muricholate contributed for 5.4% and 18.3 % (P , 0.01) to by guest, on July 24, 2017 the pool in control and EE-treated rats, respectively, but study (10), we found that conversion of endocytozed lipo- could not be detected in bile after exhaustion of the pool. A protein cholesterol to cholate was completely abolished in clear reduction of BS synthesis was found in bile-diverted EE-treated rats. The metabolic basis for the changes in BS rats treated with EE, yet biliary BS composition was only synthesis induced by EE are largely unknown. Earlier stud- minimally affected. Activity of CYP7A was decreased by EE ies have shown that EE inhibits the activity of the choles- in both intact and bile-diverted rats, whereas the activity of terol 7a-hydroxylase (CYP7A) (11, 12), the enzyme cata- the CYP27 was not affected. Hepatic mRNA levels of CYP7A lyzing the first step of the so-called neutral pathway of BS were significantly reduced by EE in bile-diverted rats only; biosynthesis. Since then, however, it has become clear that CYP27 mRNA levels were not affected by EE. In addition, an acidic pathway, initiated by 27-hydroxylation of choles- mRNA levels of sterol 12a-hydroxylase and lithocholate 6b- hydroxylase were increased by bile diversion and sup- terol by the mitochondrial sterol 27-hydroxylase (CYP27), pressed by EE. This study shows that 17a-ethinylestradiol represents a quantitative important route for BS synthesis (EE)-induced intrahepatic cholestasis in rats is associated (13–15). Recently, data have been reported to indicate with selective inhibition of the neutral pathway of bile salt that in situations where the ‘classical’ neutral pathway is (BS) synthesis. Simultaneous impairment of other enzymes specifically suppressed, the acidic pathway becomes more in the BS biosynthetic pathways may contribute to overall important for maintenance of hepatic BS synthesis (16, effects of EE on BS synthesis.—Koopen, N. R., S. M. Post, 17). It is not known whether EE differentially affects both H. Wolters, R. Havinga, F. Stellaard, R. Boverhof, F. Kuipers, pathways. In addition, peroxisomal formation of a D22 iso- and H. M. G. Princen. Differential effects of 17a-ethinyl- estradiol on the neutral and acidic pathways of bile salt syn- mer of muricholate has been suggested as a novel further thesis in the rat. J. Lipid Res. 1999. 40: 100–108. downstream pathway in BS synthesis in rats (18, 19). Whether and to what extent formation of this species is af- Supplementary key words cholestasis • estrogens • bile • D22-b-muri- fected under cholestatic conditions is not known. Finally, cholate • cholesterol-7a-hydroxylase • sterol-27-hydroxylase • enterohe- patic circulation Abbreviations: EE, 17a-ethinylestradiol; BS bile salt; Ch, cholate; CDC, chenodeoxycholate; UDC, ursodeoxycholate; LC, lithocholate; MC-b, muricholate; HC, hyocholate; BSDF, bile salt-dependent bile The synthetic estrogen 17a-ethinylestradiol (EE) in- flow; BSIDF, bile salt-independent bile flow. duces cholestasis in rodents, mainly by reducing the bile 1To whom correspondence should be addressed. 100 Journal of Lipid Research Volume 40, 1999 EE has strong impact on hepatic cholesterol synthesis vals. After the bile sampling, the animals were anesthetized with (e.g., 20–23). As the contribution of newly synthesized halothane. Blood was sampled by means of a cardiac puncture cholesterol to formation of individual BS may vary under and the liver was removed for isolation of total RNA, microsomes different conditions (24–26), altered cholesterol synthesis and mitochondria. may also affect BS synthesis. Analyses To assess the quantitative contribution of the major BS in plasma and bile were determined by an enzymatic fluori- pathways to hepatic BS synthesis in EE-treated rats, we re- metric assay (28). Plasma triglycerides, plasma and hepatic choles- lated in vivo BS synthesis to the specific activities and ex- terol were measured enzymatically using commercially available pression levels of CYP7A and CYP27. In addition, mRNA kits (Boehringer Mannheim, Mannheim, Germany). Aspartate levels of sterol 12a-hydroxylase and lithocholate 6b- transaminase (AST), alanine transaminase (ALT), and bilirubin hydroxylase, key enzymes in the formation of cholate and in plasma were assessed by standard laboratory techniques. b-muricholate, respectively, were determined, as well as Bile salt composition was studied by gas chromatography and gas chromatography–mass spectrometric techniques as described those of HMG-CoA synthase as a key enzyme in choles- earlier for human bile (29). Briefly 5–50 ml bile was subjected to terol synthesis. Experiments were performed in rats with enzymatic hydrolysis with cholylglycine hydrolase. The free bile an intact enterohepatic circulation and in rats with pro- acids formed were extracted with C18 solid phase extraction, me- longed bile diversion. Bile diversion leads to pool deple- thylated, and silylated. The methyl-TMS derivatives were sepa- tion and to up-regulation of hepatic BS synthesis, thereby rated on a 25 m 3 0.25 mm OV-1701 column (CP Sil19 CB, enabling us to directly assess the effects of EE on synthesis Chrompack Int., Middelburg, The Netherlands). As a modifica- of the individual BS species and to relate these effects to tion, coprostanol was used as internal standard for the purpose hepatic enzyme activities. of quantitation applying GC only. Identification of bile acids was performed by GC/MS (SSQ7000, Finnigan MAT, San Jose, CA) using the same GC separation system. Full scan data were re- corded from m/z 50–850 and mass spectra were compared with Downloaded from MATERIALS AND METHODS reference spectra for definitive identification. In the absence of reference spectra, a tentative identification was done based on Materials spectral information (19). 17a-Ethinylestradiol (EE) was purchased from Sigma Chemi- cals (St. Louis, MO). NADPH, isocitrate-dehydrogenase was ob- Preparation of microsomes and mitochondria tained from Boehringer Mannheim (Mannheim, Germany). For the isolation of microsomes and mitochondria, livers were www.jlr.org Cholesterol oxidase was obtained from Calbiochem (La Jolla, perfused with cold saline, removed, and 5 g of liver tissue was CA). All other chemicals were of reagent grade or the highest pu- stored in 250 mm sucrose, 10 mm Tris, 1 mm EDTA, pH 7.4. All rity commercially available. procedures were carried out at 48C. Livers were cut into small pieces with scissors and homogenized in the same buffer using a by guest, on July 24, 2017 Animals Potter-Elvehjem homogenizer. The homogenate was centrifuged Male Wistar rats (Harlan Laboratories, Zeist, The Nether- for 10 min at 800 g and the supernatant was then centrifuged 12 lands) weighing 290–330 g were used for these studies. Animals min at 8500 g. The supernatant thus obtained was used for isola- were kept in a light- and temperature-controlled environment tion of microsomes and the pellet was used for the isolation of and had free access to lab chow and tap water throughout the ex- mitochondria. periments. The animals received humane care and experimental For the isolation of microsomes, the supernatant was centri- protocols complied with the local guidelines for use of experi- fuged for 70 min at 100000 g. The pellet was resuspended by mental animals. means of a Potter-Elvehjem homogenizer in 100 mm sucrose, 100 To study the effects of EE on bile formation and composition mm potassium phosphate, 2 mm EDTA, and 5 mm DTT, pH 7.4, under conditions with an intact enterohepatic circulation, rats and centrifuged for 1 h at 100000 g. Microsomes were resus- were equipped with permanent catheters in bile duct and duode- pended in the same buffer and frozen quickly in fluid N2 in small num as described in detail elsewhere (27). Both catheters were aliquots and stored at 2808C. immediately connected to each other to maintain an intact en- The mitochondrion-enriched pellet was resuspended by homo- terohepatic circulation.