Human cytosolic sulphotransferases: genetics, characteristics, toxicological aspects.

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.

Sulfotransferases: genetics and role in toxicology.

The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.

The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.

Sulfation of thyroid hormone by estrogen sulfotransferase.

Sulfation is one of the pathways by which thyroid hormone is inactivated. Iodothyronine sulfate concentrations are very high in human fetal blood and amniotic fluid, suggesting important production of these conjugates in utero. Human estrogen sulfotransferase (SULT1E1) is expressed among other tissues in the uterus. Here we demonstrate for the first time that SULT1E1 catalyzes the facile sulfation of the prohormone T4, the active hormone T3 and the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2) with preference for rT3 approximately 3,3'-T2 > T3 approximately T4. Thus, a single enzyme is capable of sulfating two such different hormones as the female sex hormone and thyroid hormone. The potential role of SULT1E1 in fetal thyroid hormone metabolism needs to be considered.

Activation of propane 2-nitronate to a genotoxicant in V79-derived cell lines engineered for the expression of rat hepatic sulfotransferases.

2-Nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound has been attributed to a sulfotransferase-mediated formation of DNA-reactive species from the anionic form of 2-NP, propane 2-nitronate (P2N). Several observations have suggested that sulfotransferases (SULTs) 1A1 and/or 1C1 may be important in the activation of P2N to a genotoxicant in rat liver, but a definite proof is lacking. In order to identify the sulfotransferase(s) of rat liver that are capable of activating P2N, we have investigated the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of rat hepatic sulfotransferases. Genotoxicity was assessed by measuring the induction of DNA repair synthesis. 1-Hydroxymethylpyrene (HMP), which is metabolically activated by most sulfotransferases, served as a positive control. Neither P2N nor HMP induced DNA repair in the parental V79-MZ cells, which do not show any sulfotransferase activity. P2N was also inactive in V79-rHSTa and V79-rHST20 cells, which express specific hydroxysteroid sulfotransferases. By contrast, a clear and concentration-dependent induction of repair synthesis by P2N was observed in V79-rPST-IV and V79-rST1C1 cells, which express rat SULT1A1 and SULT1C1, respectively. HMP was genotoxic in all sulfotransferase-expressing cell lines. Acetone oxime (AO), the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results corroborate the essential role of sulfotransferases in the metabolic activation of P2N to genotoxic products and identify two rat sulfotransferases which are capable of catalyzing the activation step.

Chiral inversion of 1-hydroxyethylpyrene enantiomers mediated by enantioselective sulfotransferases.

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity for S-1-HEP, whereas hHST strongly preferred the R-enantiomer. The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (beta-estradiol) and hHST (dehydroepiandrosterone). Moreover, after a 30-min incubation of S-1-HEP with hEST, 95% of the recovered alcohol showed the R-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems.

Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures. Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his- Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells. Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected with N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene (in the presence of co-expressed rat cytochrome P450 1A2), hycanthone, 1'-hydroxysafrole, alpha-hydroxytamoxifen and various benzylic alcohols derived from polycyclic aromatic hydrocarbons. In several cases, it was critical that the reactive sulfuric acid conjugates were formed directly within the indicator cells, owing to the inefficient penetration of cell membranes. In other cases, spontaneous benzylic substitution reactions with medium components, such as halogenide ions or amino acids, led to secondary, membrane-penetrating reactive species. Different sulfotransferases, including related forms from rat and human, substantially differed in their substrate specificity towards the investigated promutagens. It is known that some sulfotransferases are expressed with high tissue and cell type specificities. This site-dependent expression together with the limitations in the distribution of reactive sulfuric acid conjugates may explain organotropic effects of compounds activated by this metabolic pathway.

Expression of human estrogen sulfotransferase in Salmonella typhimurium: differences between hHST and hEST in the enantioselective activation of 1-hydroxyethylpyrene to a mutagen.

Various human sulfotransferases (hP-PST, hM-PST, hHST) and rat sulfotransferases (rPST-IV, rHSTa) have already been expressed in Ames' Salmonella strains (in particular in TA1538). Now a further strain, TA1538-hEST, which expresses the human estrogen sulfotransferase (hEST), has been constructed. This strain activated the primary benzylic alcohol 1-hydroxymethylpyrene (1-HMP) and the secondary benzylic alcohol 1-hydroxyethylpyrene (1-HEP) to mutagens. Human sulfotransferases hEST and hHST both activated 1-HEP, but they differed substantially in their enantioselectivity for this compound.