Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Abstract

Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Delta4-isomer are sulfated at the 17beta-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3alpha-OH and 3beta-OH Tib can form both 3- and 17-monosulfates as well as disulfates. Only the 3beta-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4'-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.

Figure 1

Structures of tibolone, raloxifene and arzoxifene.

Figure 2

Sulfation and desulfation of Tib and metabolites. The formation of Tib and the Δ4 monosulfates was carried using SULT2A1 and the 3α- and 3β-monosulfates were generated using SULT1E1 in the presence of ³⁵S-PAPS. The four monosulfates were then tested for sensitivity for hydrolysis by STS. Panel A is the autoradiograph of the formation and hydrolysis of the four monosulfates. Panel B shows the selective hydrolysis of Tib 3β-monosulfate by STS. Each reaction was run in quadruplicate and the mean ± s.d. of the ³⁵S-Tib metabolites generated or remaining after STS incubation is shown.

Figure 3

Hydrolysis of the 3α- and 3β-Tib mono- and disulfates by STS. The 3α- and 3β-Tib monosulfates and disulfates were synthesized using SULT1E1 and ³⁵S-PAPS. The levels of the synthesized sulfates were quantified by resolution of an aliquot of the reaction by TLC, autoradiography and scintillation spectroscopy. Aliquots of the reactions were then extracted with chloroform and the Tib sulfates used as substrates for STS. After 60 min incubations the reactions were resolved by TLC and the Tib sulfates quantified by scintillation spectroscopy. The mean ± s.d. of four reactions is shown. An asterisk (*) indicates a significant difference between the assays with and without STS (p<0.01).

Figure 4

Time course of tibolone monosulfate and disulfate hydrolysis by STS. The tibolone monosulfates and disulfate were generated using SULT2A1 and 10 μM 3α-OH or 3β-OH tibolone and ³⁵S-PAPS. The 3-OH tibolone sulfates were extracted with chloroform and hydrolyzed with STS as described in Methods. Panel A shows the results of the hydrolysis of the mono- and disulfates formed with 3α-OH tibolone by STS. Panel B shows the results of the hydrolysis of the 3β-OH tibolone mono- and disulfates by STS. Aliqouts were taken from the STS reactions at different times, resolved by TLC and quantified. The mean ± s.d. of four reactions is shown.

Figure 5

Sulfation and desulfation of raloxifene. Raloxifene monosulfate was formed using SULT2A1 and both raloxifene monosulfates and disulfate were synthesized using SULT1E1 and ³⁵S-PAPS. As reported previously (12), panel A shows that using SULT1E1, raloxifene disulfate formation at 2.5 μM is favored over monosulfate synthesis although monosulfate formation is greater at the 10 μM concentration. Panel B shows the sensitivity of the raloxifene monosulfates and disulfate for sensitivity to hydrolysis by STS. The products of the STS reactions were resolved by TLC and the products quantified by scintillation spectroscopy Each reaction was run in quadruplicate and the mean ± s.d. of the ³⁵S-raloxifene sulfates generated or remaining after STS incubation is shown.

Figure 6

Time courses of the hydrolysis of raloxifene mono- and disulfate by human placental sulfatase (STS). Expressed human SULT2A1 and SULT1E1 were used to generate the two raloxifene monosulfates and the disulfate radiolabeled with ³⁵S-PAPS. The ³⁵S-labeled raloxifene products were then used as substrates for hydrolysis by STS. The formation and hydrolysis of the raloxifene monosulfates and disulfates were analyzed by HPLC. Panel A; STS hydrolysis of the single raloxifene monosulfate formed by SULT2A1. Panel B; STS hydrolysis of the monosulfates and disulfate of raloxifene formed by SULT1E1 at a raloxifene concentration of 10 μM. SULT1E1 with 10 μM raloxifene forms mostly monosulfates with a ratio of approx. 8:1, and only a relatively low level of the disulfate; Panel C. STS hydrolysis of the monosulfates and disulfate of raloxifene formed by SULT1E1 at a raloxifene concentration of 2.5 μM. SULT1E1 with 2.5 μM raloxifene forms mostly the disulfate.

Figure 7

HPLC analysis of the hydrolysis of the raloxifene monosulfates and disulfate by human placental STS. Raloxifene monosulfates and disulfate were synthesized using SULT2A1 and 1E1 as described in Figure 6. Panel A shows the elution of the monosulfates formed by SULT1E1 before and after STS hydrolysis. Panel B shows the elution of the monosulfate formed by SULT2A1 before and after STS hydrolysis.

Figure 8

Hydrolysis of arzoxifene sulfate by human placental STS. Arzoxifene possesses a single benzothiophene hydroxyl group capable of being sulfated. The figure shows the monosulfates formed by SULT2A1 with raloxifene (Ral) and arzoxifene (Arz), respectively, with and without subsequent incubation with placental STS (+ STS).