Inhibition of glucuronosyl transferase by steroid hormones

ARCHIVES

OF BIOCHEMISTRY

inhibition DAVID

AND BIOPHYSICS

103, 181-185 (1963)

of Glucuronosyi

Transferase

Y. Y. HSIA, SVETISLAV

RIABOV

From the Departments

of Pediatrics

and Medicine, Received

April

by Steroid AND

ROBERT

Northwestern

Hormones’ M. DOWBEN

University,

Chicago, Illinois

22, 1963

Pregnanediol, A’-17a-methyltestosterone, testosterone, estriol, 17cr-methyltestosterone, progesterone, 17or-ethyl-19-nortestosterone, and Gor-methylprogesterone inhibited guinea pig liver glucuronosyl transferase using o-aminophenol, p-nitrophenol, and 4-methylumbelliferone as aglycones, while cholesterol, pregnanedione, pregnenolone, 17a-hydroxyprogesterone, cortisone, estradiol, estrone, and methylandrostenediol did not. Pregnanediol glucuronide and estriol glucuronide also were inhibitors. Microsomes prepared as described contained little or no P-glucuronidase or uridine diphosphoglucuronic acid pyrophosphatase activity. Kinetic studies gave a K, for glucuronosyl transferase of 1.92 X lo+ using o-aminophenol as aglycone. The kinetics of pregnanediol glucuronide and 17a-ethyl-19.nortestosterone inhibition were mainly competitive in type with Ki of 3.24 X 1OV and 2.52 X lo+, respectively. INTRODUCTION

It has long been thought that steroid hormones may exert some of their profound physiological actions by modifying certain (1). Estrogens may enaymic reactions transfer hydrogen between diphosphopyridine nucleotide and triphosphopyridine nucleotide (2, S), and steroids inhibit the eneymic oxidation of reduced diphosphopyridine nucleotide (4). Dehydroisoandrosterone, pregnenolone, and related steroids inhibit mammalian glucose-6-phosphate dehydrogenase (5). Estrogens appear to increase mammalian liver glucuronidase activity (6), while testosterone increases renal p-glucuronidase, an action which is prevented by estrogens (7). Estrogenic steroids inhibit glutamic dehydrogenase and 1 This project was supported by a grant from the Association for the Aid to Crippled Children and U. S. Public Health Service Grants A-4055 and A-2222. 2 A portion of this study was carried out as a Lalor Fellow at the Marine Biological Laboratory; Woods Hole, Massachusetts. Present address: Biology Department, Massachusetts Institute of Technology, Cambridge 39, Massachusetts. 181

activate alanine dehydrogenase by promoting the dissociation of these enzymes into subunits (4). Recently, Lathe and Walker (8) reported inhibition of bilirubin conjugation in rat liver slices by serum from pregnant women. Evidence was presented in a preliminary paper (9) from this laboratory that some natural progestins and some synthetically prepared steroids inhibit glucuronosyl transferase activity of rat and guinea pig liver microsomes. This communication is a more detailed report of our studies. EXPERIMENTAL MATERIALS

Uridine diphosphoglucuronic acid (UDPGA), tris- (hydroxymethyl)aminomethane (tris), phenolphthalein glucuronide, pregnanediol glucuronide, estriol glucuronide, nicotinamide, o-aminophenol (OAP), and p-nitrophenol (PNP) were purchased from the Sigma Chemical Co., St. Louis. The latter two compounds were recrystallized from ethanol-water immediately before use. The steroids were gifts of Dr. Frank B. Colton, G. D. Searle and Co., Skokie, Illinois or were purchased from the California Corp. for Biochemical Research, Los Angeles. The borne01 glucuronide was a gift of Dr. Smith Freeman.

182

HSIA,

RIABOV

Adult guinea pigs were sacrificed by stunning and decapitation. The livers were removed quickly, weighed, and placed in 4 volumes of ice cold 0.10 M tris buffer at pH 7.65 containing 0.004 J4 MgClz and 0.008 M nicotinamide. The tissue was homogenized in a loose (1 mm. clearance) all glass Potter-Elvehjem tissue grinder immersed in ice. Microsomal preparations were obtained by fractionation in a Spinco model L preparative ultracentrifuge. Cell debris, nuclei, and mitochondria were removed after centrifugation at 8,000 g for 15 minutes. The microsomal fraction, collected after subsequent centrifugation at GO,OOOg for 1 hr., was suspended in a quantity of tris buffer equivalent to the volume of the homogenate from which they were obtained.

ASSAY

OF GLUCURONOSYL ACTIVITY

TRANSFERASE

The assay of glucuronosyl transferase activit’y using OAP as aglycone was modified from the procedure of Levvy and Storey (10). The incubation mixture, final volume 2.3 ml., contained 1.0 pmoles OAP, 2.0 pmoles ascorbic acid, 1.0 rmole UDPGA, and homogenate or microsomes corresponding to 0.1 g. fresh liver in 0.05 M tris buffer at pH 7.65 containing 0.004 M MgCl, and 0.008 M nicotinamide. After incubation at 37°C. for 30 minutes, the mixture was deproteinized and color developed with the Bratton-Marshall reagent (10). After 45 minutes, the absorbance was measured at 560 rnp. Glucuronosyl transferase was assayed using PNP as aglycone by a modification of Isselbacher’s (If), and using 4MU as aglycone by the procedure of Arias (12).

MEASUREMENT

OF

INHIBITOR

ACTIVITY

The steroids were dissolved in 0.1 ml. 1:l ethanol-ethylene glycol and added to the assay Comparative assays were performed mixture. using solvent alone. Steroid glucuronides were added directly to the assay mixture. Half inhibition concentrations were determined graphically from plots of per cent inhibition at several inhibitor concent,rations on log-probability paper. Formal kinetic studies were done at two inhibitor concentrations using pregnanediol gluand 17a-ethyl-19-nortestosterone as curonide inhibitors. The latter steroid was dissolved in 0.1 ml. ethylene glycol monomethyl ether and added to the incubation mixtures. Assay

OF OTHER ENZYME ACTIVITY

&Glucuronidase activity was assayed using phenolphthalein glucuronide by the procedure of Fishman et al. (1s) at pH’s 5.0 and 7.65. Uridine

AND

DOWBEN TABLE I CONCENTRATION OF SEVERAL STEROIDS PRODUCING 50% INHIBITION OF C;I,ECURONOSYL TRANSFERASE ACTIVITY Aglycone

used in assay

Inhibitor OAP

PSP

4MU

(‘Mx Al*) Pregnanediol-3a, 17a AL-17cY-methyltestosterone Testosterone Estriol Pregnanediol glucuronide 17a-Methyltestosterone Progesterone Desoxycorticosterone 17a-Ethyl-19nortestosterone Estriol glucuronide 17a-Ethyl-As(‘o)-19norandrosterone 6a-Methylprogesterone

0.42 0.46 0.83 1.32

1.52

1.42 1.28 1.80

1.80 2.9 3.4 7.5

3.0

3.6

4.6 5.5

6.0

diphosphoglucuronic acid pyrophosphatase was measured by the method of Hollman and Touster (14). RESULTS

AND

DISCUSSION

The concentrations of a number of steroids which resulted in a 50 % inhibition of glucuronosyl transferase activity are listed in Table I. The order of addition of compounds to the incubation mixture did not affect the degree of inhibition. Cholesterol, pregnanedione, pregnenolone, 17a-hydroxyprogesterone, cortisone, estradiol, estrone, and methylandrostenediol did not result in significant inhibition of glucuronosyl transferase activity in concentrations as great as 10-a M. The glucuronides of pregnanediol and estriol also inhibited glucuronosyl transferase activity, but a slightly higher concentration of glucuronide was required to produce inhibition comparable to that produced by the corresponding free steroid. Assay of the microsomal preparation for p-glucuronidase activity showed approximately 170 U in microsomes corresponding to 1 g. liver at pH 5.0, but less than 10 U at pH 7.65. Plaut and Fishman (15) have found recently

INHIBITION

OF GLUCURONOSYL

that /3-glucuronidase occurs in a distinctive type of granule. It appears that glucuronosyl transferase was separated from the P-glucuronidase containing granules by the preparative technique employed in these studies. It seems unlikely that the steroid glucuronides are hydrolyzed to free steroids under the conditions of the assay; both free steroids and steroid glucuronides appear to possess inhibitory activity. Furthermore, while use of the described conditions of differential centrifugation resulted in little loss of glucuronosyl transferase activity,

183

TRANSFERASE

no uridine diphosphoglucuronic acid pyrophosphatase activity could be demonstrated in these preparations. The rates of OAP and PNP conjugation were measured at several substrate concentrations without inhibitor and in the presence of two concentrations of pregnanediol glucuronide or of 17cll-ethyl-19-nortestosterone as inhibitors. Data from two typical experiments are given in Tables II and III. The values of K, and V, were calculated from a statistical analysis of the data by the method of Wilkinson (16). Lineweaver-

TABLE

II

OAP GLUCURONIDE FORMATION AT VARIOUS SUBSTRATE CONCENTRATIONS IN THE PRESENCE AND ABSENCE OF PREGNANEDIOL GLUCURONIDE (EXPT. 5) Substrate

(@M/L)

870 435 218 109 59

KPb S. E. Kp VP S. E. VP

Velocity’ 17.5,,M/L PG

No PG

35.0

j&I/L PG

0.740 0.630 0.555 0.330 0.210

0.530 0.490 0.270 0.140

0.500 0.350 0.270 0.170 0.105

168.0 x 10-G 27.6 X 1OW 0.892 0.053

209.7 x 10-G 81.7 X 1O-6 0.838 0.173

349.5 x 10-e 61.4 X 10-C 0.682 0.054

y Change in optical density using the OAP assay described in the text. b These values were obtained by statistical analysis of the data (16). TABLE

III

OAP GLUCURONIDE FORMATION AT VARIOUS SUBSTRATE CONCENTRATIONS IN THE PRESENCE AND ABSENCE OF 1701.ETHYL-l%NORTESTOSTERONE (EXPT. 6) Substrate (PM/L)

480 320 160 80

KPb S. E. K,

VP S. E. VP

Velocity” No ENTC

266

PM/LENT

532

pM,JL ENT

0.253 0.231 0.148 0.103

0.089 0.074 0.046 0.024

0.064 0.048 0.031 0.015

219.8 X lo-” 29.4 x 10-C 0.273 0.019

462.1 X lo-” 51.6 X 1O-6 0.177 0.012

674.5 X 1O-6 151.9 x 10-6 0.153 0.023

a Change in optical density using the OAP assay described in the text. b These values were obtained by statistical analysis of the data (16). c ENT = 17a-ethyl-19.nortestosterone.

184

HSIA,

I/(Sx

RIABOV

103)

AND

DOWBEN

(S x 104)

FIG. 1. Lineweaver-Burk

and Dixon-Webb plots of glucuronosyl transferase activity using OAP as aglycone, alone (0-O) and with 0.266 pmole per ml. (O--C!) or 0.532 pmole per ml. (A--A) 17a-ethyl-19nortestosterone. The assay was performed as described in the text except that each flask contained 0.1 ml. ethylene glycol monomethyl ether. I’is optical density. S is molar OAP concentration. Lines are regressions statistically calculated by the method of Wilkinson (16).

Burk (17) (l/V against l/S) and DixonWebb (18) (S against S/V) plots for the latter experiment is depicted in Fig. 1. The mean K, (Michaelis-Menten) for glucuronosyl transferase calculated from four experiments using OAP as aglycone was 1.92 X 10h4 and from two experiments using PNP as aglycone was 4.88 X 10p4. These values compare closely to the value of 6.2 X 1O-4 reported by Isselbacher et al. U9)*

The characteristic configuration of the plotted data as well as the near constancy of V, and the progressive decrease of K, with increasing concentrations of inhibitor indicate that the mode of inhibition of glucuronosyl transferase by pregnanediol glucuronide and 1701.ethyl-19-nortestosterone is largely competetive in nature. The Ki for pregnanediol glucuronide, the mean of three experiments using two concentrations of inhibitor in each, was 3.24 X 10-4. While the steroid glucuronides are usually water soluble, free steroids usually are not. The 17a-ethyl-19-nortestosterone was added to the incubation mixture in 0.1 ml. ethylene glycol monomethyl ether. The mean K, of glucuronosyl transferase with the solvent added to the incubation mixture in four experiments using OAP as aglycone was 2.45 X 1w4. Using this incubation mixture

plus solvent as control, the Ki for 17aethyl-19-nortestosterone, the mean of three experiments using two concentrations of inhibitor in each, was 2.52 X 10p4. The inhibitory activity seemed to cross the usual hormonal groupings of steroids. Thus, while pregnanediol and progesterone were potent inhibitors, the related steroids, pregnanedione and pregnenolone were not. The latter steroids, however, are physiologically less active progestins than the former. Among the estrogens, estriol was an inhibitor of glucuronosyl transferase while estradiol and estrone were not. In contrast to the progestins, estradiol and estrone are physiologically more active estrogens than estriol. Furthermore, testosterone, 17amethyltestosterone and A’-17~u-methyltestosterone, steroids with androgenic activity, inhibited glucuronosyl transferase. 17CYEthyl-19-nortestosterone has multiple hormonal actions; not only is it an androgen and anabolic steroid but it is also a potent progestin and also has estrogenic activity. Brown and Zuelzer (20) have suggested that the hyperbilirubinemia of the newborn infant may result from a functional deficiency of glucuronosyl transferase, since indirect reacting bilirubin is converted to bilirubin glucuronide by means of this enzyme (H). Lathe and Walker (8) reported inhibition of bilirubin conjugation in rat

INHIBITION

OF GLUCURONOSYL

liver slices by serum from pregnant women. Lucey et al. (22) found that serum from pregnant mothers of kindreds with transient neonatal familial hyperbilirubinemia was three to five times a more potent inhibitor of bilirubin conjugation in rat liver slices than serum from normal pregnant women. While Isselbacher et al. (19) have obtained evidence that the same enzyme is involved in the formation of both ether and ester glucuronides, the K, of glucuronosyl transferase using bilirubin as aglycone has not yet been determined and the potency of progestational steroids as inhibitors using bilirubin as aglycone is not yet known. While 17cr-methyltestosterone, 17a-ethylAl-1701.methyltestos19-nortestosterone, terone, and 17a-ethyl-A5Q0)-lg-norandrosterone were all inhibitors of glucuronosyl transferase using OAP, PNP, and 4MU as aglycones, it is not possible to assess the physiological role of this inhibition in the clinical jaundice which often follows the administration of 17-alkylated steroids. Abnormal liver excretory function is also associated with this type of jaundice and may be more important physiologically than glucuronosyl transferase inhibition. ACKNOWLEDGMENTS The authors are indebted to Dorothy Parker, Mary Ann Cooley, and Genevieve Barrieux for technical assistance. REFERENCES 1. DORFMAN, R. I., AND GOLDSMITH, E. D., EDS., “The Influence of Hormones on Enzymes.” Bnn. N. Y. Acad. Sci. 64, 531 (1951). 2. TALALAY, P., AND WILLIAMS-ASHMAN, H. G., Res. 16, 1 (1960). Rec. Progr. Hormone

TRANSFERASE

185

3. VILLEE, C. A., HAGERMAN, D. D., AND JOEL, P. B., Rec. Progr. Hormone Res. 16,49 (1960). 4. TOMKINS, G. M., AND YIELDING, K. L., Cold Spring Harbor Symp. Quant. Biol. 26, 331 (1961). 5. MARKS, P. A., AND BANKS, J., Proc. Xatl. Acad. Sci. U. S. 46, 447 (1960). 6. FISHMAN, W. H., AND FARMELSNT, M. H., Endocrinology 63, 536 (1953). 7. FISHMAN, W. H., in “Mechanism of Hormone Action” (C. L. Villee, and L. L. Engel, eds), p. 157. Pergamon Press, New York, 1961. G. H., AND WALKER, M., QUUT~.J. 8. LITHE, Exptl. Physiol. 43, 257 (1958). 9. HSIA, D. Y. Y., DOWBEN, R. M., SHAW, R., AND GROSSMSN, A., Nature 187, 693 (1960). 10. LEVVY, G. A., AND STOREY, I. D. E., Biochem. J. 44, 295 (1949). K. J., Rec. Progr. Hormone Res. 11. ISSELBACHER, 12, 134 (1956). 12. ARIAS, I. M., Advan. Clin. Chem. 3, 35 (1960). 13. FISHMAN, W. H., SPRINGER, B., AND BRUNETTI, R., J. Biol. Chem. 173, 449 (1948). 14. HOLLMAN, S., AND TOUSTER, O., Biochim. Biophys. Acta 62, 338 (1962). W. H., J. Cell 15. PL~UT, A. G., A4~~ FISHMAN, Biol. 16, 253 (1963). 16. WILKINSON, G. N., Biochem. J. 60, 324 (1961). 17. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 18. DIXON, M., .~ND WEBB, E. C., “Enzymes,” pp. 171 ff. Academic Press, New York, 1959. 19. ISSELBACHER, K. J., CHRABAS, M. F., AND QUINN, R. C., J. Biol. Chem. 237, 3033 (1962). 20. BROWN, A. K., BND ZUELZER, W. W., J. Clin. Invest. 37, 332 (1958). 21. SCHMID, R., Science 124, 76 (1956). 22. LUCEY, J. F., ARI.~s, I. M., AND McKay, R. J., Am. J. Dis. Child. 100, 787 (1960).