Hansruedi Glatt

Sulfotransferases in the bioactivation of xenobiotics

Conjugation of xenobiotics is often associated with detoxification. However, this traditional view is one-sided. In particular, numerous compounds are known that are metabolized to chemically reactive metabolites via sulfation (O-sulfonation). This can be rationalized by the fact that the sulfate group is electron-withdrawing and may be cleaved off heterolytically in appropriate molecules, thus leading to the formation of a strongly electrophilic cation. The heterologous expression of sulfotransferases in indicator cells of standard mutagenicity tests has substantially improved the accessibility of this activation pathway. The use of this technology is important, since many reactive sulfate conjugates only show strong toxicological effects if they are generated directly within the indicator cell, due to their insufficient penetration of cell membranes. Xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a superfamily (SULT). Eleven distinct human SULT forms are known, which strongly differ in their tissue distribution and their substrate specificity. Common functionally relevant genetic polymorphisms of the transcribed region are known for two of the forms, SULT1A1 and 1A2. Studies using recombinant test systems demonstrate that many promutagens are activated with high selectivity by an individual SULT form. Pronounced differences in promutagen activation were detected between the different human forms, including their allelic variants, and also between orthologous SULTs from different species. Therefore, SULTs may be involved in the individual genetic disposition, species differences, and organotropisms for toxicological effects of chemicals. Activation by SULTs differs from other activation pathway in its cyclic nature: reaction of a sulfuric acid ester with water usually regenerates the hydroxylated compound, which becomes available for a new cycle of activation. SULT-mediated reactivation may even occur if another initial reactive species, e.g. an epoxide, has reacted with water.

Vitamin E activates gene expression via the pregnane X receptor.

Tocopherols and tocotrienols are metabolized by side chain degradation via initial omega-oxidation and subsequent beta-oxidation. omega-Oxidation is performed by cytochrome P450 (CYP) enzymes which are often regulated by their substrates themselves. Results presented here show that all forms of Vitamin E are able to activate gene expression via the pregnane X receptor (PXR), a nuclear receptor regulating a variety of drug metabolizing enzymes. In HepG2 cells transfected with the human PXR and the chloramphenicol acetyl transferase (CAT) gene linked to two PXR responsive elements, CAT activity was most strongly induced by alpha- and gamma-tocotrienol followed by rifampicin, delta-, alpha- and gamma-tocopherol. The inductive efficacy was concentration-dependent; its specificity was underscored by a lower response when cotransfection with PXR was omitted. Up-regulation of endogenous CYP3A4 and CYP3A5 mRNA was obtained by gamma-tocotrienol, the most potent activator of PXR, with the same efficacy as with rifampicin. This points to a potential interference of individual forms of Vitamin E with the metabolism and efficacy of drugs.

The suggested physiologic aryl hydrocarbon receptor activator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in humans

Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.

Potent Inhibition of Estrogen Sulfotransferase by Hydroxylated Metabolites of Polyhalogenated Aromatic Hydrocarbons Reveals Alternative Mechanism for Estrogenic Activity of Endocrine Disrupters

Polyhalogenated aromatic hydrocarbons (PHAHs), such as polychlorinated dibenzo-p-dioxins and dibenzofurans, polybrominated diphenylethers, and bisphenol A derivatives are persistent environmental pollutants, which are capable of interfering with reproductive and endocrine function in birds, fish, reptiles, and mammals. PHAHs exert estrogenic effects that may be mediated in part by their hydroxylated metabolites (PHAH-OHs), the mechanisms of which remain to be identified. PHAH-OHs show low affinity for the ER. Alternatively, they may exert their estrogenic effects by inhibiting E2 metabolism. As sulfation of E2 by estrogen sulfotransferase (SULT1E1) is an important pathway for E2 inactivation, inhibition of SULT1E1 may lead to an increased bioavailability of estrogens in tissues expressing this enzyme. Therefore, we studied the possible inhibition of human SULT1E1 by hydroxylated PHAH metabolites and the sulfation of the different compounds by SULT1E1. We found marked inhibition of SULT1E1 by various PHAH-OHs, in particular by compounds with two adjacent halogen substituents around the hydroxyl group that were effective at (sub)nanomolar concentrations. Depending on the structure, the inhibition is primarily competitive or noncompetitive. Most PHAH-OHs are also sulfated by SULT1E1. We also investigated the inhibitory effects of the various PHAH-OHs on E2 sulfation by human liver cytosol and found that the effects were strongly correlated with their inhibitions of recombinant SULT1E1 (r = 0.922). Based on these results, we hypothesize that hydroxylated PHAHs exert their estrogenic effects at least in part by inhibiting SULT1E1-catalyzed E2 sulfation.

The apparent ubiquity of epoxide hydratase in rat organs.

Abstract Using the recently developed sensitive assay with [3H] benzo [a] pyrene 4,5-oxide as substrate, epoxide hydratase was shown to be present in 26 rat (Sprague-Dawley) organs and tissues investigated. Only blood showed no detectable activity, which indicates that the low enzyme activity found in some organs is not due to the presence of blood components in the tissues. In earlier studies with a less sensitive assay, epoxide hydratase activity was detected only in rat liver and kidney but not in organs such as muscle, spleen, heart and brain. Epoxide hydratase was also measured in 6 organs of the mouse (NMRI). The distribution pattern was quantitatively quite different in the two species. The sp. act. in the rat were in the order liver > testis > kidney > lung > intestine ∼- skin. In the mouse, very surprisingly, testis had the highest specific epoxide hydratase activity. Moreover, the order of sp. act. in the mouse organs was remarkably different from that in the rat, namely testis > liver > lung > skin > kidney > intestine. The fact that the sp. act. in kidney was much lower than in lung or skin is most striking. Pretreatment of rats with Aroclor 1254 (a mixture of polychlorinated biphenyls) increased the epoxide hydratase activity in the liver to 175 per cent of the control level. However, the enzyme activity in the 13 extrahepatic tissues investigated was not significantly changed. In organs possessing sufficiently high enzyme levels, epoxide hydratase activity was also measured with styrene oxide as substrate. The ratio of the sp. act. of the two substrates was very similar in rat liver, kidney, lung and lestis. This supports the assumption that in these organs a single enzyme is responsible for the hydration of both substrates—as was earlier shown by several methods for the rat liver.

Identification and localization of soluble sulfotransferases in the human gastrointestinal tract

Soluble SULTs (sulfotransferases) are important in the regulation of messenger molecules and the elimination of xenobiotics. However, sulfo-conjugation of various substrates can also lead to the formation of reactive metabolites that may induce cancer and cause other damage. The aim of the present study was to identify the SULT forms expressed in the human gastrointestinal tract, especially the colon and rectum (common sites for cancer), and to determine their cellular localization. Normal colonic or rectal tissue, resected with tumours, was obtained from 39 subjects. For comparison, we additionally studied one to four samples from stomach, jejunum, ileum, cecum and liver. SULTs were detected by immunoblotting, immunohistochemistry and measurement of enzyme activities. SULT1A1, 1A3 and 1B1 were found in all parts of the gastrointestinal tract, often exceeding levels in liver (where these forms were present at high, undetectable and low levels respectively). They were predominantly localized in differentiated enterocytes. SULT1E1 and 2A1 were only detected in liver, jejunum, ileum and cecum. SULT1C1 was readily found in stomach, but was negligible elsewhere. SULT1A2 was present at low levels in individual samples. The remaining forms were not detected with the limitation that only high levels could be recognized with the antisera used. In conclusion, SULTs are abundant in the gastrointestinal tract of man. We suspect that they are involved in the presystemic elimination of bioactive food-borne components, including aglycones released by gut microbiota, as well as the bioactivation of some procarcinogens.

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.

Pharmacogenetics of soluble sulfotransferases (SULTs)

Soluble sulfotransferases (SULTs) transfer the sulfo group from the cofactor 5’-phosphoadenosine-3’-phosphosulfate (PAPS) to nucleophilic sites of relatively small acceptor molecules including various hormones and numerous xenobiotics. Sulfo conjugation of xenobiotics can lead to the formation of polar, excretable products as well as reactive, potentially mutagenic and carcinogenic metabolites. Ten SULT genes encoding 11 proteins have been identified in the human. They differ in substrate specificity and tissue distribution. Genetic polymorphisms have been detected in all human SULT genes. The functional significance of any polymorphisms that do not affect the amino acid sequence has not yet been studied. Non-synonymous single-nucleotide exchanges have been observed in SULT1A1, 1A2, 1B1, 1C1, 1C2 and 2A1. Functional consequences have primarily been explored using cDNA-expressed alloenzymes. Furthermore, an Arg213His polymorphism in SULT1A1 has a strong influence on the level of enzyme protein and activity in platelets, which have been widely used for phenotyping. Compared to other xenobiotic-metabolizing enzymes, only few studies have been conducted on associations of SULT genotypes with diseases and other health-related parameters. Statistically significant associations were observed between the SULT1A1 genotype (Arg213His) and age, obesity and certain neoplasias (mammary, pulmonary, esophageal and urothelial cancer). However, these findings require corroboration and specification. The association with neoplasias appears to be complex and varies between subgroups. This is not surprising, as SULTs are involved in the activation of some carcinogens, in the inactivation of other carcinogens, and the regulation of many hormones. It is important to study these functions of SULTs in more detail and to take into account the corresponding environmental and endogenous exposures in epidemiological studies.

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

Abstract A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [3H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol(trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).

Dual role of epoxide hydratase in both activation and inactivation of benzo(a)pyrene

The effect of epoxide hydratase upon the mutagenicity of benzo(a)pyrene was investigated using two Salmonella typhimurium strains (TA 1537 and TA 98). These two bacterial strains were found to differ characteristically in their susceptibility to different mutagens biologically produced from benzo(a)pyrene providing a diagnostic tool to investigate which types of mutagenic metabolites were produced in various metabolic situations. The results showed that the pattern of mutagenic metabolites produced by microsomes from methylcholanthrene-treated mice was very different from that produced by microsomes from phenobarbital-treated or untreated mice. However in all cases at least two mutagenic metabolites were produced. Epoxide hydratase was very efficient at reducing the mutagenic effect when benzo(a)pyrene was activated by microsomes from untreated or phenobarbital-treated mice. However, when microsomes from methylcholanthrene-treated mice were used the effect of hydratase depended upon the benzo(a)pyrene concentration. At low concentrations the mutagenicity was increased by addition of epoxide hydratase and decreased by inhibition of the hydratase. At high concentrations the reverse was true. These findings indicate that when microsomes from untreated and phenobarbital-treated mice were used the main contributors to the mutagenicity were simple epoxides (or compounds arising non-enzymically from them). The activation of dihydrodiols must, however, contribute to a significant extent when microsomes from methylcholanthrene-treated mice were used. Thus the role of epoxide hydratase was determined by the monooxygenase form present in the microsomes in the activating system.ZusammenfassungDie Rolle der Epoxidhydratase wurde untersucht in bezug auf die Mutagenität von Benzo(a)pyren. Benzo(a)pyren wurde mit Lebermikrosomen aktiviert. Mutagene wurden festgestellt anhand der Reversion der his−Salmonella typhimurium-Stämme TA 1537 und TA 98. Die beiden Stämme wurden sehr unterschiedlich durch verschiedene mutagene Benzo(a)pyren-Metabolite rückmutiert. Es zeigte sich, daß das Muster der mutagenen Metabolite, die durch Mikrosomen von Methylcholanthren-behandelten Mäusen aus Benzo(a)pyren gebildet wurden, sehr verschieden war vom Muster bei Aktivierung durch Mikrosomen von Kontroll-oder von Phenobarbital-behandelten Mäusen. Jedoch trugen in allen drei Fällen wenigstens zwei verschiedene mutagene Metabolite signifikant zur Mutagenität bei. Epoxidhydratase reduzierte sehr effektiv die Mutagenität, wenn Benzo(a)pyren durch Mikrosomen von Kontroll-oder von Phenobarbital-behandelten Mäusen aktiviert wurde. Wenn jedoch Mikrosomen von Methylcholanthren-behandelten Tieren verwendet wurden, war der Effekt der Epoxidhydratase stark von der Benzo(a)pyren-Konzentration abhängig. Bei niedriger Konzentration erhöhte Zugabe von Epoxidhydratase und erniedrigten Epoxidhydratasehemmstoffe die Mutagenität. Bei hohen Konzentrationen wurde das Umgekehrte festgestellt.Diese Befunde wurden dahingehend interpretiert, daß bei der Aktivierung mit Mikrosomen von unbehandelten und von Phenobarbital-induzierten Mäusen einfache Epoxide (oder Substanzen, die nicht-enzymatisch daraus gebildet wurden) hauptsächlich für die Mutagenität verantwortlich waren, daß dagegen Mutagene, die über Dihydrodiole gebildet wurden, bedeutend zur Mutagenität beitrugen, wenn Mikrosomen von Methylcholanthren-behandelten Mäusen verwendet wurden.Die Rolle der Epoxidhydratase, ob aktivierend oder inaktivierend, wird demnach bestimmt durch die Form der Monooxygenase, die an der Aktivierung beteiligt ist.