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Doxorubicin is a chemotherapeutic drug widely utilized in cancer treatment. An enzyme critical to doxorubicin metabolism is the cytosolic sulfotransferase (SULT) SULT1C4. This study investigated the functional impact of SULT1C4 single nucleotide polymorphisms (SNPs) on the sulfation of doxorubicin by SULT1C4 allozymes. A comprehensive database search was performed to identify various SULT1C4 SNPs. Ten nonsynonymous SULT1C4 SNPs were selected, and the corresponding cDNAs, packaged in pGEX-2TK expression vector, were generated via site-directed mutagenesis. Respective SULT1C4 allozymes were bacterially expressed and purified by affinity chromatography. Purified SULT1C4 allozymes, in comparison with the wild-type enzyme, were analysed for sulphating activities towards doxorubicin and 4-nitrophenol, a prototype substrate. Results obtained showed clearly differential doxorubicin-sulphating activity of SULT1C4 allozymes, implying differential metabolism of doxorubicin through sulfation in individuals with distinct SULT1C4 genotypes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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.

Sulphated cholesterol, like its unsulphated counterpart, is known to be biologically active and serves a myriad of biochemical/physiological functions. Of the 13 human cytosolic sulphotransferases (SULTs), SULT2B1b has been reported as the main enzyme responsible for the sulphation of cholesterol. As such, SULT2B1b may play the role as a key regulator of cholesterol metabolism. Variations in the sulphating activity of SULT2B1b may affect the sulphation of cholesterol and, consequently, the related physiological events. This study was designed to evaluate the impact of the genetic polymorphisms on the sulphation of cholesterol by SULT2B1b. Ten recombinant SULT2B1b allozymes were generated, expressed, and purified. Purified SULT2B1b allozymes were shown to display differential cholesterol-sulphating activities, compared with the wild-type enzyme. Kinetic studies revealed further their distinct substrate affinity and catalytic efficiency toward cholesterol. These findings showed clearly the impact of genetic polymorphisms on the cholesterol-sulphating activity of SULT2B1b allozymes, which may underscore the differential metabolism of cholesterol in individuals with different SULT2B1b genotypes.

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.

The cytosolic sulphotransferase SULT1C3 remained the most poorly understood human SULT. The SULT1C3 gene has been shown to contain alternative exons 7 and 8, raising the question concerning their evolutionary origin and implying the generation of multiple SULT1C3 variants. Two SULT1C3 splice variants, SULT1C3a and SULT1C3d, were investigated to verify the impact of alternative C-terminal sequences on their sulphating activity. Sequence homology and gene location analyses were performed to verify the orthology of the SULT1C3 gene. The SULT1C3 gene appears to be present only in humans and other primates, but alternative exons 7b and 8b share high degrees of homology with corresponding regions of rodent SULT1C1 genes, implying their evolutionary origin being from a defunct human SULT1C1 gene. Purified recombinant SULT1C3a and SULT1C3d were analyzed for sulphating activities toward a variety of endogenous and xenobiotic compounds. While SULT1C3a displayed weaker activities and strict substrate specificity toward hydroxyl-chlorinated biphenyls, SULT1C3d exhibited broader substrate specificity toward bile acids and thyroid hormones as well as hydroxyl-chlorinated biphenyls. Molecular docking simulation suggested that Tyr249 and Met257 may play an important role in substrate recognition by SULT1C3d. Alternative splicing of exons 7 and 8 sequences resulted in differential catalytic properties of SULT1C3 variants., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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.

Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo. This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3-glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0-8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.

Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.

Sulforaphane is a compound widely present in consumed vegetables, particularly broccoli. Previous studies have demonstrated that sulforaphane has many physiological effects including anti-cancer, anti-oxidation, and detoxification. In this study, we found that sulforaphane inhibited melanogenesis and tyrosinase expression. The inhibitory effect of 5 microM sulforaphane on melanogenesis was determined to be equivalent to that of 100 microM arbutin. In addition, sulforaphane induced phosphorylated extracellular signal-regulated kinase (ERK) and inhibited phosphorylated p38. It has been reported that the phosphorylated mitogen-activated protein (MAP) kinase family (ERK and p38) controls tyrosinase expression. Our data indicate that sulforaphane inhibited melanogenesis and tyrosinase expression by affecting the phosphorylated MAP kinase family. These findings indicate that sulforaphane might be an effective skin-whitening agent.

By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT-PCR. While human SULT6B1 was specifically expressed in kidney and testis, mouse SULT6B1 was detected in brain, heart, kidney, thymus, lung, liver and testis. Further studies are needed in order to clarify the role of SULT6B1 in the metabolism of thyroxine and possibly some xenobiotics in mouse.

Protein tyrosine sulfation is emerging as a widespread post-translational modification in multicellular eukaryotes. The responsible enzyme, named tyrosylprotein sulfotransferase (TPST), catalyzes the sulfate transfer from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues of proteins. Two distinct TPSTs, designated TPST-1 and TPST-2, had previously been identified. In the present study, we cloned human TPST-1 and TPST-2 expressed and characterized the recombinant enzymes using peptide substrates. These enzymes displayed distinct acidic pH optima and stimulatory effects of Mn(2+). Additionally, the activity of TPST-2, but not TPST-1, was stimulated in the presence of Mg(2+). Compared with TPST-2, TPST-1 displayed considerably lower K(m) and V(max) for the majority of the tested peptide substrates, implying their differential substrate specificity. Quantitative real-time PCR analysis showed that although the two TPSTs were co-expressed in all 20 human tissues examined, the levels of expression of TPST-1 and TPST-2 varied significantly among different tissues. These latter findings may imply distinct physiological functions of TPST-1 and TPST-2.

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.

Cytosolic sulfotransferases (STs), traditionally viewed as Phase II drug-metabolizing or detoxifying enzymes, are increasingly being implicated in the metabolism of endogenous biologically-active molecules. Except for studies on changes in their levels of expression and activity in the early stage of development in mammals, very little is known about how these enzymes are regulated. In this study, the regulatory effects of divalent metal cations on the activity of human cytosolic STs were quantitatively evaluated. Results obtained indicate that all nine human cytosolic STs examined are partially or completely inhibited/stimulated by the ten divalent metal cations tested at 10 mM concentration. Compared with the other metal cations, the inhibitory or stimulatory effect of Mg2+ and Ca2+ on the activities of the human cytosolic STs appeared to be relatively smaller. Concentration-dependent effects of the divalent metal cations were further examined. The IC50 or EC50 values determined for different divalent metal cations were mostly above their normal physiological concentration ranges. In a few cases, however, IC50 values close to the physiological concentrations of certain divalent metal cations were observed. Using the monoamine (M)-form phenol ST (PST) as a model, it was demonstrated that the K(m) for dopamine changed only slightly with increasing concentrations of Cd2+, whereas the V(max) was dramatically decreased.