Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase.
Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17beta-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 [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) or radiolabeled substrate [(3)H]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/microg 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 (17beta-estradiol, 17alpha-estradiol, genistein, 17alpha-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.
Reinen
Jelle
J
LACDR-Department of Pharmacochemistry, Division of Molecular Toxicology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
Vriese
Eveline
E
Glatt
Hansruedi
H
Vermeulen
Nico P E
NP
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004054:Diethylstilbestrol / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D005453:Fluorescence; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D011721:Pyrenes / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013431:Sulfates / Q000032:analysis / Q000737:chemistry; D015238:Sulfotransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004791:Enzyme Inhibitors; D011721:Pyrenes; D013431:Sulfates; D004054:Diethylstilbestrol; D015238:Sulfotransferases; C020122:estrone sulfotransferase; C033146:1-hydroxypyrene