Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase

Abstract

Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17β-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [ 35S]3′-phosphoadenosine 5′-phosphosulfate (PAPS) or radiolabeled substrate [ 3H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined ( K m = 6.4 ± 0.8 nM and V max = 158 ± 19 pmol/min/μg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis–Menten kinetics involving substrate inhibition. IC 50 values have been determined for eight known SULT1E1 inhibitors or competing substrates (17β-estradiol, 17α-estradiol, genistein, 17α-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.