Potential Involvement of Sulfotransferase in the Mechanism of Lamotrigine-induced Skin Rash.

The mechanism of drug-induced skin rash is not well understood. Circumstantial evidence suggests that the covalent binding of a reactive metabolite is involved in the mechanism of most idiosyncratic drug reactions. However, there is a limited quantity of drug metabolizing enzymes in the skin, except for sulfotransferases. It is possible that some drugs are metabolized to reactive sulfate metabolites that are responsible for skin rashes. For example, nevirapine-induced skin rash involves metabolism of nevirapine to 12-hydroxy-nevirapine, which is further metabolized by sulfotransferase in the skin to a reactive benzylic sulfate that covalently binds to proteins. The working hypothesis is that lamotrigine, valdecoxib, and sertraline skin rashes involve the formation of reactive sulfate in the skin. Lamotrigine-N-oxide, hydroxy-valdecoxib, and hydroxy-sertraline were tested as substrates with known human sulfotransferases. Hydroxy-valdecoxib and the benzylic alcohol metabolite of sertraline were not substrates for human sulfotransferases. Therefore, this pathway is presumably not involved in the mechanism by which they cause skin rashes. In contrast, lamotrigine-N-oxide is a substrate for several human sulfotransferases and the sulfate is chemically reactive. Furthermore, lamotrigine-N-sulfate not only alkylates proteins as we described previously but also forms the sulfate of tyrosine, suggesting another possible mechanism for protein modification. This study has further added to the understanding of the potential of the sulfotransferase pathways and protein sulfation to play a role in drug-induced skin rash.

Sulfation of hyperoside by the human cytosolic sulfotransferases (SULTs): impact of genetic polymorphisms on hyperoside-sulfating activity of SULT1C4 allozymes.

This study aimed to identify human cytosolic sulfotransferases (SULTs) that are capable of mediating hyperoside sulfation and examine the impact of genetic polymorphisms on their sulfating activity. Of the thirteen known human SULTs analyzed, five (1A1, 1A2, 1A3, 1C2, and 1C4) displayed sulfating activity toward hyperoside. Kinetic parameters of SULT1C4 that showed the strongest sulfating activity were determined. Ten SULT1C4 allozymes previously prepared were shown to display differential sulfating activities toward hyperoside, revealing clearly the functional impact of SULT1C4 genetic polymorphisms. These findings provided a robust biochemical foundation for further studies on the metabolism of hyperoside by sulfation.

Molecular cloning and characterization of common marmoset SULT1C subfamily members that catalyze the sulfation of thyroid hormones.

Cytosolic sulfotransferase SULT1C subfamily is one of the most flexible gene subfamilies during mammalian evolution. The physiological functions of SULT1C enzymes still remain to be fully understood. In this study, common marmoset (Callithrix jacchus), a promising primate animal model, was used to investigate the functional relevance of the SULT1C subfamily. Gene database search revealed 3 intact SULT1C genes and a pseudogene in its genome. These 4 genes were named SULT1C1, SULT1C2, SULT1C3P, and SULT1C5, according to the sequence homology and gene location. Since SULT1C5 is the orthologous gene for human SULT1C2P, we propose, here, to revisit the designation of human SULT1C2P to SULT1C5P. Purified recombinant SULT1C enzymes showed sulfating activities toward a variety of xenobiotic compounds and thyroid hormones. Kinetic analysis revealed high catalytic activities of SULT1C1 and SULT1C5 for 3,3'-T2. It appears therefore that SULT1C isoforms may play a role in the thyroid hormone metabolism in common marmoset.

Sulfation of Quercitrin, Epicatechin and Rutin by Human Cytosolic Sulfotransferases (SULTs): Differential Effects of SULT Genetic Polymorphisms.

Radix Bupleuri is one of the most widely used herbal medicines in China for the treatment of fever, pain, and/or chronic inflammation. Quercitrin, epicatechin, and rutin, the flavonoids present in Radix Bupleuri, have been reported to display anti-inflammatory, antitumor, and antioxidant biological activities among others. Sulfation has been reported to play an important role in the metabolism of flavonoids. In this study, we aimed to systematically identify the human cytosolic sulfotransferase enzymes that are capable of catalyzing the sulfation of quercitrin, epicatechin, and rutin. Of the thirteen known human cytosolic sulfotransferases, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1C2, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward quercitrin, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A3, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward epicatechin, and six (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A2, cytosolic sulfotransferase 1A3, cytosolic sulfotransferase 1B1, cytosolic sulfotransferase 1C4, and cytosolic sulfotransferase 1E1) displayed sulfating activity toward rutin. The kinetic parameters of the cytosolic sulfotransferases that showed the strongest sulfating activities were determined. To investigate the effects of genetic polymorphisms on the sulfation of quercitrin, epicatechin, and rutin, individual panels of cytosolic sulfotransferase allozymes previously prepared were analyzed and shown to display differential sulfating activities toward each of the three flavonoids. Taken together, these results provided a biochemical basis underlying the metabolism of quercitrin, epicatechin, and rutin through sulfation in humans.

Functional analysis of novel sulfotransferases in the silkworm Bombyx mori.

Sulfoconjugation plays a vital role in the detoxification of xenobiotics and in the metabolism of endogenous compounds. In this study, we aimed to identify new members of the sulfotransferase (SULT) superfamily in the silkworm Bombyx mori. Based on amino acid sequence and phylogenetic analyses, two new enzymes, swSULT ST1 and swSULT ST2, were identified that appear to belong to a distinct group of SULTs including several other insect SULTs. We expressed, purified, and characterized recombinant SULTs. While swSULT ST1 sulfated xanthurenic acid and pentachlorophenol, swSULT ST2 exclusively utilized xanthurenic acid as a substrate. Based on these results, and those concerning the tissue distribution and substrate specificity toward pentachlorophenol analyses, we hypothesize that swSULT ST1 plays a role in the detoxification of xenobiotics, including insecticides, in the silkworm midgut and in the induction of gametogenesis in silkworm ovary and testis. Collectively, the data obtained herein contribute to a better understanding of SULT enzymatic functions in insects.

Investigation of the effects of indoxyl sulfate, a uremic toxin, on the intracellular oxidation level and phagocytic activity using an HL-60-differentiated human macrophage cell model.

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations: CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine.

Impact of SULT1A3/SULT1A4 genetic polymorphisms on the sulfation of phenylephrine and salbutamol by human SULT1A3 allozymes.

OBJECTIVES: Phenylephrine and salbutamol are drugs that are used widely to treat diseases/disorders, such as nasal congestion, hypotension, and asthma, in individuals of different age groups. Human cytosolic sulfotransferase (SULT) SULT1A3 has been shown to be critically involved in the metabolism of these therapeutic agents. This study was carried out to investigate the effects of single nucleotide polymorphisms of human SULT1A3 and SULT1A4 genes on the sulfation of phenylephrine and salbutamol by SULT1A3 allozymes. MATERIALS AND METHODS: Wild-type and SULT1A3 allozymes, prepared previously by site-directed mutagenesis in conjunction with bacterial expression and affinity purification, were analyzed for sulfating activity using an established assay procedure. RESULTS: Purified SULT1A3 allozymes, in comparison with the wild-type enzyme, showed differential sulfating activities toward phenylephrine and salbutamol. Kinetic studies showed further significant variations in their substrate-binding affinity and catalytic activity toward phenylephrine and salbutamol. CONCLUSION: The results obtained showed clearly the differential enzymatic characteristics of SULT1A3 allozymes in mediating the sulfation of phenylephrine and salbutamol. This information may contribute toward a better understanding of the pharmacokinetics of these two drugs in individuals with distinct SULT1A3 and/or SULT1A4 genotypes.

Effects of genetic polymorphisms on the sulfation of dehydroepiandrosterone and pregnenolone by human cytosolic sulfotransferase SULT2A1.

The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of DHEA as well as some other hydroxysteroids such as pregnenolone. The present study was designed to investigate how genetic polymorphisms of the human SULT2A1 gene may affect the sulfation of DHEA and pregnenolone. Online databases were systematically searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non-synonymous coding SNPs identified, seven were selected for further investigation. Site-directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 Escherichia coli cells and purified by glutathione-Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward both DHEA and pregnenolone. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild-type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes.

Effects of human SULT1A3/SULT1A4 genetic polymorphisms on the sulfation of acetaminophen and opioid drugs by the cytosolic sulfotransferase SULT1A3.

Sulfoconjugation has been shown to be critically involved in the metabolism of acetaminophen (APAP), morphine, tapentadol and O-desmethyl tramadol (O-DMT). The objective of this study was to investigate the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the sulfating activity of SULT1A3 allozymes toward these analgesic compounds. Twelve non-synonymous coding SNPs (cSNPs) of SULT1A3/SULT1A4 were investigated, and the corresponding cDNAs were generated by site-directed mutagenesis. SULT1A3 allozymes, bacterially expressed and purified, exhibited differential sulfating activity toward each of the four analgesic compounds tested as substrates. Kinetic analyses of SULT1A3 allozymes further revealed significant differences in binding affinity and catalytic activity toward the four analgesic compounds. Collectively, the results derived from the current study showed clearly the impact of cSNPs of the coding genes, SULT1A3 and SULT1A4, on the sulfating activity of the coded SULT1A3 allozymes toward the tested analgesic compounds. These findings may have implications in the pharmacokinetics as well as the toxicity profiles of these analgesics administered in individuals with distinct SULT1A3 and/or SULT1A4 genotypes.

The critical role of His48 in mouse cytosolic sulfotransferase SULT2A8 for the 7α-hydroxyl sulfation of bile acids.

Members of the cytosolic sulfotransferase (SULT) SULT2A subfamily are known to be critically involved in the homeostasis of steroids and bile acids. SULT2A8, a 7α-hydroxyl bile acid-preferring mouse SULT, has been identified as the major enzyme responsible for the mouse-specific 7-O-sulfation of bile acids. Interestingly, SULT2A8 lacks a conservative catalytic His residue at position 99th. The catalytic mechanism underlying the SULT2A8-mediated 7-O-sulfation of bile acids thus remained unclear. In this study, we performed a mutational analysis in order to gain insight into this yet-unresolved issue. Results obtained revealed two amino acid residues, His48 and Leu99, that are unique to the mouse SULT2A8, but not other SULTs, are essential for its 7-O-sulfating activity toward bile acids. These findings suggested that substitutions of two amino acids, which might have occurred during the evolution of the mouse SULT2A8 gene, endowed mouse SULT2A8 the capacity to catalyze the 7-O-sulfation of bile acids.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.

A reappraisal of the 6-O-desmethylnaproxen-sulfating activity of the human cytosolic sulfotransferases.

In this study, we aimed to obtain a comprehensive account of the human cytosolic sulfotransferases (SULTs) that are capable of sulfating 6-O-desmethylnaproxen (O-DMN), a major metabolite of naproxen. Of the 13 known human SULTs tested, 7 (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT1E1) displayed O-DMN-sulfating activity, when analyzed using an elevated substrate concentration (500 µmol·L-1) together with 14 µmol·L-1 of the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). At 10 µmol·L-1 O-DMN concentration, however, only SULT1A1 and SULT1A3 displayed detectable activity, with the former being nearly 2 orders of magnitude more active than the latter. A pH-dependence study indicated that SULT1A1 exhibited a broad pH optimum spanning pH 5.5-7. Kinetic parameters of the sulfation of O-DMN by SULT1A1 were determined. The production and release of sulfated O-DMN was demonstrated using cultured human HepG2 hepatoma cells and Caco-2 colon carcinoma cells. Moreover, assays using human organ specimens revealed that the O-DMN-sulfating activities present in the cytosols of liver and small intestine (at 502.5 and 497.2 pmol·min-1·(mg protein)-1, respectively) were much higher than those detected for the cytosols of lung and kidney. Taken together, these results provided relevant information concerning the sulfation of O-DMN both in vitro and in vivo.

Updated perspectives on the cytosolic sulfotransferases (SULTs) and SULT-mediated sulfation.

The cytosolic sulfotransferases (SULTs) are Phase II detoxifying enzymes that mediate the sulfate conjugation of numerous xenobiotic molecules. While the research on the SULTs has lagged behind the research on Phase I cytochrome P-450 enzymes and other Phase II conjugating enzymes, it has gained more momentum in recent years. This review aims to summarize information obtained in several fronts of the research on the SULTs, including the range of the SULTs in different life forms, concerted actions of the SULTs and other Phase II enzymes, insights into the structure-function relationships of the SULTs, regulation of SULT expression and activity, developmental expression of SULTs, as well as the use of a zebrafish model for studying the developmental pharmacology/toxicology.

A novel procedure for the assessment of the antioxidant capacity of food components.

Carbonylation, an oxidative modification of the amino group of arginine and lysine residues caused by reactive oxygen species, has emerged as a new type of oxidative damage. Protein carbonylation has been shown to exert adverse effects on various protein functions. Recently, the role of food components in the attenuation of oxidative stress has been the focus of many studies. Most of these studies focused on the chemical properties of food components. However, it is also important to determine their effects on protein functions via post-translational modifications. In this study, we developed a novel procedure for evaluating the antioxidant capacity of food components. Hydrogen peroxide (H2O2)-induced protein carbonylation in HL-60 cells was quantitatively analyzed by using fluorescent dyes (Cy5-hydrazide dye and IC3-OSu dye), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence determination. Among a panel of food components tested, quinic acid, kaempferol, saponin, squalene, trigonelline, and mangiferin were shown to be capable of suppressing protein carbonylation in HL-60 cells. Our results demonstrated that this fluorescence labeling/SDS-PAGE procedure allows for the detection of oxidative stress-induced protein carbonylation with high sensitivity and quantitative accuracy. This method should be useful for the screening of new antioxidant food components as well as the analysis of their suppression mechanism.

Human Cytosolic Sulfotransferase SULT1A3 Mediates the Sulfation of Dextrorphan.

Dextrorphan, an active metabolite of the antitussive dextromethorphan, has been shown to be subjected to sulfation by several zebrafish cytosolic sulfotransferases (SULTs). We were interested in finding out which of the human SULT(s) is(are) capable of catalyzing the sulfation of dextrorphan, and to verify whether sulfation of dextrorphan may occur in cultured human cells and human organ cytosols. Data from the enzymatic assays showed that, of all thirteen known human SULTs, SULT1A3 displayed the strongest dextrorphan-sulfating activity. Cell culture experiments using HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells incubated with [(35)S]sulfate together with varying concentrations of dextrorphan revealed indeed the production and release of [(35)S]sulfated dextrorphan in a concentration-dependent manner. Additionally, significant dextrorphan-sulfating activity was detected in human liver, small intestine and lung cytosols. Taken together, these results provided a biochemical basis for the sulfation of dextrorphan in humans.

Sulfation of 6-hydroxymelatonin, N-acetylserotonin and 4-hydroxyramelteon by the human cytosolic sulfotransferases (SULTs).

1. This study aimed to investigate the involvement of sulfation in the metabolism of 6-hydroxymelatonin (6-OH-Mel), N-acetylserotonin (NAS) and 4-hydroxyramelteon (4-OH-Ram), and to identify and characterize the human cytosolic sulfotransferases (SULTs) capable of sulfating these drug compounds. 2. A systematic analysis using 13 known human SULTs revealed that SULT1A1 displayed the strongest activity in catalyzing the sulfation of 6-OH-Mel and 4-OH-Ram, whereas SULT1C4 exhibited the strongest sulfating-activity towards NAS. pH-dependence and kinetic parameters of these SULT enzymes in mediating the sulfation of respective drug compounds were determined. A metabolic labeling study showed the generation and release of [35S]sulfated 6-OH-Mel, NAS and 4-OH-Ram by HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells labeled with [35S]sulfate in the presence of these drug compounds. Cytosols of human lung, liver, kidney and small intestine were examined to verify the presence of 6-OH-Mel-, NAS- and 4-OH-Ram-sulfating activity in vivo. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-OH-Mel-, NAS- and 4-OH-Ram-sulfating activities than those of lung and kidney. 3. Collectively, these results provided a molecular basis for the metabolism of 6-OH-Mel, NAS and 4-OH-Ram through sulfation.

Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis.

Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo.

Sulfation of benzyl alcohol by the human cytosolic sulfotransferases (SULTs): a systematic analysis.

The aim of the present study was to identify human cytosolic sulfotransferases (SULTs) that are capable of sulfating benzyl alcohol and to examine whether benzyl alcohol sulfation may occur in cultured human cells as well as in human organ homogenates. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1 SULT1A2, SULTA3, and SULT1B1 are capable of mediating the sulfation of benzyl alcohol. The kinetic parameters of SULT1A1 that showed the strongest benzyl alcohol-sulfating activity were determined. HepG2 human hepatoma cells were used to demonstrate the generation and release of sulfated benzyl alcohol under the metabolic settings. Moreover, the cytosol or S9 fractions of human liver, lung, kidney and small intestine were examined to verify the presence of benzyl alcohol sulfating activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs): A systematic analysis.

Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.