Aryl Sulfotransferase Activity Research Articles

Transcriptomic-based toxicological investigations of ethanol to human umbilical vein endothelial cells.

Ethanol has a complex effect on the cardiovascular system in humans, but the systemic effects of ethanol to endothelial cells were rarely investigated. In this study, we exposed human umbilical vein endothelial cells (HUVECs) to 5- or 50-mM ethanol and performed transcriptomics to investigate the systemic effects of ethanol. While these concentrations of ethanol did not significantly affect HUVEC viability, 5-mM ethanol significantly upregulated and downregulated 59 and 73 genes, respectively, whereas 50-mM ethanol significantly upregulated and downregulated 50 and 80 genes, respectively. Totally, 37 genes were shared by the two concentrations of ethanol. The most significantly altered gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway after 5-mM ethanol treatment were nucleic acid binding (GO:0003676) and Herpes simplex virus 1 infection (ko05168), respectively, whereas the most significantly altered GO term and KEGG pathway by 50-mM ethanol treatment were aryl sulfotransferase activity (GO:0004062) and chemical carcinogenesis (ko05204). We further verified that ethanol treatment downregulated the mRNA levels of CD38 molecule (CD38), ORAI calcium release-activated calcium modulator 2 (ORAI2), cysteinyl leukotriene receptor 2 (CYSLTR2), key genes involved in calcium signaling pathway (ko04020), as well as integrin subunit alpha 2 (ITGA2), and cAMP responsive element binding protein 3 like 2 (CREB3L2), key genes involved in PI3K-Akt signaling pathway (ko04151). The results from this study suggested that ethanol could induce systemic effects and alter signaling pathways in HUVECs.

Identification of interspecific differences in phase II reactions: determination of metabolites in the urine of 16 mammalian species exposed to environmental pyrene.

Interspecific differences in xenobiotic metabolism are a key to determining relative sensitivities of animals to xenobiotics. However, information on domesticated livestock, companion animals, and captive and free-ranging wildlife is incomplete. The present study evaluated interspecific differences in phase II conjugation using pyrene as a nondestructive biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Polycyclic aromatic hydrocarbons and their metabolites have carcinogenic and endocrine-disrupting effects in humans and wildlife and can have serious consequences. The authors collected urine from 16 mammalian species and analyzed pyrene metabolites. Interspecific differences in urinary pyrene metabolites, especially in the concentration and composition of phase II conjugated metabolites, were apparent. Glucuronide conjugates are dominant metabolites in the urine of many species, including deer, cattle, pigs, horses, and humans. However, they could not be detected in ferret urine even though the gene for ferret Uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) 1A6 is not a pseudogene. Sulfate conjugates were detected mainly in the urine of cats, ferrets, and rabbits. Interestingly, sulfate conjugates were detected in pig urine. Although pigs are known to have limited aryl sulfotransferase activity, the present study demonstrated that pig liver was active in 1-hydroxypyrene sulfation. The findings have some application for biomonitoring environmental pollution.

Aryl Sulfotransferase from Haliangium ochraceum: A Versatile Tool for the Sulfation of Small Molecules

AbstractSulfation is an important molecular modification that regulates essential cellular processes and is also implicated in numerous pathological processes. The enzymes responsible for this reaction in living organisms are sulfotransferases. The gene Hoch_5094 from Haliangium ochraceum is annotated as a putative sulfotransferase. The arylsulfotransferase codified by this gene (HocAST) was expressed heterologously in E. coli and showed aryl sulfotransferase activity. Circular dichroism analysis of HocAST showed a main α/β secondary structure that agrees with the overall structure of other cytosolic sulfotransferases. Interestingly, HocAST was able to use both p‐nitrophenyl sulfate and 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as sulfuryl donors contrary to that of aryl sulfate sulfotransferase, which cannot use PAPS as a donor. Regarding the specificity towards the acceptor, HocAST has shown quite a wide scope and was able to accept several mono‐ and dihydroxylated phenols and other phosphorylated compounds as substrates.

Pentachlorophenol inhibits micronuclei induction by 2-acetylaminofluorene but not by thioacetamide

Our study examined the capacity of pentachlorophenol (PCP) to inhibit the ability of 2-acetylaminofluorene (2-AAF) and thioacetamide (TAA) to induce micronuclei in mouse bone marrow cells in vivo. 2-AAF (5.6 mg/kg) and TAA (60 mg/kg) were administered intra-peritoneally (i.p.) to Mus musculus males (BALB/c), and the frequencies of polychromatic erythrocytes with micronuclei (PCE-MN) 24 h after injection were analyzed. Treatment with 2-AAF or TAA resulted in high PCE-MN frequencies in comparison with untreated and negative controls (19.9 and 21.6‰, respectively, versus ≈3‰). Pretreatment with a single PCP dose (44 mg/kg) 24 h prior to the 2-AAF administration virtually eliminated micronuclei formation by 2-AAF, although it had no inhibitory effect on TAA-induced micronuclei. Animals receiving cyclophosphamide (CP) served as positive control. Since PCP is known to inhibit arylsulfotransferase (AST) activity, which is involved in 2-AAF activation, this mechanism most likely produced the results with PCP and 2-AAF. Our results also are consistent with a different pathway involved in TAA induction of micronuclei, one that is not inhibited by PCP.

Arylsulfotransferase from Clostridium innocuum—A new enzyme catalyst for sulfation of phenol‐containing compounds

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.

Purification, cloning, and characterization of an arylsulfotransferase from the anaerobic bacterium Eubacterium rectale IIIH.

A bacterium, Eubacterium rectale IIIH, which possessed arylsulfotransferase (ASST) activity was isolated from human feces. The ASST gene (astA) was cloned and the corresponding protein partially characterized. This gene shows only moderate homology to the previously sequenced ASST genes of Klebsiella and Enterobacter, which are very closely related to each other.

Nonlinearities in 2-Acetylaminofluorene Exposure Responses for Genotoxic and Epigenetic Effects Leading to Initiation of Carcinogenesis in Rat Liver

The dose responses for several effects of low-level limited exposures to 2-acetylaminofluorene (AAF) in the livers of male Fischer 344 rats were measured and a subsequent phenobarbital tumor promotion regimen was used to manifest initiation of carcinogenesis. Three doses over a 10-fold range yielding cumulative total exposures of 0.126, 0.42, and 1.26 mmol AAF/kg body weight were achieved by daily intragastric instillation for up to 12 weeks with interim terminations. This was followed by 24 weeks administration of 500 ppm phenobarbital (PB) in the diet to promote liver tumor development. At 12 weeks at the end of AAF administration, all exposures produced adducts in liver DNA, measured by 32P postlabeling, and the level of adducts increased with exposure, except that the high exposure did not produce a dose proportional increase. Measurement of arylsulfotransferase activity, a key enzyme in the metabolic activation of AAF, revealed that in livers from the high exposure animals, the enzyme was inhibited. To assess for toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was quantified immunohistochemically at 12 weeks. The area of the zone was reduced in the high exposure group and there was a trend to reduction in relationship to exposure. The two lower exposures to AAF produced no increase in cell proliferation, whereas the high exposure resulted in a marked increase, about 8-fold over controls. Initiation was assessed by induction of hepatocellular altered foci (HAF) that expressed the placental form of glutathione S-transferase. AAF induced HAF in the high exposure group, 9-fold at 8 weeks and 170-fold at 12 weeks compared to controls. In rats maintained on PB for 24 weeks after exposure, the multiplicity of HAF increased in controls and comparably in the low and mid exposure groups, but remained at the about the same high level in the high exposure group. The high exposure produced a substantial incidence of benign neoplasms by 12 weeks, and with promotion by 36 weeks, all rats developed hepatocellular neoplasia. In the mid exposure group, only one adenoma occurred at 36 weeks in 17 rats, while in the low exposure group, no liver tumor occurred in 23 rats. Thus, these findings document nonlinearities for some of the effects of AAF, with supralinear effects at the high exposure for cell proliferation and induction of HAF, and a no-observed-effect level for induction of promotable liver neoplasms at the lowest cumulative exposure of 0.126 mmol/kg, in spite of the formation of DNA adducts. We conclude that the effects of this DNA-reactive hepatocarcinogen leading to initiation exhibit nonlinearities and possible thresholds.

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2-acetylaminofluorene (2AAF)-induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80-90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV activity. In the livers of rats administered only 2AAF (group 1), a general pattern of overall downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action.

Glucose and sulfate conjugations of phenol, β-naphthol and 3-hydroxybenzo[a]pyrene by the American lobster ( Homarus americanus)

Little is known of the potential for conjugation of phenolic compounds by lobsters. Studies were designed to determine the ability of American lobster tissues to conjugate the phenolic compounds phenol, β-naphthol and 3-hydroxybenzo[a]pyrene (3-OH BaP). Aryl sulfotransferase (ST) activity was present in the cytosolic fraction of the antennal gland (AG), but not hepatopancreas (HP), whereas UDP-glucosyltransferase (UDPGT) activity was found in musomal fractions of both AG and HP. We observed differences between male and female lobsters in their ability to conjugate phenols. For example, in males the apparent K m for PAPS of AG β-naphthol ST was 9.0 ± 4.2 μM (mean ± SD, n = 5) and V max was 77 ± 7 pmol/min/mg protein, whereas in females K m was 62 ± 3 μM ( n = 3) and V max was 254 ± 92 pmol/min/mg protein ( P < 0.05). The β-naphthol UDPGT exhibited different pH optima in HP (10.5) and AG (7.5) musomes, suggesting that more than one form of β-naphthol UDPGT exists in the American lobster. With few exceptions, K m values for the phenolic substrates were in the μM range for both UDPGT and ST, whereas K m values for the cosubstrates were lower for PAPS than for UDP-glucose.

Self-catalyzed irreversible inactivation of rat hepatic aryl sulfotransferase IV by Nhydroxy-2-acetylaminofluorene

Rat hepatic aryl sulfotransferase IV catalyzes the sulfonation of the hepatocarcinogen, N-hydroxy-2-acetylaminofluorene. The resulting reactive N-O-sulfate ester is believed to be the ultimate carcinogenic species responsible for the induction of hepatic neoplasia. Previous studies have shown that dietary administration of either 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene to rats is accompanied by a rapid decline in hepatic aryl sulfotransferase activity in vivo. In the present study, preincubation of purified rat hepatic aryl sulfotransferase IV with N-hydroxy-2-acetylaminofluorene resulted in rapid, time-dependent enzyme inactivation. This in vitro inactivation was not reversed by dialysis or gel filtration. Inclusion of excess nucleophile, methionine, resulted in considerable but not complete protection from inactivation. The inactivation was PAPS dependent and blocked by the sulfotransferase inhibitor, pentachlorophenol. The above observations and the apparent pseudo first-order kinetics observed suggest that the inactivation was in part mechanism based. Mechanism-based inactivation of the aryl sulfotransferases has not been previously reported. Furthermore, the results of the present study indicate that the previously reported in vivo decline in rat hepatic aryl sulfotransferase activity may be attributable in part to enzyme inactivation by its own reactive product.

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 ± 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.

Further characterization of the ability of hepatocarcinogens to lower rat liver aryl sulfotransferase activity.

Aryl sulfotransferase (AST) activity in rat liver is thought to be a primary pathway in the bio-activation of various hepatocarcinogens to forms which act as ultimate carcinogens in chemical hepatocarcinogenesis. In an effort to understand the significance of rapid and sustained decreases in liver AST that accompany dietary administration of hepatocarcinogens and to further assess its relationship to carcinogenic processes, we determined the abilities of various xenobiotics known to be hepatocarcinogens or non-hepatic carcinogens to lower AST activity. We also determined whether the co-administration of the AST enzyme inhibitor, pentachlorophenol, with hepatocarcinogens will abrogate the lowering of AST activity caused by hepatocarcinogens which do not utilize AST for bio-activation versus hepatocarcinogens which can utilize AST. Among carcinogens tested thus far, we have found the AST activity of liver cytosols to be lowered by the hepatocarcinogens 2-acetylaminofluorene, ethionine, 3'-methyl-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1, diethylnitrosamine and benzidine, but not by the non-hepatic carcinogens 2-acetylaminophenanthrene or 3-methylcholanthrene. Pentachlorophenol reversed activity losses when co-administered with all carcinogens which lowers AST activity with the exception of ethionine and thioacetamide. We suggest that AST activity lowering is relatively specific for liver carcinogens and involves two different mechanisms.

The role of intestinal flora in metabolism of phenolic sulfate esters

Arylsulfotransferase activity was found in the feces of human (14.74 ± 2.674) and rat (7.37 ± 1.126 μmol/hr/g wet feces). In the case of the rat, arylsulfotransferase activity was markedly and rapidly decreased by the treatment with antibiotics mixture, but restored to the original activity 3 weeks after stopping the administration of antibiotics. The excretion of the sulfate esters derived from p-nitrophenylsulfate was enhanced by the administration of acetaminophen but not by the treatment with antibiotics. Furthermore, in rats treated with antibiotics, inorganic sulfate excretion was severely decreased. When only acetaminophen was administered, the excretion of acetaminophen- O-sulfate showed a 10–15% decrease in rats treated with antibiotics compared with conventional rat.

Stimulation of arylsulfotransferase activity by progestins in human endometrium in vitro.

The effect of progestin on the rate of estradiol (E2) sulfurylation in human endometrium was studied in two culture systems: 1) in intact endometrial tissue fragments and 2) in isolated endometrial epithelial glands and stromal cells. Human endometrial tissue fragments were cultured in medium in the presence and absence of progesterone (P) for 24-48 h. The arylsulfotransferase activity was determined in the cytosol of the tissue homogenate by measuring the rate of conversion of E2 to E2-3 sulfate (E2S) in the presence of an excess amount of substrate and cofactor (adenosine-3'-phosphate-5'-phosphosulfate). The enzyme activity was found to be stimulated in the sample cultured with P. The increase in activity correlated with the dose of P and period of culture. Stimulation of endometrial estradiol dehydrogenase activity by P, which has been reported in a series of previous publications, was also evident in the current study. Human endometrial glandular epithelial and stromal cells were cultured separately in medium in the presence and absence of medroxyprogesterone acetate (MPA). The cultured cells were incubated with E2 (0.4-0.6 nM) for 2 h. The formation of estrogen sulfates, [estrone sulfate (E1S) and E2S] was greatly increased in all the proliferative glandular epithelial cells cultured with MPA. However, the rate of sulfurylation was not appreciably affected by MPA in stromal cells and in glandular epithelial cells from secretory endometrium. The oxidation of E2 in these cultured cells was also influenced by P but not to the extent of sulfurylation. These results indicate that arylsulfotransferase in endometrium originates from glandular epithelial cells and can be stimulated by progestin.