Elsevier

Steroids

Volume 71, Issue 5, May 2006, Pages 343-351
Steroids

Sulfation of tibolone metabolites by human postmenopausal liver and small intestinal sulfotransferases (SULTs)

https://doi.org/10.1016/j.steroids.2005.11.003Get rights and content

Abstract

Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3α-hydroxytibolone (3α-OH-tibolone) and 3β-hydroxytibolone (3β-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n = 5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 ± 0.3 units/mg protein, n = 8, mean ± SEM), and activities with 3α-OH-tibolone (0.6 ± 0.1, n = 8) and 3β-OH-tibolone (0.9 ± 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 ± 0.01 with 3α-OH-tibolone and 0.04 ± 0.01 with 3β-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.

Introduction

Tibolone (Livial) is a synthetic steroid that has been used extensively for the prevention of postmenopausal osteoporosis [1], [2] and for the treatment of climacteric symptoms [3]. It is the prototype of a compound that acts as a selective tissue estrogen activity regulator (STEAR) [4]. A key element in this tissue specific regulation of estrogen activity is mediated through enzymatic mechanisms that activate or inactivate estrogenic compounds prior to their interaction with a steroid receptor [5]. After oral administration, tibolone is metabolized into three biologically active metabolites. The 3α-hydroxytibolone (3α-OH-tibolone) and 3β-hydroxytibolone (3β-OH-tibolone) metabolites have estrogen agonist properties, and the Δ4-ketoisomer has progestogenic and androgenic effects. Sulfation of the hydroxytibolone metabolites by sulfotransferase enzymes (SULTs) renders these metabolites receptor-inactive and reduces their estrogenic effects. SULT activity may account for the clinical effects of tibolone treatment that include an unchanged mammographic breast density in most women, less symptoms related to the breast, and less vaginal bleeding when compared with usual hormone replacement therapy [6], [7], [8], [9], [10]. An additional protective effect of tibolone in these tissues is the inhibition of sulfatase activity by the tibolone metabolites in breast and endometrial cells [5], [11]. By contrast, the lack of steroid SULT activity and minimal inhibition of sulfatase activity by tibolone in bone cells allow free hydroxytibolone metabolites to activate the estrogen receptor (α) in bone and to decrease bone resorption [5], [11]. Thus, tibolone, as a STEAR, does not behave as a selective estrogen receptor modulator (SERM), but rather acts by enzymatic pre-receptor regulation of the estrogenic active metabolites.

Sulfated hydroxytibolone metabolites have been found in the circulation, supporting the involvement of SULTs in the regulation and metabolism of tibolone. In the cytosolic SULT superfamily there are at least 10 human SULT genes [12]. Fusion proteins of two steroid SULTs expressed in bacteria have been shown to use tibolone metabolites as substrates [13]. However, the exact SULT isozymes involved in the sulfation of the active tibolone metabolites in postmenopausal human liver and other tissues have yet to be fully characterized.

In this study we tested samples of human premenopausal and postmenopausal liver and small intestine for SULT activities with tibolone metabolites. We then used postmenopausal liver to establish biochemical properties and radiochemical assays with the substrates 3α-OH-tibolone and 3β-OH-tibolone. These tools allowed us to document the postmenopausal liver contribution to the sulfation of hydroxytibolone metabolites. Because the cytosolic liver preparation represented a composite of SULT activities, we also compared the kinetic parameters of cDNA expressed SULT activities to the liver SULT activities when tested with the tibolone metabolites.

Section snippets

Materials

[35S] Phosphoadenosine-5-phosphosulfate ([35S]-PAPS, specific activity from 2.4 to 3.0 Ci/mmol) was purchased from Perkin-Elmer LAS, Shelton, CT. 2-Difluoro-methyloestrone-3-O-sulphamate (EMATE), the hydroxytibolone metabolites and their respective sulfated compounds were provided by Dr. H.J. Kloosterboer (Organon, Oss, The Netherlands). Dithiothreitol (DTT) was purchased from CalBiochem, La Jolla, CA. Bovine serum albumin (BSA), sodium chloride (NaCl), 2,6-dichloro-4-nitrophenol (DCNP) and

RT-PCR detection of human SULT expression

Reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed with total RNA extracted from individual premenopausal and postmenopausal human livers and SULT gene-specific primers for SULT1A1, 1A3, 1E1, 2A1, 2B1_v1 and 2B1_v2. Liver and small intestinal samples were tested to confirm whether mRNA predicted the presence of activity for SULT1E1 and SULT2A1. SULT1A1, SULT1E1 and SULT2A1 mRNAs were expressed in all 10 postmenopausal liver samples (Fig. 1). In separate studies

Discussion

Tibolone represents the initial steroid in the class of agents identified as selective tissue estrogen activity regulators (STEAR) [4]. This study is the first systematic investigation of human postmenopausal tissue sulfation of tibolone metabolites as one of the tissue-specific components in the pre-receptor enzymatic regulation of the estrogenic effects of tibolone. We have identified human premenopausal and postmenopausal liver and small intestine SULT activities involved in the sulfation of

Acknowledgments

We thank Dr. Helenius Kloosterboer, N.V. Organon, Oss, The Netherlands, for the tibolone metabolites and sulfated metabolites for HPLC standards, Dr. Richard M. Weinshilboum, Mayo Foundation, Mayo Clinic, Rochester, MN, for the listed SULT cDNAs, and Cindy Pietsch, VA Medical Center, Omaha, NE, for her assistance in the preparation of this manuscript.

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    This work was supported by a grant from N.V. Organon Inc. and the VA Medical Research Service.

    1

    Present address: Department of Obstetrics and Gynecology, The Second Affiliated Hospital, China Medical University, Shenyang, PR China.

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