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.     

Chenodeoxycholic acid-mediated activation of the farnesoid X receptor negatively regulates hydroxysteroid sulfotransferase

Hydroxysteroid sulfotransferase catalyzing bile acid sulfation plays an essential role in protection against lithocholic acid (LCA)-induced liver toxicity. Hepatic levels of Sult2a is up to 8-fold higher in farnesoid X receptor-null mice than in the wild-type mice. Thus, the influence of FXR ligand (chenodeoxycholic acid (CDCA) and LCA) feeding on hepatic Sult2a expression was examined in FXR-null and wild-type mice. Hepatic Sult2a protein content was elevated in FXR-null and wild-type mice fed a LCA (1% and 0.5%) diet. Treatment with 0.5% CDCA diet decreased hepatic Sult2a to 20% of the control in wild-type mice, but increased the content in FXR-null mice. Liver Sult2a1 (St2a4) mRNA levels were reduced to 26% in wild-type mice after feeding of a CDCA diet, while no decrease was observed on Sult2a1 mRNA levels in FXR-null mice after CDCA feeding. A significant inverse relationship (r(2)=0.523) was found between hepatic Sult2a protein content and small heterodimer partner (SHP) mRNA level. PCN-mediated increase in Sult2a protein levels were attenuated by CDCA feeding in wild-type mice, but not in FXR-null mice. Human SULT2A1 protein and mRNA levels were decreased in HepG2 cells treated with the FXR agonists, CDCA or GW4064 in dose-dependent manners, although SHP mRNA levels were increased. These results suggest that SULT2A is negatively regulated through CDCA-mediated FXR activation in mice and humans.     

Sex-dependent regulation of hepatic cytochrome P-450 by DDT.

Dichlorodiphenyltrichloroethane (DDT) is a well-known inducer of microsomal monooxygenase systems in rodent liver. However, little information is available on its effects on the sex-dependent regulation of CYPs preferentially affected. Therefore, our objective was to evaluate the effects of DDT on the sexual expression pattern of some hepatic P-450 isozymes. Single doses of technical DDT (0, 0.1, 1, 5, 10, or 100 mg/kg body wt) were administered by gavage to Wistar rats. The effects on CYPs 1A1, 2B11/2B2, 2C11, 2E1, 3A1, and 3A2, were assessed 24 h later by means of CYP protein content determined by Western blotting and/or enzyme activities participating in alkoxyresorufin and pnitrophenol metabolism. The highest dose induced 18-fold the expression of CYP3A2 in female rats without producing significant induction (< 3-fold) in males. The effects on this isozyme, which is not normally expressed in females, suggest that DDT is able to modulate sexual metabolic dimorphism, as 3A2 expression is androgen dependent. DDT did not significantly alter CYP3A1 in males, suggesting that DDT is not a pure phenobarbital (PB)-type inducer. The effects on CYP2B1/2B2 protein (19-fold) and associated enzyme activities indicated that males had a lower response threshold than females, but that the latter were able to reach a higher relative induction. The preferential induction of CYPs 2B and 3A by DDT in a sex-related manner suggest that CYP regulation could play an important role in endocrine disruption.     

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 liver cytosolic hydroxysteroid sulfotransferase (sulfotransferase a) catalyzing the formation of reactive sulfate esters from carcinogenic polycyclic hydroxymethylarenes

Female rat liver cytosol contained at least three sulfotransferases (STs) that were separable on a DEAE-Sephadex A-50 column and transformed the carcinogen 5-hydroxymethylchrysene (5-HCR) to the potent mutagen 5-HCR sulfate. The STs also catalyzed sulfation of dehydroepiandrosterone (DHA), a typical substrate for hydroxysteroid STs. Of these three isozymes, the one (STa) with the highest 5-HCR-sulfating activity was isolated and purified (100-fold) as a homogeneous protein, in 15% yield, by successive column chromatography on agarose modified with 3'-phosphoadenosine 5'-phosphate as an affinity ligand and on Sephadex G-100. Purified STa was classified as a hydroxysteroid ST because the 5-HCR- and DHA-sulfating activities were inseparable from each other throughout the purification steps. Sulfation of 5-HCR by purified STa was competitively inhibited by DHA. STa also catalyzed sulfation of other potent carcinogens, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a], anthrocene, to produce sulfate esters with high reactivity and mutagenicity. However, STa had no activity with 4-nitrophenol, a typical substrate for phenol STs, or with N-hydroxy-2-acetylaminofluorene. STa had a pl value of 6.4 and existed on a gel filtration column as a homooligomer of a subunit protein with Mr 30,500, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of STa was as follows: Pro-Asp-Tyr-Thr-Trp-Phe-Glu-Gly-Ile-Pro-Phe-Pro-Ala-Phe-Gly-Ile- Pro-Lys-Glu-Thr-. Immunoblot analysis of female and male rat liver cytosol, carried out by using rabbit antiserum raised against the purified enzyme STa and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicated that the female liver contained a much higher level of the enzyme than did the male liver. The marked sex difference in STa level was in good accordance with the previous demonstration that cytosol from the liver of female rats catalyzed sulfation of 5-HCR to a greater extent than did cytosol from the liver of male rats.     

Enantioselectivity of human hydroxysteroid sulfotransferase ST2A3 with naphthyl-1-ethanols.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of several endogenous steroids and many hydrophobic xenobiotic alcohols. The substrate stereoselectivities of sulfotransferases may be critically important in determining their overall roles in metabolism of drugs, carcinogens, and other xenobiotics. In the present work, stereoselectivity of the human hydroxysteroid sulfotransferase ST2A3 (also variously named as SULT2A1 or human DHEA-ST) was examined through analysis of its catalytic activities with the enantiomers of 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol. The kcat/Km value for sulfation of the R-(+)-enantiomer of 1-naphthyl-1-ethanol catalyzed by ST2A3 was 3.3 min-1mM-1, whereas the S-(-)-enantiomer was not a substrate for the enzyme. S-(-)-1-naphthyl-1-ethanol did however interact with ST2A3 as an inhibitor of the sulfation of dehydroepiandrosterone. This substrate stereospecificity was not present with the enantiomers of 2-naphthyl-1-ethanol, since both were substrates for the enzyme. Such differences between the sulfation of 1- and 2-naphthyl-1-ethanol are consistent with the importance of steric interactions between the ethanol group and a hydrogen atom at the peri-position (C8) on the naphthyl ring in 1-naphthyl-1-ethanol that combine with the topology of the enzyme's active site to determine stereospecificity.     

Hydroxylated polychlorinated biphenyls are substrates and inhibitors of human hydroxysteroid sulfotransferase SULT2A1

Polychlorinated biphenyls (PCBs) are important persistent environmental contaminants. PCBs can be metabolically converted to their hydroxylated metabolites (OHPCBs), and in recent years, these OHPCBs have been observed to inhibit human sulfotransferases (SULTs) such as the phenol SULTs (SULT family-1) involved in the metabolism of estrogen and various other endogenous and xenobiotic phenols. In the present study, we have investigated the hypothesis that OHPCBs interact with family 2 hydroxysteroid (alcohol) SULTs (e.g., human SULT2A1), enzymes that are physiologically important for the metabolic transformations of several key endogenous hydroxysteroids as well as xenobiotic alcohols. We have examined the interactions of three OHPCBs with purified recombinant human SULT2A1 (also known as either human DHEA-ST or ST2A3). These studies with SULT2A1 were carried out on 4'-hydroxy-2,5-dichlorobiphenyl (4'-OH PCB 9), 4-hydroxy-2',3,5-trichlorobiphenyl (4-OH PCB 34), and 4'-hydroxy-2,3',4,5'-tetrachlorobiphenyl (4'-OH PCB 68). Our results showed that 4-OH PCB 34 and 4'-OH PCB 68 were substrates for SULT2A1, and 4-OH PCB 34 exhibited substrate inhibition similar to that seen with the physiological substrate dehydroepiandrosterone (DHEA). Although the sulfation of 4-OH PCB 34 and 4'-OH PCB 68 represents a potential metabolic route for these compounds, these OHPCBs may also compete with other xenobiotic substrates as well as endogenous substrates for SULT2A1. The third OHPCB studied, 4'-OH PCB 9, was not a substrate for SULT2A1 but was an inhibitor of the enzyme. Thus, the interactions of OHPCBs with human SULT2A1 represent both a potential route of metabolism and a possible source of interference with sulfation reactions catalyzed by this enzyme.     

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.     

Sex-dependent control of murine emotional-affective behaviour in health and colitis by peptide YY and neuropeptide Y

BACKGROUND AND PURPOSE

Peptide YY (PYY) and neuropeptide Y (NPY) are involved in regulating gut and brain function. Because gastrointestinal inflammation is known to enhance anxiety, we explored whether experimental colitis interacts with genetic deletion (knockout) of PYY and NPY to alter emotional-affective behaviour.

EXPERIMENTAL APPROACH

Male and female wild-type, NPY (NPY^−/−), PYY (PYY^−/−) and NPY^−/−; PYY^−/− double knockout mice were studied in the absence and presence of mild colitis induced by ingestion of dextran sulphate sodium (2%) in drinking water. Anxiety-like behaviour was tested on the elevated plus maze and open field, and depression-like behaviour assessed by the forced swim test.

KEY RESULTS

In the absence of colitis, anxiety-like behaviour was increased by deletion of NPY but not PYY in a test- and sex-dependent manner, while depression-like behaviour was enhanced in NPY^−/− and PYY^−/− mice of either sex. The severity of DSS-induced colitis, assessed by colonic myeloperoxidase content, was attenuated in NPY^−/− but not PYY^−/− mice. Colitis modified anxiety- and depression-related behaviour in a sex-, genotype- and test-related manner, and knockout experiments indicated that NPY and PYY were involved in some of these behavioural effects of colitis.

CONCLUSIONS AND IMPLICATIONS

These data demonstrate sex-dependent roles of NPY and PYY in regulation of anxiety- and depression-like behaviour in the absence and presence of colitis. Like NPY, the gut hormone PYY has the potential to attenuate depression-like behaviour but does not share the ability of NPY to reduce anxiety-like behaviour.