Ethanol Sulfation by the Human Cytosolic Sulfotransferases: a Systematic Analysis
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2180 Regular Article Biol. Pharm. Bull. 35(12) 2180–2185 (2012) Vol. 35, No. 12 Ethanol Sulfation by the Human Cytosolic Sulfotransferases: A Systematic Analysis Katsuhisa Kurogi,a Garrett Davidson,a Yasir Ihsan Mohammed,a Frederick Edward Williams,a Ming-Yih Liu,b Yoichi Sakakibara,c Masahito Suiko,c and Ming-Cheh Liu*,a a Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo; Toledo, OH 43614, U.S.A.: b National Synchrotron Radiation Research Center; Hsinchu 30076, Taiwan, ROC: and c Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki; Miyazaki 889–2192, Japan. Received June 20, 2012; accepted September 7, 2012 Ethyl sulfate, a minor and direct ethanol metabolite in adult human body, has been implicated as a biomarker for alcohol consumption and in utero exposure to ethanol. To understand better the physiological relevance of the sulfation of ethanol, it is important to clarify the cytosolic sulfotransferase (SULT) enzymes that are responsible for ethanol sulfation. The present study aimed to identify the major ethanol-sulfating human SULTs and to investigate the sulfation of ethanol under the metabolic setting. A systematic analysis revealed four ethanol-sulfating SULTs, SULT1A1, SULT1A2, SULT1A3, and SULT1C4, among the eleven human SULT enzymes previously prepared and purified. A metabolic labeling study demonstrated the gen- eration and release of ethyl [35S]sulfate in a concentration-dependent manner by HepG2 human hepatoma cells labeled with [35S]sulfate in the presence of different concentrations of ethanol. Cytosol or S9 fractions of human lung, liver, and small intestine were examined to verify the presence of ethanol-sulfating activity in vivo. Of the three human organs, the small intestine displayed the highest activity. Key words ethanol; ethyl sulfate; sulfation; cytosolic sulfotransferase In humans and other vertebrates, ethanol is generally containing either hydroxyl or amino groups.20) Sulfate conju- known to be metabolized sequentially through acetaldehyde gation by the SULT enzymes generally leads to the inactiva- to acetate, under the actions of alcohol dehydrogenase and tion of biologically active compounds and/or the increase in aldehyde dehydrogenase.1,2) A lesser known and, in fact, minor their water-solubility, thereby facilitating their removal from metabolic fate of ethanol is the conjugation with sulfate or the body.17–19) In humans, eleven SULTs that fall into three glucuronic acid, forming ethyl sulfate and ethyl glucuro- distinct gene families have been identified and character- nide.3,4) Upon ethanol ingestion, ethyl sulfate and ethyl gluc- ized.21,22) Seven of the eleven human SULTs that belong to uronide are excreted for a considerably longer period of time the SULT1 gene family are: SULT1A1 and SULT1A2 (both than ethanol, hence allowing urine testing of these minor eth- believed to be general detoxifying enzymes), SULT1A3 (do- anol metabolites as a sensitive method to screen for alcohol in- pamine/catecholamine SULT), SULT1B1 (thyroid hormone gestion.3,5–7) More recently, ethyl sulfate and ethyl glucuronide SULT), SULT1C2 and 1C4 (hydroxyarylamine SULTs), and have also been implicated as biomarkers for in utero exposure SULT1E1 (estrogen SULT). Three that belong to the SULT2 to ethanol.8–10) While the physiological relevance of ethanol gene family are: SULT2A1 (dehydroepiandrosterone SULT), sulfation remains unknown, the capacity of metabolizing SULT2B1a (pregnenolone SULT), and SULT2B1b (cholesterol ethanol to acetate had been shown to be much lower in fetuses SULT). The remaining human SULT, a neuronal/brain SULT, than in adults due to the low level of the activity of alcohol belongs to the SULT4 gene family. In a previous study, it dehydrogenase.11,12) In contrast, the levels of expression and/or was demonstrated that several human SULTs, expressed in activity of cytosolic sulfotransferases (SULTs), of which some Salmonella typhimurium, displayed sulfating activity toward may be capable of catalyzing the sulfation of ethanol forming ethanol.16) To better define their ethanol-sulfating activity, ethyl sulfate, in fetuses had been shown to be higher than or however, it is necessary to use human SULTs in purified form. comparable to those in adults.13–15) It is therefore an intriguing We report in this communication a systematic investiga- question whether sulfate conjugation may constitute an im- tion of the ethanol-sulfating activity of eleven human SULTs portant route for the metabolism and detoxification of ethanol previously expressed and purified. A metabolic labeling study early on during the developmental process. To date, however, using HepG2 human hepatoma cells labeled with [35S] sulfate there have been few studies on the sulfation of ethanol by in the presence of different concentrations of ethanol was human SULTs.16) performed. Moreover, cytosol or S9 fractions of human lung, In vertebrates, sulfate conjugation as catalyzed by the cy- liver, kidney, and small intestine were examined to verify the tosolic sulfotransferases (SULTs) is known to be involved in presence of ethanol-sulfating activity in human tissues. the biotransformation and excretion of xenobiotics as well as the homeostasis of key endogenous compounds such as MATERIALS AND METHODS steroid and thyroid hormones, catecholamines, cholesterol, and bile acids.17–19) The SULTs catalyze the transfer of a sul- Materials Adenosine 5′-triphosphate (ATP), 3′-phospho- fonate group from the active sulfate, 3′-phosphoadenosine adenosine-5′-phosphosulfate (PAPS), 3-(N-morpholino) pro- 5′-phosphosulfate (PAPS), to an acceptor substrate compound pane sulfonic acid (Mops), Trizma base, dithiothreitol (DTT), Nonidet P-40 (NP-40), ethyl alcohol (absolute), minimum The authors declare no conflict of interest. essential medium (MEM), penicillin G, and streptomycin * To whom correspondence should be addressed. e-mail: [email protected] © 2012 The Pharmaceutical Society of Japan December 2012 2181 sulfate were products of Sigma Chemical Co. (St. Louis, MO, Thereafter, the radioactive spots were cut out from the plate U.S.A.). Protease inhibitor cocktail, ethylenediaminetetra- and the materials therein were eluted and counted using a acetic acid (EDTA)-free, was purchased from Roche Diagnos- liquid scintillation counter. To examine the pH-dependence tics (Indianapolis, IN, U.S.A.). Carrier-free sodium [35S] sulfate of sulfation of ethanol by human SULT1A1 and SULT1C4, and Ecolume scintillation cocktail were obtained from MP 50 mM different buffers (sodium acetate at 4.5 or 5.5; Mes at Biomedicals, Inc. (Irvine, CA, U.S.A.). Cellulose thin-layer 5.5, 6.0, or 6.5; Mops at 6.5, 7.0, or 7.5; Hepes at 7.0, 7.5, or chromatography (TLC) plates were from EMD Chem. Inc. 8.0; Taps at 8.0, 8.5, 9.0; Ches at 9.0, 9.5, or 10.0; and Caps (Gibbstown, NJ, U.S.A.). Ultrafree-MC 5000 NMWL filter at 10.0, 10.5, or 11.5), instead of 50 mM Mops (pH 7.0), were units were products of Millipore (Bedford, MA, U.S.A.). Fetal used in the reaction. To assay for ethanol-sulfating activity of bovine serum was from Biomeda (Foster City, CA, U.S.A.). human tissue cytosol or S9 fraction and HepG2 cell lysate, the HepG2 human hepatoma cell line (ATCC HB-8065) was reaction mixture mentioned above was employed. The reaction obtained from American Type Culture Collection (Manas- was started by the addition of the cytosol (50 µg), S9 fraction sas, VA, U.S.A.). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (50 µg), or lysate (50 µg), and allowed to proceed for 30 min, tetrazolium bromide (MTT) cell proliferation assay kit was a followed by the TLC analysis and scintillation described product of Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). above. Each experiment was performed in triplicate, together Pooled human lung S9 fraction from a mixed-gender group with a control without enzyme. The results obtained were of 4 donors (Lot No. 0710281), liver cytosol from 50 donors calculated and expressed in nanomoles of sulfated product (Lot No. 09103970), small intestine (duodenum and jejunum) formed/min/mg purified enzyme. S9 fraction from 18 donors (Lot No. 0710351), and kidney Metabolic Labeling of HepG2 Human Hepatoma Cells S9 fraction from 8 donors (Lot No. 0510093) were purchased HepG2 cells were maintained, under a 5% CO2 atmosphere from XenoTech, LLC (Lenexa, KS, U.S.A.). All other chemi- at 37°C, in MEM supplemented with 10% FBS, penicillin G cals were of the highest grade commercially available. (30 µg/mL), and streptomycin sulfate (50 µg/mL). Confluent Preparation of Purified Human SULTs Recombi- cells grown in individual wells of a 24-well culture plate, pre- nant human P-form (SULT1A1 and SULT1A2) and M- incubated in sulfate-free (prepared by omitting streptomycin form (SULT1A3) phenol SULTs, thyroid hormone SULT sulfate and replacing magnesium sulfate with magnesium (SULT1B1), two SULT1Cs (SULT1C2 and SULT1C4), es- chloride) MEM with 10% dialyzed FBS for four hours, were trogen SULT (SULT1E1), dehydroepiandrosterone (DHEA) labeled with 0.25 mL aliquots of the same medium containing SULT (SULT2A1), and a neuronal SULT (SULT4A1), ex- [35S] sulfate (0.3 mCi/mL) plus different concentrations (0, 1, pressed using pGEX-2TK prokaryotic expression system, and 10, 100, 500, 1000 mM) of ethanol. At the end of a 18-h label- two SULT2B1s (designated a and b) expressed using pET23c ing period, the media were collected, spin-filtered to remove expression system, were prepared and purified as previously high-molecular weight [35S] sulfated macromolecules, and sub- described.23–26) jected to thin-layer analysis for ethyl [35S] sulfate-based on the Preparation of HepG2 Cell Lysate Confluent HepG2 procedure described above. cells grown in a 25 cm2 culture flask were lysed in 300 µL of Miscellaneous Methods PAP[35S] was synthesized from 35 a lysis buffer containing 1% (w/w) NP-40, 0.02 M potassium ATP and carrier-free [ S] sulfate using the recombinant human phosphate buffer, pH 7.5, 0.15 M NaCl, 5 mM EDTA, 50 mM bifunctional PAPS synthase and its purity was determined sodium fluoride, and protease inhibitor cocktail.