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 metabolite in adult human body, has been implicated as a biomarker for 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 , 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 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. HepG2 cell as previously described.28) The PAP[35S] synthesized was lysate prepared was cleared by centrifugation at 25000×g for adjusted to the required concentration and a specific activity 30 min at 4°C, and the supernatant collected was used in the of 15 Ci/mmol at 1.4 mM by the addition of unlabelled PAPS. SULT assay. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was SULT Assay The ethanol-sulfating activity of the re- performed on 12% polyacrylamide gels using the method of combinant human SULTs was assayed using PAP[35S] as the Laemmli.29) Protein determination was based on the method sulfonate group donor. The PAP[35S] synthesized was adjusted of Bradford,30) with bovine serum albumin as the standard. to the required concentration and a specific activity of 15 Ci/ Trypan blue exclusion cell viability assay was performed 31) mmol at 1.4 mM by the addition of cold PAPS. The standard based on an established procedure. MTT cell viability assay assay mixture, in a final volume of 20 µL, contained 50 mM was carried out according to the manufacture’s protocol and 35 of Mops buffer at pH 7.0, 1 mM DTT, 14 µM PAP[ S], and absorbance at 570 nm was measured using a SpectraMax M2 the substrate, ethanol. Stock solutions of ethanol, prepared in microplate reader (Molecular Devices, CA, U.S.A.). water at 10 times the final concentration in the assay mixture, were used in the enzymatic assay. A control with water alone RESULTS AND DISCUSSION was also prepared. The reaction was started by the addition of the SULT enzyme (1 µg), allowed to proceed for 10 min at The present study was designed to identify the major 37°C, and terminated by placing the thin-walled tube contain- human SULTs that are responsible for the sulfation of ethanol ing the assay mixture on a 100°C heating block for 3 min. and to investigate the sulfation of ethanol under the metabolic The precipitates were cleared by centrifugation at 14000×g setting and in human tissues. It should be pointed out that, in for 3 min, and the supernatant was subjected to the analysis contrast to a previous study on ethanol sulfation that had been of [35S]sulfated product using the previously established TLC performed using recombinant human SULTs expressed in Sal- procedure with n-butanol–isopropanol–88% formic acid–water monella typhimurium,16) purified recombinant human SULTs, (3 : 1 : 1 : 1; by volume) as the solvent system.27) Upon comple- with purities greater than 95% as judged by SDS-PAGE,23–26) tion of TLC, an autoradiograph was taken from the TLC plate were used in the present study. to reveal radioactive spots corresponding to ethyl [35S]sulfate. Differential Sulfating Activities of the Human SULTs 2182 Vol. 35, No. 12

Fig. 1. pH-Dependence of the Sulfating Activity of Human SULT1A1 (A) and SULT1C4 (B) with Ethanol as Substrate The enzymatic assays were carried out under standard assay conditions as described in Materials and Methods, using different buffer systems as indicated. Data shown represent calculated mean±S.D. derived from three independent analyses. toward Ethanol To identify the SULT enzyme(s) that are in S. typhimurium demonstrated a proportionate increase in involved in the sulfation of ethanol, eleven purified human the ethanol-sulfating activity with increasing concentrations 16) SULTs were examined for sulfating activity with ethanol as (ranging 10–1000 mM) of ethanol. In our study, dramatically substrate. Activity data compiled in Table 1 indicated that lower ethanol-sulfating activity was detected for SULT1A1, four (SULT1C2, SULT2B1a, SULT2B1b, and SULT4A1) of SULT1C4, SULT1A3, and SULT1A2, when the concentration the eleven SULTs displayed no detectable activities. The other of ethanol tested was 10 mM (data not shown). These results seven SULTs (SULT1A1, SULT1A2, SULT1A3, SULT1B1, therefore indicated that human SULTs have a low affinity for

SULT1C4, SULT1E1, and SULT2A1) exhibited differential ethanol, with Km values possibly well over 10 mM. Moreover, sulfating activity toward ethanol. Among these seven SULTs, in a concentration-dependent experiment, the sulfating activ- SULT1A1 displayed the strongest ethanol-sulfating activity (at ity of SULT1A1, SULT1C4, SULT1A3, and SULT1A2 in- 4.07 nmol/min/mg enzyme), followed by SULT1C4, SULT1A3, creased with increasing concentrations (ranging 10–1000 mM) and SULT1A2 (at 2.42, 1.84, 1.60 nmol/min/mg enzyme, re- of ethanol, indicating a typical Michaelis–Menten kinetics and spectively). These results indicated that these latter four SULT showing no evidence of substrate inhibition or activation. To enzymes may primarily be responsible for the sulfation of further characterize the SULT-mediated sulfation of ethanol, ethanol in humans. It is noted that in a previous study using the pH-dependence of the sulfation reactions was analyzed. In recombinant human SULTs expressed in S. typhimurium, the pH-dependence study, the two strongest ethanol-sulfating four SULTs which were found to display ethanol-sulfating SULTs, SULT1A1 and SULT1C4, were analyzed. As shown in activities were, in decreasing order, SULT1A2, SULT1C4, the Fig. 1, SULT1A1 displayed a distinct pH optimum at 6.0 SULT1A3, and SULT1B1.16) These latter results, however, with ethanol as a substrate. In contrast, SULT1C4 showed a might have been affected by undetermined levels of the ex- broad pH optimum spanning pH 6.5 to 9.5, with a maximum pression of different SULTs in S. typhimurium cells and/or sulfating activity at 6.5. the inhibitory/stimulatory effects of compounds present in Generation and Release of Ethyl [35S]Sulfate by HepG2 the crude cell homogenates used as the source of the SULT Cells Labeled with [35S]Sulfate in the Presence of Ethanol enzyme(s) in the assay for sulfation of ethanol. It is noted that HepG2 human hepatoma cells (ATCC HB-8065) were used to SULT2A1, while previously referred to as alcohol sulfotrans- investigate whether sulfation of ethanol occurs in the meta- ferase,32) displayed much lower ethanol-sulfating activity than bolic setting. Confluent HepG2 cells grown in individual wells did members of the phenol sulfotransferase family including of a 24-well plate were labeled with [35S]sulfate in sulfate-free SULT1A1, SULT1A2, SULT1A3, and SULT1C4. While the medium containing different concentrations of ethanol. TLC rationale for the low ethanol-sulfating activity of SULT2A1 analysis of the labeling media collected at the end of an 18-h remains to be clarified, previous studies have demonstrated labeling period revealed indeed the presence of ethyl [35S]- that SULT2A1 displayed much lower activity and affinity sulfate in a concentration-dependent manner. As shown in Fig. toward ethanol than other alcohols including butanol, hexa- 2A, a significant amount of ethyl [35S]sulfate was detected in 32,33) nol, cyclohexylmethanol, and benzyl alcohol. Moreover, medium containing 1 mM ethanol, which continued to increase SULT2A1 showed higher catalytic efficiency toward primary with higher concentrations of ethanol. These results indicated aliphatic alcohols with increasing carbon chain length.33) It clearly that sulfation of ethanol may occur in cells under the is therefore possible that the short carbon chain length of metabolic setting. Since previous studies indicated that etha- ethanol may be the reason for its not being a preferable nol may exert cytotoxicity,34,35) cell viability assay based on substrate for SULT2A1. Another issue is with regard to the trypan blue exclusion or MTT reduction was performed to affinity of the SULT enzymes toward ethanol as a substrate. verify the viability of HepG2 cells used in the metabolic label- Previous study using recombinant human SULTs expressed ing experiments. As shown in Fig. 2B, trypan blue exclusion December 2012 2183

Table 1. Specific Activities of the Human SULTs with Ethanol as Substratea)

Specific activity (nmol/min/mg)

SULT1A1 SULT1A2 SULT1A3 SULT1B1 SULT1C4 SULT1E1 SULT2A1

Ethanol 4.07±0.05 1.60±0.01 1.84±0.01 0.13±0.01 2.42±0.06 0.30±0.01 0.41±0.03 Standardb) 36.54±0.21 21.93±0.13 34.55±0.55 3.50±0.01 33.53±0.76 45.83±0.30 47.70±0.51 (pNP) (pNP) (DA) (pNP) (pNP) (E2) (DHEA)

a) Data represent mean±S.D. derived from three determinations. The concentration of the substrate used in the assay mixture was 100 mM. b) Standard refers to represen- tative substrate for each of the seven SULTs. p-Nitrophenol (pNP) was used for SULT1A1, SULT1A2, SULT1B1, and SULT1C4. Dopamine (DA), 17β-estradiol (E2), and dehydroepiandrosterone (DHEA) were used for SULT1A3, SULT1E1, and SULT2A1, respectively.

Fig. 2. Analysis of the Ethyl [35S]Sulfate Generated and Released by HepG2 Human Hepatoma Cells Labeled with [35S]Sulfate in the Presence of Ethanol (A) The figure shows the autoradiograph taken from the TLC plate used for TLC analysis of the labeling media. Confluent HepG2 cells were labeled with [35S]sulfate for 18 h in the presence of 6 different concentrations (0, 1, 10, 100, 500, or 1000 mM) of ethanol. The figure is representative of three independent experiments. (B) Cell viability assay on HepG2 cells treated by ethanol. Cells were treated with 6 different concentrations (0, 1, 10, 100, 500, or 1000 mM) of ethanol for 18 h and followed by trypan blue exclusion assay or MTT assay. Data shown represent calculated mean±S.D. (%) of the unstained cell numbers (Trypan blue exclusion assay) and the absor- bance measured against 0 mM ethanol concentration (MTT assay) derived from three independent analyses. Statistical significance versus the control, 0 mM of ethanol, are indicated by * p<0.05 or ** p<0.01, as analyzed by one-way ANOVA with Dunnett’s test. (C) Ethanol-sulfating activity of HepG2 cell lysate. Assays were performed using 1, 10, 100, 500, or 1000 mM ethanol as substrate. Data shown represent calculated mean±S.D. derived from three independent analyses. N.D. refers to the specific activity below the detection limit (estimated to be ca. 1.00 pmol/min/mg). assay demonstrated 95, 87, and 81% cell viability when the majority of HepG2 cells, nevertheless, survived and were able 35 cells were incubated with 100, 500 or 1000 mM of ethanol, to generate and release ethyl [ S] sulfate under the conditions whereas MTT assay showed 101, 97, and 73% of cell viability adopted in the metabolic labeling experiments. To verify the at the same concentrations of ethanol. Therefore, although ethanol-sulfating activity in the cells, HepG2 cell lysate was higher concentrations of ethanol showed cytotoxic effects, the prepared and tested in the SULT assay. As shown in Fig. 2C, 2184 Vol. 35, No. 12

tissues/organs including brain, skin, and placenta.41–43) Taking the tissue distributions of the major ethanol-sulfating SULTs into consideration, the sulfation of ethanol may possibly occur in not only liver and small intestine but also other tissues/or- gans. For SULT1C4, another major ethanol-sulfating SULT, its expression was mainly detected in the fetal tissues/organs in- cluding lung, liver, small intestine, and kidney.14,24) In the fetal tissues, the expression of SULT1A1 and SULT1A3, at levels comparable to or higher than those in postnatal and adult tis- sues,13–15) has also been detected in brain, lung, liver, intestine, and kidney. Interestingly, ethyl sulfate has been detected in the placenta and fetal tissues, as well as neonatal meconium of pregnant woman.8–10) It is worthwhile mentioning that previ- ous studies showed alcohol dehydrogenase activity was pres- ent at very low level (less than 3–4%) in fetal liver and did not reach the adult level until 5 years of age.11,12) Although there Fig. 3. Ethanol-Sulfating Activities of Human Tissues Cytosol Frac- is little available information on the ontogeny of aldehyde tions from Lung, Liver, and Small Intestine dehydrogenases in humans, the levels of its mRNA expres- The sulfating activity assays with 50, 100, or 500 mM ethanol as a substrate were sion in mouse livers had been reported to be undetectable or carried out using human tissues cytosol or S9 fractions. Data shown represent calculated mean±S.D. derived from three independent analyses. N.D. refers to the extremely low at prenatal stage and gradually increased until specific activity below the detection limit (estimated to be ca. 1.00 pmol/min/mg). 45 d after birth.44) It is therefore an intriguing issue whether sulfate conjugation may constitute an important route for the metabolism and detoxification of ethanol early on during the no ethanol-sulfating activity was detected at lower concen- developmental process. trations of ethanol (1, 10 mM). At concentrations higher than To summarize, our results revealed the four major human 100 mM, a proportionate increase in ethanol-sulfating activ- ethanol-sulfating SULTs, SULT1A1, SULT1A2, SULT1A3, ity was detected. It is noted that previous studies have dem- and SULT1C4, the occurrence of the sulfation of ethanol onstrated the expression of major ethanol-sulfating SULTs, under the metabolic setting, and the presence of ethanol-sul- SULT1A1, SULT1A2, and SULT1A3, in HepG2 cells.36,37) fating activity in human lung, liver, and small intestine. More Moreover, in relation to the production of ethyl sulfate in vivo, work is warranted in order to fully elucidate the biochemical a previous study demonstrated that ethyl sulfate, with concen- and physiological relevance of the sulfation of ethanol. trations ranging from 1 to 7 µM, was detected in blood samples of drinkers with blood alcohol concentration ranging from Acknowledgment This work was supported in part by a 5) 0.02 to 0.08% (equivalent to 4.3 to 17.4 mM). 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