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

doi:10.1016/0926-6917(95)90002-0

European Journal of Pharmacology: Environmental Toxicology and Pharmacology

Volume 293, Issue 2, 1 July 1995, Pages 173-181

https://doi.org/10.1016/0926-6917(95)90002-0 Get rights and content

Abstract

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 4H-cyclopental[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 4H-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.

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Our results implied the significant functional difference on the isoenzymes of human SULT2A1 in various animal species. In addition to responsible for sulfation of hydroxysteroids, SULT2A1 also plays a vital role in the metabolism of xenobiotics36–39. That is, the activity levels of SULT2A1 regulate the system and local exposure of endogenous and exogenous substances.
Human cytosolic sulfotransferase 2A1 (SULT2A1) is an important phase II metabolic enzyme. The detection of SULT2A1 is helpful for the functional characterization of SULT2A1 and diagnosis of its related diseases. However, due to the overlapping substrate specificity among members of the sulfotransferase family, it is difficult to develop a probe substrate for selective detection of SULT2A1. In the present study, through characterization of the sulfation of series of bufadienolides, arenobufagin (AB) was proved as a potential probe substrate for SULT2A1 with high sensitivity and specificity. Subsequently, the sulfation of AB was characterized by experimental and molecular docking studies. The sulfate-conjugated metabolite was identified as AB-3-sulfate. The sulfation of AB displayed a high selectivity for SULT2A1 which was confirmed by in vitro reaction phenotyping assays. The sulfation of AB by human liver cytosols and recombinant SULT2A1 both obeyed Michaelis-Menten kinetics, with similar kinetic parameters. Molecular docking was performed to understand the interaction between AB and SULT2A1, in which the lack of interaction with Met-137 and Tyr-238 of SULT2A1 made it possible to eliminate substrate inhibition of AB sulfation. Finally, the probe was successfully used to determine the activity of SULT2A1 and its isoenzymes in tissue preparations of human and laboratory animals.
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It is well established that various endocrine disrupting compounds (EDCs) can inhibit human estrogen sulfotransferase (SULT1E1). In this study, we investigate murine SULT1E1 inhibition in vitro and in silico and compare this to data for the human enzyme. 34 potential EDCs were screened for their ability to inhibit both murine and human SULT1E1 and IC₅₀ values were determined for 14 of the inhibitory EDCs. Only estrone, dienestrol and enterolactone showed significant differences in affinity between the human and murine SULT1E1. Extensive molecular modelling was performed using molecular dynamics (MD) simulations. During the MD simulations the ligands moved away from the catalytically active position, something which was not observed when simulating the unit cell of the crystal structure. This finding suggests that catalytically inactive binding modes, other than the one observed in the crystal structures, are possible in SULT1E1. The ligands stayed longer in the catalytically active position in mSULT1E1, which is likely a result of simultaneous hydrogen bond formation on both sides of the binding pocket, which does not seem to be possible in hSULT1E1. The ligands in the human protein moved to a sub-pocket near the entrance of the active site, which offers hydrogen bond formation possibilities with Asp22 and Lys85 as well as favourable hydrophobic interactions. The ligands moved more randomly in mSULT1E1. These observations offer a possible explanation for the substrate inhibition only observed in hSULT1E1.
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The transformed bacterial strains were grown overnight in the presence of ampicillin (100 μg/ml) to reinforce the maintenance of the recombinant plasmid. Bacterial cytosol was prepared by ultrasonication and dialyzed for 4 h against a 100-fold excess of buffer (150 mM KCl in 10 mM sodium phosphate buffer, pH 7.4) [41,43]. The use of Salmonella cytosol has advantages over the use of purified SULTs.
Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17β-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [³⁵S]3′-phosphoadenosine 5′-phosphosulfate (PAPS) or radiolabeled substrate [³H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined (K_m = 6.4 ± 0.8 nM and V_max = 158 ± 19 pmol/min/μg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis–Menten kinetics involving substrate inhibition. IC₅₀ values have been determined for eight known SULT1E1 inhibitors or competing substrates (17β-estradiol, 17α-estradiol, genistein, 17α-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.
Sulfotransferases and acetyltransferases in mutagenicity testing: Technical aspects
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Identification of the critical form may be strived for by comparing the effect of preparations from tissues differing in the forms expressed or by using selective inhibitors. Likewise, individual cDNA‐expressed and/or purified enzymes may be employed (Glatt et al., 1995). Tissue levels of PAPS and acetyl‐CoA are low.
Sulfotransferases (SULTs) and N‐acetyltransferases (NATs) mediate the terminal activation step of various mutagens and carcinogens. Target cells of standard in vitro mutagenicity tests do not express any endogenous SULTs. NATs are expressed in some cells, but may not reflect the substrate specificity of human NATs. External activating systems usually lack the cofactors for these enzymes. Upon addition of the cofactor, the ultimate mutagen may be formed, but especially sulfo conjugates—anions—may not reliably penetrate into the target cells. This chapter presents methods used to incorporate these enzyme systems into in vitro mutagenicity test systems and to identify the critical human forms. The method of choice is direct expression of the enzymes in target cells. We present procedures on how this can be reached in bacteria and in mammalian cell lines in culture. Furthermore, genetically manipulated mouse models are a very promising perspective for answering open questions.
Human cytosolic sulphotransferases: Genetics, characteristics, toxicological aspects
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Cytosolic sulphotransferases transfer the sulpho moiety from the cofactor 5′-phosphoadenosine-3′-phosphosulphate (PAPS) to nucleophilic groups of xenobiotics and small endogenous compounds (such as hormones and neurotransmitters). This reaction often leads to products that can be excreted readily. However, other sulpho conjugates are strong electrophiles and may covalently bind with DNA and proteins. All known cytosolic sulphotransferases are members of an enzyme/gene superfamily termed SULT. In humans, 10 SULT genes are known. One of these genes encodes two different enzyme forms due to the use of alternative first exons. Different SULT forms substantially differ in their substrate specificity and tissue distribution. Genetic polymorphisms have been described for three human SULTs. Several allelic variants differ in functional properties, including the activation of promutagens. Only initial results are available from the analysis of SULT allele frequencies in different population groups, e.g. subjects suffering from specific diseases and corresponding controls.
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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.

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^☆: Dedicated to Prof. Christian Barth, founding director of the Deutsche Institut für Ernährungsforschung, on the occasion of his 60th birthday.

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

Abstract

Chem.-Biol. Interact.

Trends Pharmacol. Sci.

Arch. Biochem. Biophys.

J. Steroid. Biochem. Mol. Biol.

Toxicol. Lett.

Mutation Res.

Chem.-Biol. Interact.

Biochem. Biophys. Res. Commun.

Chem. Biol. Interact.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Chem.-Biol. Interact

Hydroxylation and conjugation at the benzylic carbon atom: a possible mechanism of carcinogenic activation for some methyl-substituted aromatic hydrocarbons

Purification and characterization of a rat liver sulfotransferase activating benzylic alcohols to mutagens

Naunyn-Schmied. Arch. Pharmacol.

Aryl sulfotransferase IV catalyzed sulfation of 1-naphthalenemethanol

Synthesis of 11 benzylic sulfate esters, their bacterial mutagenicity and its modulation by chloride, bromide and acetate anions

Polycycl. Arom. Comp.