Aguilino, AM

Barone, M

DiLeo, A

Eagon, PK

Fanizza, G

Ferrara, R

Francavilla, A

Giangaspero, A

Guglielmi, FW

Polimeno, L

Starzl, TE

Zizza, G

English

Name not available

Effects of antialdosteronics on plasmatic hormonal levels and an hepatic sexual steroid receptors in rat

D-Scholarship@Pitt

Effects of Antialdosteronics on Plasmatic Hormonal Levels and on Hepatic Sexual Steroid Receptors in Rat A. Di Leo!, P.K. Eagon3, L. Polimeno!, F.W. Guglielmini!, G. Fanizza2 , M. Barone!, G. Zizza!, A.M. Aquilino!, R. Ferrara!, A. Giangasperol, T.E. Starzl4 and A. Francavilla! 1 Dept. of Gastroenterology and 2 Dept. of Obstetrics and Gynecology, University of Bari, Bari, Italy 3 Dept. of Medicine and 4 Dept. of Surgery, Univesity of Pittsburgh, Pittsburgh, Pennsylvania Spironolactone and potassium canrenoate are diuretics widely used for management of cirrhotic ascites. The administration of spironolactone frequently leads to feminization, which has been noted less frequently with the use of potassium canrenoate, a salt of the active metabolite of spironolactone. The use of these two drugs has been associated with decreases in serum testos-terone levels and in case of spironolactone with a reduction in androgen receptor (AR) activity (8,10,19,20,23,25-28). Considering the critical role of the liver in sex hormone me-tabolism the study about the effects of these drugs on liver sex hormone receptor state, could add new informations for under-standing the mechanism of feminization by these two drugs. We report here the effect of spironolactone and potassium canrenoate treatment on the sex hormone receptor and androgen responsive protein status of the liver and the effects of these treatments on serum estrogen and testosteron levels. MATERIALS AND METHODS Animals Three groups of adult male Sprague Dawley rats (240 gr b.w.) were studied. Animals in the control group (group 1) were I i' 242 ANTIALDOSTERONIC DIURETICS AND RAT LIVER injected intraperitoneally with 0.5 ml of a solution of physiologic salin with TWEEN 80 (0.1% v/v). The rats in the other groups received 5 mg of spironolactone (group 2) or 5 mg of potassium canrenoate (group 3) dissolved in the same vehicle. All animals were injected daily for 21 days and killed by decapita-tion after that time. Materials Estradiol (E2) and Dihydrotestosterone were purchased from Steraloids, Wilton, NH. Spironolactone and Potassium Canrenoate were from SPA, Milan, Italy. Diethylstilbestrol (DEB), Leupeptin, Benzamidine, Bovine serum Albumin, Sodium Molybdate, Protamine Sulfate and other buffer component were purchased from Sigma Chemical Co., St. Louis, MO. Norit A and Dextran C were obtained from Fisher Scientific Co., Pittsburgh, PA. Radioactive (2,4,6,7,16,17)- 3H -E2 (131 Ci/rnrnol), 17 -methyl- 3H -methyltrienolone (3H -RI881) (79 Ci/rnrnol) and nonradioactive R1881 were obtained from New England Nuclear, Boston, MA. In-stagel scintillation fluid was purchased from Packard Instrument Co., Downers Grove, IL. The radiolabeled steroids used in these studies were assayed periodically for purity by thin layer chromatography on silica gel G in ethyl acetate/hexane/ethanol (85:10:5), and were used only if purity was> 95%. Assay of nuclear and cytosolic Sex-Steroid receptors Nuclear and cytosolic Sex-Steroid receptors were evaluated by the method previously described by us (11,14,16-18). Assay of androgen-responsive male estrogen binding protein Assay for the determination of cytosolic content of a male specific estrogen binding protein (MEB) has been described pre-viously (12,34). Briefly, cytosol is pre incubated for 1 h at 0 °c with· 500 nM DES to block the estrogen receptor. The cytosol is then incubated with 5 nM 3H-E2 in the absence and presence of 5 M unlabeled E2 for 2 h a 0 °c. Bound and free steroids are separated by centrifuge-assisted BioGel P-6 chromatography (34). This assay is quantitative for MEB and is linear over a broad range of protein concentration. ANTIA. Protein conCt DNA concentr. method of Burto Cytosolic r the basis of 3 rected for yel receptor conten bound/mg DNA re rected for the animal, the tot the cytosolic a Cytosolic cont activity of ale and the washe 0.5% of that f recovery of D the homogenate. Serum testos mined by specif Equilibrium tions of bindin (35) . Statistical program. Radioactivit ard TriCarb 543 In spironol decrease in tes hormone levelt' trol animals. observed in c· significant red Cytosolic At; renoate treate control animal" variations in groups studied 0.25+0.08 nM tively (Tab I). Il'ER solution of " in the other or 5 mg of <2 vehicle. All by decapita-'urchased from Lum Canrenoate l), Leupeptin, ~eI Protamine sed from Sigma were obtained Radioactive -methyl- 3H -:lonradioactive :on, MA. In-lrd Instrument used in these 1y thin layer :exane/ethanol ; evaluated by ~ng protein of a male :escribed pre-r 1 h at 0 °c he cytosol is presence of steroids are 19raphy (34). - over a broad . - ~ .. ~"D:~"I_~~:ty!D ">-ANTIALDOSTEIWNIC D/URETICS AND l?/IT LIVER 2-13 Other methods Protein concentrations: method of Bradford (5). DNA concentrations of homogenates and nuclear preparations: method of Burton (6). Cytosolic the basis of receptor content for each animal was calculated on 3H -steroid bound/mg cytosol protein, and then cor-rected for yeld of cytosolic protein for gram of liver. Nuclear receptor content was calculated on the basis of 3H -steroid bound/mg DNA recovered in the nuclear preparation, and then cor-rected for the DNA content of the original homogenate. For each animal, the total liver content of receptor represents the sum of the cytosolic and nuclear content based on 1 gram of liver. Cytosolic contamination of nuclei was assayed by determining the activity of alcohol dehydrogenase (37) in the homogenate, cytosol and the washed nuclei preparation and was found to be less than 0.5% of that found in the nuclear preparation. The average recovery of DNA in the nuclear preparation was 72.8% of that in the homogenate. Serum testosterone, estradiol and other hormones were deter-mined by specific radioimmunoassays as described previously (38). Equilibrium dissociation constants (Kd's) and the concentra-tions of binding sites were calculated by the method of Scatchard (35) • Statistical analyses were performed using the Student's t-test program. Radioactivity content of samples was determined using a Pack-ard TriCarb 5430 Liquid Scintillation System. RESULTS In spironolactone treated animals there is a significant decrease in testosterone levels; all other gonadal and pituitary hormone levels in this group are comparable to those of the con-trol animals. In potassium canrenoate treated rats no change was observed in either testosterone or estradiol levels, although significant reductions in progesterone and DHEAS was found. Cytosolic AR levels (cAR) are significantly higher in K can-renoate treated rats as compared with those observed in either control animals or spironolactone treated ones. There were no variations in the affinity of the cAR for its ligand in any groups studied. The Kd values for cAR were 0.26+0.06 nM, 0.25+0.08 nM and 0.31+0.04 nM for group I, II and III respec-tively (Tab I). 244 ANTIALDOSTERONIC DIURETICS AND RAT LIVER The activity of nuclear androgen receptor (nAR) , however, presents a markedly different picture, and is significantly decreased in the animals treated with either spironolactone or potassium canrenoate. As a result of these variations, the total AR content of the liver changed significantly only in those animals treated with spironolactone (50% decrease from normal levels). Interestingly, the activity of total androgen receptors in the spironolactone treated animals shows a decrease proportional to that of plasma testosterone. Tab I: Variation in specific 3H -R1881 in rat liver CONTROL SPIRO K-CAN 3H -R1881 specific binding (fmol/g liver) TOTAL 364+62 170+38 (P<0.05) 347+61 NUCLEUS 202+46 26+7 (P<0.05) 21+8 (P<0.05) CYTOSOL 162+9.9 144+24 326+42 (P<0.05) PLASMA TESTOSTERONE (NG/ML) 1. 77+0.23 0.78+0.20 (P<0.05) 1. 53+0. 32 As total hepatic androgen receptor activity and plasma testosterone values were reduced in the spironolactone-treated animals, the activity of a hepatic androgen responsive protein, a male specific estrogen binder (MEB), was also assessed (Tab II). Tab II: Activity of an androgen responsive protein in rat liver CONTROL SPIRO K-CANREO MEB (pmol/mg protein) 2.41+1 o 6.3+1.5 (p<0.05) The activity of this protein is undetectable in the livers of the spironolactone treated animals; this loss of MEB activity is comparable to that seen in rats castrated at least 15 days prior ANTIA to sacrifice (1-ra ts is ac tuall-To determin· competing for a-they were cap an in vitro ass Spironolac to' effective as cytosol. Howeve-effective comp, canrenoate is i; any concentrati\ The activit: were virtually of cER for E2 v, were 0.26-+{).07 1 and III respect: (nER) was decree treated animal~ content does not latter finding values in these Tab III: Variati liver 31-TOTAl CONTROL 623+11 SPIRO 549+8' K-CAN 650+2:': Sexual dimorp \ animals and huma) In male rat: . LIVER nAR) , however, Ls significantly Lronolactone or R content of the ,; treat.ed with Interestingly, spironolactone u that of plasma ver iver) TESTOSTERONE ~d/MiF 77+0.23 78+0.20 ~MKMRF 53+0.32 i plasma Jlactone-treated ~sive protein, a ~ssed (Tab II). ~ in rat liver n the livers of MEB activity is ~t 15 days prior ANTIALDOSTERONIC DIURETICS AND RAT LIVER 245 to sacrifice (14,36). However MEB level in k-carenoate treated rats is actually higher than that observed in the control tats. To determine whether either of these drugs might interfer by competing for androgen binding to its receptor, we tested whether they were capable of competing for androgen receptor binding in an in vitro assay. Spironolactone, at a 100 and 1000-fold excess, is partially effective as a competitor for 3H - R188l binding in rat liver cytosol. However, the strong androgen R188l and DHT are much more effective competitors than is spironolactone. In contrast, k-canrenoate is incapable of competing for 3H - R1881 binding at any concentration tested. The activity of cytosolic estrogen receptors (cER) (Tab IIi) were virtually unchanged among the 3 groups, nor did the affinity of cER for E2 vary among the groups of animals (Kd values for cER were 0.26i9.07 nM, 0.45i9.l2 nM and 0.36!0.08 nM for group I, II and III respectively). The activity of nuclear estrogen receptor (nER) was decreased significantly only in the spironolactone treated animals. However, the total hepatic estrogen receptor content does not vary in the 3 groups of animals studied. This latter finding is consistent with the similar plasma estradiol values in these three groups of animals. Tab III: Variation in hepatic estrogen receptor activity in rat liver CONTROL SPIRO K-CAN 3H -E2 specific binding (pmol/g liver) TOTAL 623+115 549+89 650+222 NUCLEUS 178+33 16+12 (P<0.05) 209+44 CYTOSOL 445+61 533+150 441+94 DISCUSSION PLASMA ESTRADIOL (PG/ML) 9.52+1.73 7.82+3.04 9.91+1.13 Sexual dimorphism of liver function has been observed in both animals and humans (reviewed in 11). In male rats, androgen is the major steroid hormone deter-Ii \.' :j , I ,;' ,i 246 ANTIALDOSTERONIC DIURETICS AND RAT LIVER minant of the stimulates the masculine pattern of liver function. Androgen expression of both androgen receptor and certain enzymes and proteins such as the male specific estrogen binder MEB (14,36). This masculine pattern can be feminized by certain changes in serum hormone levels occuring after castration (14,36), chronic alcohol ingestion (13), estrogen treatment (21), portal vein ligation (15) and during liver regeneration (16,18). Our results present that administration of spironolactone, resulted in a significant decrease in plasma of testosterone levels and hepatic androgen receptors (AR), and the complete loss of activity of the hepatic androgen responsive protein (MEB), that is a cytosolic protein which has a moderate affinity for estradiol, high binding capacity, and specificity for steroid estrogen (12,34,36). Its function in the cytosol of male rats is unknown, but may be that of an intracellular estrogen scavenger (12,34). The complete disappearance of MEB in rats treated with spironolactone is significant, first, in that its loss represents interruption of hepatic androgen response, and second, that its absence might lead to an increased intracellular estrogen concentration. Plasma estradiol, estriol content, and the total ER activity in liver don't result to be influenced by spironolactone. It is possible that our treatment period was too short to produce a feminization of (i.e. an increase in) these activities. It is more likely, however, that in liver disease patients, feminiza-tion occurs because spironolactone treatment interferes in main-taining the androgenic influence necessary to sustain a masculine pattern of liver function. Such interference, coupled with an al-ready compromised ability to excrete estrogens, may permit full feminization of liver and other peripheral tissues, such as the breast. Patients with liver disease may also fail to clear efficiently othe~ hormones such as prolactin which may enhance devolopment of gynecomastia. However, our experimental animals display no liver diseases; thus, treatment of normal male animals with spironolac-tone may not result in the pronounced feminization that has been noted in some liver Our result in firmed the clinical disease patients. the potassium canrenoate treated animals con-data indicating that this drug has less anti-androgenic effect than spironolactone. It has been demonstrated, in castrated animal, that potassium canrenoate does not inhibit cytochrome p-450 as spironolactone does, and produces sig-nificantly less androgenic antagonism. No decrease of plasma ANTIA testosterone, hibition of and in the liver of In conclusi that spironola testosterone, (low levels of MEB). We also d, any of the ant liver, suggest-patients treat, spironolactone Also, our resu liver disease, estrogen levels ACKNOWLEDGEMENT: technical assis; 1. Armanini D., Invest (1983) 2, Aten R.F., Di 103: 1629-35 3. Bannister P., 1985); 23: 49 4, Bellati G., ] 5. Bradford M.M. 6. Burton K.: Me 7. Chopra 1.J., 79: 198-203 8. Clark E.: JA!-9. Duffy H.J., 1 10. Dupont A.: 1 11. Eagon P. K. , D.H.: Semin 12, Eagon P. K. , 1980); 201: 13. Eagon P.K., (1981); 211: 14. Eagon P. K. , Hepatology ( 15. Farrel G.C., ,. LIVER :lction. Androgen ~ptor and certain estrogen binder Lnized by certain tter castration 11 treatment (21), ~ration (16,18). spironolactone, 3. of testosterone the complete loss ve protein (MEB), te affinity for icity for steroid ,1 of male rats is ,strogen scavenger oats treated with :s loss represents second, that its :ellular estrogen total ER activity nolactone. It is -t to produce a lctivities. It is :ients, feminiza-terferes in main-:stain a masculine upled with an al-may permit full ,ues, such as the clear efficiently lce devolopment of i display no liver , with spironolac-~on that has been cated animals con-~ug has less anti-)een demonstrated, does not inhibit and produces sig-,crease of plasma ANT1ALDOSTERONIC DIURETICS AND RAT LIVER 2.J7 testosterone, total hepatic androgen receptor or MEB, and no in-hibition of androgen binding to its receptor were found to occur in the liver of the potassium canrenoate treated animals. In conclusion, the data reported in this paper demonstrate that spironolactone treatment of male rats decreased plasma testosterone, and exhibited a powerful anti-androgenic effect (low levels of hepatic androgen receptor and disappearance of MEB). We also demonstrated that potassium canrenoate did not show any of the anti-androgenic actions of spironolactone in the liver, suggesting that the clinical improvement reported in patients treated with potassium canrenoate as compared to spironolactone is the result of this biochemical difference. Also, our results indicate that in animals with no underlying liver disease, feminization, defined as an increase in plasma estrogen levels and hepatic ER content, does not occur. ACKNOWLEDGEMENT: The authors thank Francesco Ardito for excellent technical assistance REFERENCES 1. Armanini D., Karbowiak I., Goi A., Mantero F.: J Endocrino Invest (1983); 6 (suppl 1): 115 2. Aten R.F., Dickson R.B., Einsenfeld A.J.: Endocrinology 1978); 103: 1629-35 3. Bannister P., Sheridan P., Losowsky M.S.: Clin Endocrinol 1985); 23: 495-502 4. Bellati G., Ideo G.: Lancet (1986); 1: 626 5. Bradford M.M.: Anal Biochem (1976); 72: 248-54 6. Burton K.: Methods Enzymol (1968); 12: 163-66 7. Chopra I.J., Tulchinsky D., Greenway F.L.: Ann Int Med (1973); 79: 198-203 8. Clark E.: JAMA (1965); 193: 163-64 9. Duffy M.J., Duffy G.J.: J Steroid Biochem (1978); 9: 233-35 10. Dupont A.: Lancet (1985); II: 731 11. Eagon P.K., Porter L.E., Francavilla A., DiLeo A., Van Thiel D.H.: Semin Liver Dis (1985); 5: 59-69 12. Eagon P.K., Fisher S.E., Imhoff A.F.: Arch Biochem Biophys 1980); 201: 486-99 13. Eagon P.K., Zdunek J.R., Van Thiel D.H.: Arch Biochem Biophys (1981); 211: 48-54 14. Eagon P.K., Seguiti S.M., Willet J.E., Rogerson B.J.: Hepatology (1985); 5: 1046 (Abstract 397) 15. Farrel G.C., Koltai A.,Zaluzny L., Murray M.:Gastroenterology 248 ANTJALDOSTERONIC DIURETICS AND RAT LIVER (1986); 90: 299-305 16. Francavilla A., DiLeo A., Eagon P.K.: Gastroenterology (1984); 86: 552-57 17. Francavilla A., Eagon P. K. , DiLeo A. : Gastroenterology 1986) ; 91: 182-88 18. Francavilla A., Eagon P. K., DiLeo A. : Gastroenterology (1986); 91: 1263-70 19. Fromatin M.: Mises Jour Cardiol (1980); 19: 95-98 20. Greenblatt D.J., Koch-Weser J.: JAMA (1973); 225: 40-43 21. Gustafsson J.A., Mode A., Norstedt G., Skett P.: Annu Rev Physiol (1983); 45: 51-60 22. Gustafsson J.A., Mode A., Norstedt G.: Biochem Action Horm (1980); 7: 47-69 23. Huffman D.H., Kampmann J.P., Hignite C.E., Azarnoff D.L.: Clin Pharm Ther (1978); 24: 465-73 24. Kneifel R., Katzenellenbogen B.S.: Endocrinology (1981); 108: 545-52 25. Loriaux D.L., Menard R., Taylor A., Pita J., Sarton R.: Ann Int Med (1976); 85: 630-36 26. Marco J., Constans R., Alibelli M.J., Baradat G., Dardenne: Nouv Presse Med (1978); 7: 3668-74 27. Menard R.H., Stripp B., Gillette J.R.: Endocrinology (1974); 94: 1628-36 28. Niewoehner C.B., Nuttal F.Q.: Am J Med (1984); 37: 633-38 29. Norstedt G., Wrange 0., Gustafsson J.A.: Endocrinology (1981); 108: 1190-96 30. Porter L.E., Elm M.S., Van Thiel D.H, Dugas M.C., Eagon P.K.: Gastroenterology (1983); 84: 704-12 31. Porter L.E., Elm M.S., Van Thiel D.H.,Eagon P.K.: Gastroenterology (in press) 32. Powell-Jones W., Thompson C., Nayfeh S.N., Lucier G.W.: J Steroid Biochem (1980); 13: 219-29 33. Rifka S.M., Pita J.C., Vigersky R.A., Wilson Y.A., Loriaux D.L.: J Clin Endo Metab (1978); 46: 338-44 34. Rogerson B.J., Eagon P.K.: Arch Eiochem Biophys (in press) 35. Scatchard G.: Ann N Y Acad Sci (1949); 51: 660-72 36. Turocy J.F., Chiang A.N., Seeley D.H., Eagon P.K.: Endocrinology (1985); 117:1953-61 37. Vallee B.L., Hoch F.L.: Proc Natl Acad Sci USA (1955); 41: 327-34 38. Van Thiel D.H., Gavaler J.S., Cobb C.F., Sherins R.J., Lester R.: Endocrinology (1979); 105: U~U-94 qhi~ study was supported by Grant 85.2165.44 from Consiglio Nazionale delle Ricerche Italy Adren, Pos L. Demelia, R Istitll Hypogonadi sm 2 with liver cirrr observed in chroni Moreover, marked c hypotha 1 ami c and patients (18,20). or increased estr SHBG (5) levels presence and the c in non alcoholic n (7,21,23). The aim of our cortex on the met, alcoholic cirrhot1 Eleven male nor HBsAg +, in Chi 1 gynecomastia, and studied. In each (DHEA), Dehydr Androstenedione ( Luteinizing Hormor in basal state an ACTH (Synacthen C above the basal Vi 

http://d-scholarship.pitt.edu/4186/1/31735062129527.pdf

Effects of antialdosteronics on plasmatic hormonal levels and an hepatic sexual steroid receptors in rat

Abstract

Similar works

Full text

Available Versions

Name not available

Name not available

D-Scholarship@Pitt