Rat Hydroxysteroid Sulfotransferase Research Articles

Caffeine induction of sulfotransferases in rat liver and intestine

Sulfotransferases (SULTs) are important phase II drug-metabolizing enzymes. Regulation of SULTs by hormones and other endogenous molecules is relatively well understood, while xenobiotic induction of SULTs is not well studied. Caffeine is one of the most widely consumed psychoactive substances. However, SULT regulation by caffeine has not been reported. In this report, male and female rats were treated with different oral doses of caffeine (2, 10, 50 mg kg⁻¹ per day) for 7 days. Western blot and real-time RT-PCR were used to investigate the changes in SULT protein and mRNA expression following the caffeine treatment. Caffeine induced both rat aryl sulfotransferase (rSULT1A1, AST-IV) and rat hydroxysteroid sulfotransferase (rSULT2A1, STa) in the liver and intestine of female rats in a dose-dependent manner. Caffeine induction of rSULT1A1 and rSULT2A1 in the female rat intestine was much stronger than that in the liver. Although caffeine induced rSULT1A1 significantly in the male rat liver, it did not significantly induce rSULT2A1. In male rat intestine, caffeine significantly induced rSULT2A1. The different SULTs induction patterns in male and female rats suggest that the regulation of rat SULTs by caffeine may be affected by different hormone secretion patterns and levels. Our results suggest that consumption of caffeine can induce drug metabolizing SULTs in drug detoxification tissues.

Interactions of the stereoisomers of alpha-hydroxytamoxifen with human hydroxysteroid sulfotransferase SULT2A1 and rat hydroxysteroid sulfotransferase STa.

Tamoxifen (TAM) is a nonsteroidal antiestrogenic drug that is widely used for the treatment of estrogen receptor-dependent breast cancer. An increased risk of endometrial cancer in some patients treated with TAM has been linked to the metabolic formation of alpha-hydroxytamoxifen (alpha-OHTAM) and its subsequent sulfation. Alpha-OHTAM has been found to be a substrate for rat and human hydroxysteroid sulfotransferases (STa and SULT2A1, respectively). Since stereochemistry plays an important role in the interactions of hydroxysteroid sulfotransferases with their substrates, we have now investigated the interactions of each of the stereoisomers of alpha-OHTAM with highly purified recombinant STa and SULT2A1. Methods for the preparation of the enantiomers of E- and Z-alpha-OHTAM were developed. When each of the four enantiomers was examined with rat STa, E-(+)-alpha-OHTAM was the only substrate for the enzyme, whereas E-(-)-alpha-OHTAM, Z-(+)-alpha-OHTAM, and Z-(-)-alpha-OHTAM were inhibitors of the sulfation of E-(+)-alpha-OHTAM catalyzed by STa. The dissociation constants for the alpha-OHTAM enantiomers indicated that they bound to STa with similar affinity, but only the E-(+)-enantiomer was a substrate. In contrast to the results obtained with rat hydroxysteroid sulfotransferase STa, all enantiomers of alpha-OHTAM were substrates for the human SULT2A1. Moreover, kcat/Km values with SULT2A1 were higher with the Z enantiomers than with the E enantiomers. As a result of the potential for interconversion of the E and Z geometric isomers upon metabolism, the sulfation of the Z isomers may be of greater concern in human tissues than has been previously assumed.

Protective Role of Hydroxysteroid Sulfotransferase in Lithocholic Acid-induced Liver Toxicity

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

4-Hydroxytamoxifen sulfation metabolism.

Tamoxifen (TAM) is an important chemotherapeutic agent for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease. The increased risk of endometrial cancer in women has raised concerns in the use of the drug. Tamoxifen has also been shown to be a potent hepatocarcinogen in rats. The oxidative metabolites of TAM include alpha-hydroxytamoxifen (alpha-OH-TAM) and 4-hydroxytamoxifen (4-OH-TAM). The studies on the sulfation of these metabolites are very limited. It has been reported that alpha-OH-TAM is a substrate for rat hydroxysteroid sulfotransferase a (STa). Our studies on the sulfation of 4-OH-TAM demonstrated that 4-hydroxytamoxifen can be sulfated by human liver and human intestinal cytosols. Human phenol-sulfating sulfotransferase and human estrogen sulfotransferase are the major enzymes for the sulfation of 4-OH-TAM. Human dopamine-sulfating sulfotransferase also has sulfation activity for 4-OH-TAM. In contrast, rat liver and intestine cytosols have no detectable sulfation activity for 4-OH-TAM. The results suggest that the alpha-OH-TAM sulfation pathway leads to bioactivation of TAM, and the 4-OH-TAM sulfation pathway leads to detoxification of TAM. This agrees with the fact that TAM is more toxic for rats than for human beings.

The mutational signature of alpha-hydroxytamoxifen at Hprt locus in Chinese hamster cells.

The anti-oestrogen tamoxifen is very effective in the treatment and prevention of breast cancer. Tamoxifen is not a pure antagonist, but possesses weak oestrogenic activity that may contribute to a slightly increased risk of endometrial cancer. Whilst this can be incorporated into risk-benefit analysis for the use of the drug, residual concerns exist over the exact mechanism of formation of these tumours. Tamoxifen is a potent hepatocarcinogen in the rat, probably via a genotoxic mechanism. Whilst tamoxifen does not appear to cause liver tumours in humans, DNA adducts have been found in endometrial tissue of women receiving the drug. Hence, there is still a need to establish the mechanism of formation of these tumours. We have therefore determined the molecular nature of mutations induced in vitro by alpha-hydroxytamoxifen, the putative proximate genotoxic metabolite, in a mammalian cell line (V79-rHSTa) with stable expression of rat hydroxysteroid sulfotransferase a, which catalyses the further metabolism of alpha-hydroxytamoxifen to its ultimate genotoxic product. DNA sequence alterations were examined at the Hprt gene in 50 mutant clones. Simple base substitutions, mainly GC-->TA transversions, predominated. However, single G:C base pair deletions and partial/complete exon skippings were also observed. All but one of the mutations involved guanine bases on the non-transcribed strand, probably indicating preferential repair of alpha-hydroxytamoxifen-induced guanine adducts from the transcribed strand. Nearest neighbour analysis of the mutations (on the non-transcribed strand) indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours, with adenines or guanines the most frequent 3' neighbours. Many of the mutations occurred at TTGA/G sequences. Three mutational hot spots accounted for 11 GC-->TA transversions and another site for two single G:C base pair deletions. A search for these characteristic mutations in tumour-related genes of treated rats and humans should help in understanding the mechanism(s) of tamoxifen-induced carcinogenicity.

Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa.

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.

Current status of the cytosolic sulfotransferases in the metabolic activation of promutagens and procarcinogens.

Cytosolic sulfotransferases (SULT) catalyze the sulfation of structurally diverse drugs, endogenous compounds and xenobiotics. These reactions involve the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl/amino groups of acceptor molecules. Although sulfate conjugation is generally considered as a detoxication pathway producing more water-soluble and often less toxic metabolites, sulfation of certain classes of compounds produce sufficiently electrophilic metabolites that can covalently bind to cellular macromolecules, DNA and RNA. The important roles of electrophilic sulfate ester metabolites in the metabolic activation, mutagenicity and ultimate carcinogenicity of many xenobiotics have been considerably elucidated. Examples include the class of hydroxymethyl polycyclic aromatic hydrocarbons, allylic alcohols, N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Results obtained by many scientists during the last two decade correlate with a hypothesis that electrophilic sulfate esters may be the major ultimate carcinogenic forms of many, if not most, procarcinogens derived from benzylic/allylic alcohols and hydroxy arylamines. Careful analysis of these results suggest that the activities of human hydroxysteroid sulfotransferase (hHST), and a related form in rat liver, rat hydroxysteroid sulfotransferase a (STa), as well as aryl sulfotransferases both from rat and human liver, account for a substantial portion of the activation of benzylic/allylic alcohols in these species. Moreover, aryl sulfotransferases have also been indicated as the responsible SULT family in the bioactivation of hydroxy arylamines in the liver of different species including human. Molecular cloning of the individual sulfotransferases and expression of these individual forms in heterologous expression systems have allowed us to better understand the role of SULTs in the bioactivation of different procarcinogens and the form of sulfotransferase involved in their bioactivation. Additional structure-activity studies with homogeneous forms of rat liver STa and AST IV have also yielded comparative insight into some of the parameters important in recognition of substrates and inhibitors by these enzymes.

Importance of peri-interactions on the stereospecificity of rat hydroxysteroid sulfotransferase STa with 1-arylethanols.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of polycyclic aromatic hydrocarbons (PAHs) that contain benzylic hydroxyl functional groups. This metabolic reaction is often a critical step in the activation of a hydroxyalkyl-substituted PAH to form an electrophilic metabolite that is capable of forming covalent bonds at nucleophilic sites on DNA, RNA, and proteins. Since hydroxyalkyl-substituted PAHs are often metabolically formed by the stereoselective enzymatic hydroxylation of a benzylic position on an alkyl-substituted PAH, we have investigated the possibility that the sulfation of hydroxyalkyl aromatic hydrocarbons is also stereoselective. Homogeneous preparations of rat hepatic hydroxysteroid (alcohol) sulfotransferase STa were utilized to investigate the stereoselectivity of its catalytic function with the enantiomers of model 1-arylethanols. While only minimal stereoselectivity was observed for the catalytic efficiency of STa with the enantiomers of 1-(2-naphthyl)ethanol and 1-acenaphthenol, the enzyme was stereospecific for (R)-(+)-1-(1-naphthyl)ethanol, (R)-(+)-1-(1-pyrenyl)ethanol, and (R)-(+)-1-(9-phenanthryl)ethanol as substrates. Moreover, (S)-(-)-1-(1-naphthyl)ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)- 1-(9-phenanthryl)ethanol were competitive inhibitors of STa. Structural and conformational analyses of these 1-arylethanols indicated that steric interactions between the substituents on the benzylic carbon and the hydrogen in the peri-position on the aromatic ring system were important determinants of the stereospecificity of the enzyme with these molecules. The findings presented here have implications for the more accurate prediction of the ability of hydroxyalkyl-substituted PAHs to be activated via metabolic formation of electrophilic sulfuric acid esters.

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S. typhimurium. None activated alpha-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

On the nature of rat hepatic and mouse olfactory sulfotransferases

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimeric enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3→q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat ST1C1 in amino acid sequences.

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli. Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA 98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[ a]anthracene and 4 H-cyclopenta l[ def]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[ a]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4 H-cyclopental[ def]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in

Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli . Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA 98. All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[ a ]anthracene and 4 H -cyclopenta l [ def ]chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated. However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz[ a ]anthracene was 27-fold more efficiently activated by the rat enzyme. The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated. The only exception was 4 H -cyclopental[ def ]chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV. We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds. The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Activation of promutagens by endogenous and heterologous sulfotransferases expressed in continuous cell cultures

Various environmental chemicals are metabolised to chemically reactive sulfuric acid esters, which may covalently bind to cellular macromolecules and induce mutations and tumours. This activation pathway is usually not taken into account in external xenobiotic-metabolising systems used in short-term tests. We therefore analysed the abilities of cytosols from mammalian cell lines to activate benzylic alcohols (1-hydroxymethylpyrene and 9-hydroxymethylanthracene) to mutagens detectable in Salmonella typhimurium TA98. No activation was observed in cell lines which are commonly used in mutagenicity and cell transformation assays, and only low activities were found in epithelial cell lines in culture. We have therefore constructed Chinese hamster V79-derived cell lines which stably express a heterologous sulfotransferase, rat hydroxysteroid sulfotransferase a. Cytosol of these cells effectively activated 1-hydroxymethylpyrene and 9-hydroxymethylanthracene to mutagens detected in S. typhimurium. The hepatocarcinogen 6-hydroxymethylbenzo[ a]pyrene induced gene mutations in sulfotransferase-expressing V79-derived cells, whereas it elicited only marginal effects in sulfotransferase-deficient control cells. The new cell lines may allow the detection of novel classes of mutagens, since some externally generated reactive sulfuric acid esters may not readily penetrate target cells due to their short life span and their ionization.

Cloning and expression of cDNA encoding human placental estrogen sulfotransferase

Using two oligoprimers derived from the bovine placental estrogen sulfotransferase sequence, we amplified a probe for human placental estrogen sulfotransferase. Using this probe to screen a human placental cDNA library constructed in λgt11, we isolated a cDNA clone of 1.3 kb encoding human estrogen sulfotransferase. DNA analysis predicts a protein of 295 amino acids with a calculated molecular weight of 34199. Alignment of the amino acid sequence with other sulfotransferases indicates that human placental estrogen sulfotransferase shares 68.6, 68.2 and 65.9% similarity with bovine placental, guinea pig adrenocortical, and rat liver estrogen sulfotransferase, respectively. It shows also 95.6, 57.6, 85.3, and 54.2% similarity to human phenol, human DHEA, rat phenol, and rat hydroxysteroid sulfotransferase, respectively. Transfection of expression vectors encoding human estrogen sulfotransferase and dehydroepiandrosterone (DHEA) sulfotransferase in human adrenal adenocarcinoma SW-13 cells indicates that estrogen sulfotransferase transforms estrone more specifically, whereas DHEA sulfotransferase is more specific for DHEA and pregnenolone.

Molecular Characterization of a Human Aryl Sulfotransferase cDNA

A full-length aryl sulfotransferase cDNA was isolated from a human liver cDNA library. It was 1155 bp long containing a coding region of 885 basepairs encoding a cytosolic protein (Mr 34178 Da) of 295 amino acids. This human cDNA shared 80% homology to the rat aryl sulfotransferase cDNA, 58% to the bovine and rat oestrogen sulfotransferase cDNAs, 53% to the rat hydroxysteroid sulfotransferase cDNA and 51% to the human liver dehydroepiandrosterone sulfotransferase cDNA over its whole 885 bp coding region. The deduced amino acid sequence of this human cDNA was 79% homologous to that of the rat aryl sulfotransferase cDNA and the putative common-substrate binding site motif GXXGXXK of the sulfotransferases has been conserved in this human amino acid sequence. At least two sizes of this human aryl sulfotransferase mRNA were detected in the human liver and lung.