nitrophenols and 2-6-dichloro-4-nitrophenol

Chloronitrophenols (CNPs) constitute a group of environmental pollutants that are widely distributed in our surrounding environment due to human based activities. This group of chemicals is highly toxic to living beings due to its mutagenic and carcinogenic nature. Examples include 2-chloro-4-nitrophenol, 4-chloro-2-nitrophenol, 2-chloro-5-nitrophenol, 4-chloro-3-nitrophenol and 2,6-dichloro-4-nitrophenol. Several methods including advanced oxidation processes, adsorption and bacterial degradation have been used for degradation of CNPs. Among, bacterial degradation is an eco-friendly and effective way to degrade CNPs. Several bacterial metabolic pathways have been proposed for degradation of CNPs and their genes and enzymes have been identified in bacteria. These bacteria were able to degrade CNPs in broth culture and soil. Therefore, CNPs-degrading bacteria are suitable candidates for bioremediation of CNPs-contaminated sites. Few CNP-degrading bacteria exhibited chemotaxis towards CNPs to enhance their biodegradation. The present review summarizes recent progress in degradation of CNPs.

Topics: Biodegradation, Environmental; Humans; Nitrophenols

Aromatic amines can be metabolized by N-acetylation and N-hydroxylation to hydroxamic acids; these subsequently are conjugated to form the N,O-sulfonate and N,O-glucuronide conjugates. The N,O-sulfonates are highly labile metabolites that generate reactive intermediates involved in the covalent binding of the parent compound to protein, RNA and DNA, as well as to low molecular compounds like glutathione. This paper discusses methods used to decrease sulfation in vivo, and thereby to enhance the formation of the more stable N,O-glucuronides from N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylamino-4'-fluorobiphenyl. Acetaminophen pretreatment decreases the sulfate availability, but results in many side effects that complicate the analysis of the results. An 8% casein diet reduces the sulfate availability in the rat to approximately 20% of control and thus offers an effective approach to decrease sulfation. The most effective selective inhibition of sulfation is by pentachlorophenol, which very strongly reduces N,O-sulfonation of both hydroxamic acids, and selectively inhibits the formation of DNA adducts that have retained the N-acetyl group. This inhibitor and the related 2,6-dichloro-4-nitrophenol can be employed to study the role of sulfation of hydroxamic acids in initiation and promotion of tumor formation by aromatic amines.

Topics: Acetaminophen; Amines; Animals; Binding Sites; Biological Availability; Carcinogens; Diet; Glucuronates; Glutathione; Hydroxamic Acids; Nitrophenols; Pentachlorophenol; Rats; Sulfates; Urinary Bladder Neoplasms

Proton transfer reaction between 2-amino-4-methylpyridine (2AMP) as the proton acceptor with 2,6-dichloro-4-nitrophenol (DCNP) as the proton donor has been investigated spectrophotometrically in methanol (MeOH), acetonitrile (AN) and a binary mixture composed of 50% MeOH and 50% AN (AN-Me). The composition of the complex has been investigated utilizing Job(')s and photometric titration methods to be 1:1. Minimum-maximum absorbance equation has been applied to estimate the formation constant of the proton transfer reaction (K(PT)) where it reached high values in the investigated solvent confirming its high stability. The formation constant recorded higher value in AN compared with MeOH and mixture of AN-Me. Based on the formation of stable proton transfer complex, a sensitive spectrophotometric method was suggested for quantitative determination of 2AMP. The Lambert-Beer(')s law was obeyed in the concentration range 0.5-8 μg mL(-1) with small values of limits of detection and quantification. The solid complex between 2AMP with DCNP has been synthesized and characterized by elemental analysis to be 1:1 in concordant with the molecular stoichiometry in solution. Further analysis of the solid complex was carried out using infrared and (1)H NMR spectroscopy.

Topics: Acetonitriles; Methanol; Nitrophenols; Picolines; Protons; Spectrophotometry

We report herein a study of the hydrolysis of Tabun mimic DCNP in the presence of different amines, aminoalcohols and glycols as potential suitable organocatalysts for DCNP degradation. Experiments were performed in CD3CN in the presence of 5% D2O, which is a suitable solvent mixture to follow the DCNP hydrolysis. These studies allowed the definition of different DCNP depletion paths, resulting in the formation of diethylphosphoric acid, tetraethylpyrophosphate and phosphoramide species as final products. Without organocatalysts, DCNP hydrolysis occurred mainly via an autocatalysis path. Addition of tertiary amines in sub-stoichiometric amounts largely enhanced DCNP depletion whereas non-tertiary polyamines reacted even faster. Glycols induced very slight increment in the DCNP hydrolysis, whereas DCNP hydrolysis increased sharply in the presence of certain aminoalcohols especially, 2-(2-aminoethylamino)ethanol. For the latter compound, DCNP depletion occurred ca. 80-fold faster than in the absence of organocatalysts. The kinetic studies revealed that DCNP hydrolysis in the presence of 2-(2-aminoethylamino)ethanol occurred via a catalytic process, in which the aminoalcohol was involved. DCNP hydrolysis generally depended strongly on the structure of the amine, and it was found that the presence of the OHCH2CH2N moiety in the organocatalyst structure seems important to induce a fast degradation of DCNP.

Topics: Amines; Amino Alcohols; Catalysis; Chemical Warfare Agents; Environmental Restoration and Remediation; Glycols; Hydrolysis; Kinetics; Nerve Agents; Nitrophenols; Organophosphates

Proton transfer reaction between the proton donor 2,6-dichloro-4-nitrophenol (DCNP) with the proton acceptor 4-aminopyridine (4APy) has been investigated spectrophotometrically in different solvents included the aprotic solvent acetonitrile (MeCN), the protic one methanol (MeOH) and a mixture consists of 50% acetonitrile+50% dichloroethane (ANDC). The proton transfer complex is produced instantaneously with deep yellow color and absorption maxima in the range 395-425nm. The composition of the complex was characterized spectrophotometrically to be 1:1 in all solvent proving that the solvent has no effect on the complex stoichiometry. The proton transfer formation constant has been estimated by using Benesi-Hildebrand equation where the highest value was recorded in the mixture ANDC. This proofs the high stability of the complex in less polar solvent as a result of the high stability of the complex ground state. The solid complex has been synthesized and characterized by elemental analysis to be 1:2 [(proton donor) (proton acceptor)2]. The obtained solid complex was analyzed by infrared spectroscopy where two broad band's at 3436 and 2500cm(-1) characterized for asymmetric NHN(+) hydrogen bond were identified. Molecular modeling utilizing GAMESS computations as a package of ChemBio3D Ultra12 program was carried out where asymmetric NHN(+) was explored with NN bond distance 2.77Å. The computations showed a difference in molecular geometry of the complex compared with reactants especially bond lengths, bond angles and distances of close contact.

Topics: 4-Aminopyridine; Hydrogen Bonding; Models, Molecular; Nitrophenols; Protons; Solvents; Spectrophotometry

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Topics: Acetophenones; Benzaldehydes; Biocatalysis; Catechols; Coumaric Acids; Decision Trees; Dichlorophen; Fungal Proteins; Hydrazones; Laccase; Models, Chemical; Models, Molecular; Molecular Structure; Nitrophenols; Oxidation-Reduction; Phenols; Polyporales; Protein Conformation; Quantitative Structure-Activity Relationship; Vanillic Acid

Water-based Zn(II) bisterpyridine systems were used as fluorometric sensors for the detection of the nerve gas G mimics DMMP, DCP and DCNP. Analyte concentrations in the range of 10(-7) to 10(-6) M are detectable in solution. The utilization of a test stripe additionally allows the detection of organophosphonates from the gas phase.

Topics: Chemical Warfare Agents; Coordination Complexes; Fluorometry; Gases; Nitrophenols; Organophosphorus Compounds; Pyridines; Sensitivity and Specificity; Water; Zinc

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Abeta) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Abeta peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20muM Abeta peptide 25-35 and variable concentrations of P and PS ranging from 0.5muM to 100muM. To examine the effects of steroid treatment on Abeta peptide toxicity, 0.5muM (low) and 50muM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72h. Morphological changes of cells were also examined. The treatment with higher than 1muM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72h. The Abeta treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Abeta peptide in PC-12 cells. But its sulfate ester did not have the same effect on Abeta peptide toxicity, even it significantly decreased cell viability in Abeta-treated cells. Consequently, the discrepant effects of P and PS on Abeta peptide toxicity may provide insight on the pathogenesis of Alzheimer's disease.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Cell Survival; Cytoprotection; Dose-Response Relationship, Drug; Microscopy, Fluorescence; Nitrophenols; PC12 Cells; Peptide Fragments; Pregnenolone; Rats; Time Factors

KYNA (kynurenic acid) is an endogenous metabolite of tryptophan in the kynurenine pathway and has been characterized as an antagonist of ionotropic glutamate receptors. In addition, we have reported this endogenous compound as a potent inhibitor of SULTs (cytosolic sulfotransferases). In the present study we characterized the inhibitory effects of KYNA on several human (h) and mouse (m) recombinant SULTs. No sulfate metabolite of KYNA was detected with mouse and human SULTs examined under the conditions used, suggesting that it is a bona fide inhibitor of SULTs. Among the mouse enzymes examined, KYNA exhibited selective inhibitory effects on Sult1b1-mediated sulfation of various compounds with IC50 values in the low micromolar range (2.9-4.9 microM). KYNA also exerted an inhibitory activity towards hSULT1A1 and hSULT1B1. The inhibitory potency of KYNA for mSult1b1 was stronger than that of 2,6-dichloro-4-nitrophenol, a known non-specific SULT inhibitor, whereas the potencies of these two inhibitors for hSULT1B1 were comparable. The inhibitory characteristics of KYNA were clearly distinct from those of mefenamic acid, a selective inhibitor of SULT1A enzymes. The KYNA derivatives 5,7-dichlorokynurenic acid and L689,560 exhibited preferential inhibitory effects on hSULT1A1 and hSULT1B1 respectively. Interestingly, gavestinel, another KYNA derivative, was found to be an extremely potent inhibitor of hSULT1B1. Finally, we have demonstrated that the mechanism underlying the KYNA inhibition varied depending on the enzyme and substrate involved. Taken together, the present results unveil another distinct aspect of KYNA and its derivatives as an inhibitor of SULTs.

Topics: Animals; Enzyme Activation; Excitatory Amino Acid Antagonists; Humans; Kinetics; Kynurenic Acid; Mefenamic Acid; Mice; Mice, Inbred C57BL; Nitrophenols; Recombinant Proteins; Sulfotransferases; Tryptophan

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17beta-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent K(m) values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism.

Topics: Arylsulfotransferase; Cell Line, Tumor; Choriocarcinoma; Dopamine; Female; Humans; Isoenzymes; Nitrophenols; Placenta; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride; Substrate Specificity; Sulfotransferases; Trophoblasts

Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), potent inhibitors of phenol sulphotransferases, are frequently used in animal studies to elucidate the role of these enzymes in the biotransformation and toxicity of xenobiotics. An unexpected finding with 1-hydroxymethylpyrene--a strong decrease in the excretion of the corresponding carboxylic acid in rats concurrently treated with PCP-led us to suspect that this sulphotransferase inhibitor may also affect alcohol dehydrogenases (ADHs) and/or aldehyde dehydrogenases (ALDHs). Subsequently we investigated the influence of PCP and DCNP on the activity of cDNA-expressed human ADHs and ALDHs. PCP inhibited all four ADHs studied. The inhibition was strong for ADH3 (K(i) 1.4 microM, K(i)' 5.2 microM, mixed-type) and ADH2 (K(i) 3.7 microM, competitive), but moderate for ADH4 (K(i) 81 microM, competitive) and ADH1C (K(i)' 310 microM, uncompetitive). Activities of ALDH2 and ALDH3A1 were unaffected by PCP (used up to a concentration of 1 mM). In contrast, DCNP primarily inhibited ALDH2 (K(i)=K(i)' 7.4 microM, non-competitive), showed moderate competitive inhibition of ADH2 (K(i) 160 microM) and ADH4 (K(i) 710 microM), but did not affect the remaining enzymes (ADH1C, ADH3 and ALDH3A1). The study demonstrates that caution is required when using putative specific enzyme inhibitors in biotransformation studies.

Topics: Alcohol Dehydrogenase; Aldehyde Dehydrogenase; DNA, Complementary; Herbicides; Humans; Isoenzymes; Kinetics; Nitrophenols; Oxidation-Reduction; Pentachlorophenol; Spectrophotometry, Ultraviolet

Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3alpha-hydroxytibolone (3alpha-OH-tibolone) and 3beta-hydroxytibolone (3beta-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n=5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 +/- 0.3 units/mg protein, n = 8, mean +/- SEM), and activities with 3alpha-OH-tibolone (0.6 +/- 0.1, n = 8) and 3beta-OH-tibolone (0.9 +/- 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 +/- 0.01 with 3alpha-OH-tibolone and 0.04 +/- 0.01 with 3beta-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.

Topics: Chromatography, High Pressure Liquid; Gene Expression Profiling; Humans; Intestine, Small; Liver; Nitrophenols; Norpregnenes; Phosphoadenosine Phosphosulfate; Postmenopause; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Chloride; Sulfotransferases; Temperature

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 microM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3'-phosphoadenosine 5'-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3'-phosphoadenosine 5'-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.

Topics: Adenosine Diphosphate; Animals; Chromatography, DEAE-Cellulose; Diptera; Ecdysterone; Enzyme Inhibitors; Fat Body; Hemolymph; Larva; Naphthols; Nitrophenols; Phosphoadenosine Phosphosulfate; Photoaffinity Labels; Pupa; Sulfates; Sulfotransferases; Sulfur Radioisotopes

The present paper describes the role of conjugating enzymes in the development of hepatotoxicity after administration of repeated doses of a novel monoamine oxidase type-A (MAO-A) inhibitor, (5R)-3-[2-(( 1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011). The effects of pretreatment with three kinds of conjugating enzyme inhibitors on hepatic lesions induced by E2011 were evaluated in female Sprague-Dawley rats. The inhibitors used were 2,6-dichloro-4-nitrophenol (DCNP; inhibitor of sulfotransferase (ST)), pentachlorophenol (PCP; inhibitor of both ST and acetyltransferase (AT)) or ranitidine (inhibitor of UDP-glucuronosyltransferase (UDP-GT)). Two weeks treatment of E2011 alone at an oral dosage of 150 mg/kg induced hepatocellular changes characterized by nuclear enlargement. Daily pretreatment with DCNP (10 mg/kg, i.p.) enhanced the E2011-induced hepatocellular changes accompanied by single cell necrosis. On the other hand, the hepatotoxicity was clearly diminished by PCP (5 mg/kg, i.p.). Ranitidine pretreatment had no effect. Protection by PCP was attributed to the inhibitory effects of AT in addition to ST; it was considered that the hepatocellular changes caused by E2011 were largely dependent on the formation of acetyl conjugate(s).

Topics: Acetyltransferases; Animals; Benzothiazoles; Enzyme Inhibitors; Female; Glucuronosyltransferase; Liver; Monoamine Oxidase Inhibitors; Nitrophenols; Oxazolidinones; Pentachlorophenol; Ranitidine; Rats; Rats, Sprague-Dawley; Sulfotransferases; Thiazoles

In primary human and porcine hepatocyte cultures, we investigated the relationship between metabolism and cytotoxicity of troglitazone. Treatment of human hepatocytes for 2 h with 10, 20, 25, 35, and 50 microM troglitazone in protein-free medium resulted in concentration-dependent decreases in total protein synthesis. Decreases at 10 and 20 microM were reversible by 24 h, however protein synthesis did not recover at concentrations >/=25 microM. Troglitazone at 50 microM caused cellular death. In porcine hepatocytes, 100 microM troglitazone was lethal, whereas at 50 microM, protein synthesis completely recovered by 24 h. Recovery in protein synthesis was associated with metabolism of parent drug, whereas toxicity correlated (r(2) = 0.82) with accumulation of unmetabolized troglitazone. By 1 h, in human hepatocytes, troglitazone was metabolized to similar amounts of sulfate and quinone metabolites with little glucuronide detected. In contrast, porcine hepatocytes metabolized troglitazone to the similar amounts of glucuronide and the quinone metabolites with little sulfate detected. Exposure of human hepatocytes to a combination of 10 microM troglitazone and 10 microM 2,4-dichloro-4-nitrophenol resulted in a 70% decrease in protein synthesis, associated with 90% inhibition in the formation of troglitazone sulfate, a 4-fold increase in unmetabolized troglitazone, and no effect on formation of the quinone metabolite. Treatment with a combination of acetaminophen or phenobarbital with 20 microM troglitazone resulted in sustained decrease in protein synthesis associated with inhibition of sulfation and accumulation of troglitazone. These results suggest that inhibition of troglitazone sulfation may result in increased hepatotoxicity due to exposure to parent drug, or increased metabolism by alternate pathways.

Topics: Acetaminophen; Animals; Cells, Cultured; Chromans; Dose-Response Relationship, Drug; Hepatocytes; Humans; Nitrophenols; Pentachlorophenol; Phenobarbital; Protein Biosynthesis; Proteins; Swine; Thiazoles; Thiazolidinediones; Troglitazone

The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites have been studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with benzophenone (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by loss of intracellular ATP and depletion of adenine nucleotide pools. Benzophenone at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted to benzhydrol, p-hydroxybenzophenone and its sulfate conjugate, without marked loss of cell viability. The amounts of benzhydrol and sulfate conjugate increased with time. In contrast, addition of 2,6-dichloro-4-nitrophenol (an inhibitor of sulfotransferase; 0.1 mM), nontoxic to hepatocytes during the incubation period, enhanced benzophenone-induced cytotoxicity, and this effect was accompanied by a decrease in the formation of sulfate conjugate and increase in the amount of free p-hydroxybenzophenone. In another experiment, MCF-7 cells, estrogen-responsible breast cancer cells were cultured in estradiol free medium and then exposed to 10 nM-500 microM benzophenone or its metabolites for 6 days. Although at higher concentrations all the compounds were toxic, except for benzophenone and benzhydrol, 10-100 microM p-hydroxybenzophenone significantly increased cell proliferation. These results indicate that benzophenone is enzymaticaly converted to benzhydrol, p-hydroxybenzophenone and its sulphate conjugate in rat hepatocytes. Even if there is less free p-hydroxybenzophenone than benzhydrol and sulfate conjugate in hepatocyte suspensions, p-hydroxybenzophenone itself acts as a weak xeno-estrogen on MCF-7 cells.

Topics: Animals; Benzophenones; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Estrogens; Female; Hepatocytes; Humans; Male; Nitrophenols; Rats; Rats, Inbred F344; Tumor Cells, Cultured

Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of housekeeping genes which have canonical CpG islands at 5' promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3-7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Energy Metabolism; Enzyme Activation; Flow Cytometry; Gene Expression Regulation; Hepatocytes; Humans; Hydrogen-Ion Concentration; Mitochondria; Nitrophenols; Oligonucleotide Array Sequence Analysis

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is an acute nephrotoxicant in rats. Our previous studies suggested that sulfate conjugation of NDPS metabolites might be a bioactivation step mediating NDPS nephrotoxicity. In this study, effects of substrates and/or inhibitors of sulfation on NDPS nephrotoxicity were examined to explore further the role of sulfation in NDPS nephrotoxicity. Male Fischer rats (4-8/group) were administered one of the following intraperitoneal (ip) pretreatment (dose, pretreatment time) prior to NDPS (0.6 mmol/kg) or NDPS vehicle (sesame oil, 2.5 ml/kg): (1) no pretreatment, (2) dehydroepiandrosterone (DHEA) (0.5 mmol/kg, 1 h), or (3) 2,6-dichloro-4-nitrophenol (DCNP) (0.04 mmol/kg, 1 h). Following NDPS or NDPS vehicle administration, renal function was monitored at 24 and 48 h. Pretreatment with DHEA, a typical substrate for and an inhibitor of hydroxysteroid (alcohol) sulfotransferase, resulted in marked protection against NDPS nephrotoxicity. A selective inhibitor of phenol sulfotransferase, DCNP, afforded little attenuation in NDPS nephrotoxicity. These results suggest that alcohol sulfate conjugates of NDPS metabolites, rather than phenolic sulfate conjugates, may be a penultimate or ultimate nephrotoxicant species mediating NDPS nephrotoxicity. The marked, but not complete, protection by DHEA also suggests that there are other metabolites or mechanisms responsible for NDPS nephrotoxicity.

Topics: Adjuvants, Immunologic; Animals; Biotransformation; Dehydroepiandrosterone; Eating; Fungicides, Industrial; Kidney Diseases; Male; Nitrophenols; Rats; Rats, Inbred F344; Succinimides; Sulfuric Acid Esters

The importance of sulfotransferases in xenobiotic metabolism is gaining recognition. The gastrointestinal (GI) tract is a major portal of entry for many xenobiotics, yet little is known about the contribution of sulfotransferases to detoxication or bioactivation metabolism in these tissues. To this end, isolation and characterisation of sulfotransferases expressed in the stomach of rabbits was undertaken. A unique sulfotransferase cDNA (GenBank Accession No. AF026304) was isolated from a rabbit stomach cDNA library. This cDNA was 1439 base pairs (bp) long and has an open reading frame of 888 bp. On expression of the cDNA in both COS cells and E. coli, a protein molecular weight of 34 kDa was detected on SDS-PAGE. Immunoblotting using an antibody raised in goats against the bacterially expressed protein detected expression of the protein in GI tract tissues. The 34 kDa immunoreactive band was detected in rabbit GI tract tissues (stomach, duodenum, jejunum, ileum, colon, caecum and rectum), liver and kidneys, but not in the lungs (n = 3). The human ortholog (GenBank Accession No AF026303) of the rabbit enzyme was cloned from a human stomach cDNA library. These two enzymes share 84% amino acid sequence identity and have been termed 1C2 sulfotransferases. When functional and kinetic characterisation of the recombinant rabbit and human proteins was carried out using 16 known ST substrates, detectable sulfonation activity was observed only with p-nitrophenol (with Km values of 2.2 mM and 13.3 mM, respectively). In conclusion, we have identified a rabbit GI tract sulfotransferase belonging to a newly defined sulfotransferase subfamily.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; COS Cells; Digestive System; Enzyme Inhibitors; Enzyme Stability; Escherichia coli; Gene Expression Regulation, Enzymologic; Kinetics; Molecular Sequence Data; Nitrophenols; Rabbits; Sequence Alignment; Sulfotransferases; Transfection

The hepatocellular entry of 4-methylumbelliferyl sulfate (4MUS) a highly ionized and highly bound anion capable of futile cycling, was examined in the single-pass albumin-free perfused rat liver preparation. Desulfation of 4MUS to 4-methylumbelliferone (4MU) was verified in vitro to be a low-affinity, high-capacity process (Km = 731 micromol/L; Vmax = 414 nmol min(-1) g(-1) liver). With 4MUS given to the perfused rat liver, sulfation of 4MU, the formed metabolite, was attenuated in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a sulfation inhibitor, and when sulfate ion was substituted by chloride ion. 4MU sulfation, being a high-affinity system, was reduced most effectively at the lowest 4MUS concentration (15 micromol/L) used, evidenced by the increased (24%) net hepatic extraction ratio of 4MUS and reduced utilization (72%) of infused tracer 35SO4(2-) by 4MU for 4MU35S formation. Single-pass multiple indicator dilution (MID) studies were thus conducted under identical conditions (DCNP and absence of inorganic sulfate), with injection of [3H]4MUS and a set of noneliminated vascular and cellular reference indicators into the portal vein (prograde) or hepatic vein (retrograde), against varying background bulk concentrations of 4MUS (5 to 900 micromol/L). The steady-state removal rate of 4MUS and formation rates of 4MU and its glucuronide conjugate (4MUG) were not altered with perfusion flow direction, suggesting the presence of even or parallel distributions of 4MUS desulfation and 4MU glucuronidation activities. When the outflow dilution profile of [3H]4MUS was evaluated with the barrier-limited model of Goresky, a slight red cell carriage effect was found for 4MUS. The permeability surface area product for cellular entry for prograde showed a dramatic concentration-dependent decrease (from 0.13 to 0.01 mL sec(-1) g(-1), or 7.4 to 0.56 times the blood perfusate flow rate) and was resolved as saturable and nonsaturable components, while data for retrograde were more scattered, varying from 2.8 to 1 times the blood perfusate flow rate. Efflux (coefficient = 0.0096 +/- 0.0024 and 0.0088 +/- 0.0062 mL sec(-1) g(-1), respectively) was relatively insensitive to concentration and flow direction. The same was observed for the removal capacity for metabolism and excretion (sequestration coefficient: for prograde, 0.0056 +/- 0.0017 mL sec(-1) g(-1); for retrograde, 0.0056 +/- 0.003 mL sec(-1) g(-1)). The decrease in the apparent partition coefficient (ratio o

Topics: Animals; Drug Carriers; Hymecromone; Indicator Dilution Techniques; Liver; Male; Nitrophenols; Perfusion; Rats; Rats, Sprague-Dawley; Sulfates

Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84-148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (Km, Vmax, and Vmax/Km) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84-89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84-148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.

Topics: Amino Acid Sequence; Arylsulfotransferase; Binding Sites; Dopamine; Escherichia coli; Humans; Kinetics; Manganese; Molecular Sequence Data; Nitrophenols; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Substrate Specificity

Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 28899-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, several mutants showed the cross-resistance. These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site. All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II. Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold. Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II. Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.

Topics: Amino Acid Sequence; Benzoquinones; Cell Membrane; Drug Resistance, Microbial; Electron Transport Complex IV; Escherichia coli; Models, Molecular; Molecular Sequence Data; Nitrophenols; Phenols; Point Mutation; Pyridines

Biotransformation of xenobiotics and hormones through sulfate conjugation is an important metabolic pathway in humans. The activation of minoxidil, an antihypertensive agent and hair growth stimulator, by sulfation (sulfonation) is carried out by more than one sulfotransferase. Initially only the thermostable form of phenol sulfotransferase was thought to catalyze minoxidil sulfation. We document in this report the new finding that human liver dehydroepiandrosterone sulfotransferase (DHEAST), an hydroxysteroid sulfotransferase distinct from phenol sulfotransferases, also catalyzes the reaction. To characterize more precisely the activity of DHEA ST toward minoxidil, we used COS-1 cells to express DHEA ST from a human liver cDNA clone. The apparent Km values for minoxidil and [35S]3'-phosphoadenosine-5'-phosphosulfate were 3.9 mM and 0.13 microM, respectively. The 50% inactivation temperature of the COS-expressed enzyme was 42 degrees, and the IC50 value for 2,6-dichloro-4-nitrophenol was 1.4 x 10(-4) M. Both the thermal stability behavior and response to DCNP were similar when the cDNA encoded DHEA ST was assayed with DHEA or minoxidil as a substrate. NaCl led to a greater activation of the cDNA expressed DHEA ST when assayed with DHEA (2.5-fold) than when the same preparation was assayed with minoxidil (1.4-fold). These data indicate that DHEA ST catalyzes the sulfate conjugation of minoxidil: DHEA ST activity present in the human gut and liver would be expected to add to the overall sulfate conjugation of orally administered minoxidil. Thus, DHEA ST activity must be considered when determining the human tissue sulfotransferase contribution to minoxidil sulfation.

Topics: Animals; Chromatography, High Pressure Liquid; COS Cells; Humans; Liver; Minoxidil; Nitrophenols; Recombinant Proteins; Sodium Chloride; Sulfotransferases; Temperature; Vasodilator Agents

Mouse nasal cytosols show high sulfotransferase (ST) activities toward phenolic aromatic odorants, but have little activities for most alcoholic aromatic odorants. Most ST activities toward the phenolic odorants preferred slightly acidic pH (6.4) and were sensitive to 2,6-dichloro-4-nitrophenol, a specific inhibitor for phenol ST (P-ST) but were not inhibited by triethylamine and tetra-n-butylammonium chloride, which are specific inhibitors for hydroxysteroid ST (HS-ST). These results suggested that P-ST activities are responsible for sulfation of the phenolic odorants. The spectra of the ST activities for these odorants were similar in mouse nasal and liver cytosols, however, nasal cytosols showed much higher ST activity toward cinnamyl alcohol than liver cytosols. This activity preferred higher pH (7.4) compared to the phenolic odorant-ST activities and was inhibited by both types of inhibitors, specific for P-ST and HS-ST. These results appear to indicate the participation of multiple ST isoforms for the sulfation of odorants in mouse nasal cytosols. The existence of P-ST(s) active for the phenolic odorants in olfactory cytosols suggests a role in odorant perception, in particular, in the signal termination process.

Topics: Alcohols; Animals; Arylsulfotransferase; Cytosol; Enzyme Inhibitors; Guaiacol; Hydrogen-Ion Concentration; Isoenzymes; Liver; Male; Mice; Mice, Inbred Strains; Molecular Structure; Naphthols; Nitrophenols; Odorants; Olfactory Mucosa; Phenols; Smell; Substrate Specificity; Sulfotransferases

Phenol sulfotransferases (PSTs) represent a family of sulfotransferase enzymes that modify the biologic activities and excretion of phenolic compounds and monoamines. A novel human hippocampal PST (H-PST) cDNA with homology to phenol (P) and monoamine (M) forms of PST was previously isolated from brain. To compare the biochemical properties of H-PST with that of phenol (P-PST) and monoamine (M-PST) sulfotransferases, high level expression of recombinant H-PST was achieved in this study with the pET3c vector in BL21(DE3) Escherichia coli cells. Expression was demonstrated by isopropyl beta-D-thiogalactopyranoside induction of 34-kDa H-PST that represented 5-10% of total E. coli proteins. Purification by ion-exchange chromatography on DEAE-Sepharose yielded more than 2 mg of H-PST. Characterization showed that H-PST exists as a homodimer of 60-65 kDa by gel filtration chromatography. H-PST prefers p-nitrophenol as substrate and does not sulfate dopamine or neuropeptide substrates. Kinetic studies showed that H-PST possessed K(m(app)) and Vmax(app) values of 3 microM p-nitrophenol and 160 nmol/min/mg, respectively. H-PST was sensitive to inhibition by DCNP (2,6-dichloro-4-nitrophenol). H-PST is thermolabile since its activity was reduced upon preincubation at 37 degrees C. These results indicate that H-PST shows similarities and differences compared to P-PST and M-PST sulfotransferases. P-PST prefers p-nitrophenol as substrate, is sensitive to inhibition by DCNP, and is thermostable; in contrast, M-PST prefers monoamines as substrate, is not sensitive to DCNP, and is thermolabile. The distinct profile of biochemical properties of H-PST, and its primary sequence homology to P-PST and M-PST, suggests that H-PST represents a novel allelic variant of human phenol sulfotransferases. Importantly, this study demonstrates that high level expression of H-PST allows determination of distinguishing characteristics of variant forms of PSTs.

Topics: Alleles; Amino Acid Sequence; Arylsulfotransferase; Blood Platelets; Enzyme Inhibitors; Escherichia coli; Hippocampus; Humans; Isoenzymes; Kinetics; Liver; Molecular Sequence Data; Nitrophenols; Recombinant Proteins; Sequence Alignment

HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.

Topics: Blood Platelets; Chromatography, Thin Layer; Dihydroxyphenylalanine; Enzyme Activation; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Isoenzymes; Liver; Manganese; Nitrophenols; Stereoisomerism; Substrate Specificity; Sulfates; Sulfotransferases; Tumor Cells, Cultured; Tyrosine

1. Amine N-sulphotransferase (NST) activity with desipramine (DMI) as substrate was assayed in vitro in various areas of the rat brain. Biosynthesis of 3'-phosphoadenosine-5'-phospho35sulphate (PAPS) from sodium 35sulphate and ATP was also measured by coupling it to the sulphation of minoxidil by minoxidil sulphotransferase (MST). 2. For the DMI-NST reaction, an apparent Km = 0.5 mM was obtained for DMI and two apparent Kms = 0.3 and 1.7 microM for PAPS, whereas in the PAPS-generating reactions, Km for sodium 35sulphate = 20 microM. 3. Both the enzyme activities were widely distributed in rat brain. The rate of NST activity was 2-3 orders of magnitude lower than that of PAPS generation. N-sulphoconjugation of DMI, which is proposed as a possible biotransformation pathway of DMI in the rat brain, could conceivably be supported adequately by the 'active sulphate' generated within the same areas of the brain.

Topics: Adenosine Triphosphate; Animals; Antidepressive Agents, Tricyclic; Biotransformation; Brain; Desipramine; Dopamine; Kinetics; Male; Minoxidil; Nerve Tissue Proteins; Nitrophenols; Phosphoadenosine Phosphosulfate; Rats; Rats, Wistar; Sulfates; Sulfotransferases; Sulfur Radioisotopes; Vasodilator Agents

The natural product quercetin was a potent inhibitor of the human P-form phenolsulfo-transferase with an IC50 value of 0.10 +/- 0.03 microM (mean +/- SEM; N = 5), which was three to four orders of magnitude more potent than its inhibition of other human sulfotransferases. The inhibition was noncompetitive with a Ki value of 0.10 microM. The potency and mechanism of this inhibition appear similar to those of the current standard P-form inhibitor, 2,6-dichloro-4-nitrophenol. Among other flavonoids examined, kaempferol was found to have an IC50 value of 0.39 +/- 0.07 microM, naringenin 10.6 +/- 1.6 microM and naringin 265 +/- 90 microM (N = 3). These observations suggest the potential for clinically important pharmacologic and toxicologic interactions by flavonoid-containing foods and beverages.

Topics: Arylsulfotransferase; Dehydroepiandrosterone; Dopamine; Humans; Liver; Molecular Probes; Nitrophenols; Quercetin; Sulfotransferases

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.

Topics: Animals; Aroclors; Benzoflavones; Carcinogens; Chlorodiphenyl (54% Chlorine); Chromatography, High Pressure Liquid; Culture Techniques; Cytochrome P-450 Enzyme System; DNA; DNA Adducts; Food Contamination; Food Handling; Imidazoles; Liver; Male; Mutagens; Nitrophenols; Pentachlorophenol; Rats; Rats, Sprague-Dawley

5-Hydroxymethyl-2-furaldehyde (HMF), a ubiquitous food contaminant, has been proposed to be metabolically activated through sulfonation of its allylic hydroxyl functional group. In support of this idea, we have found the strong direct mutagenicity of chemically synthesized sulfuric acid ester, 5-sulfooxymethylfurfural (SMF), in Salmonella typhimurium TA104. The intrinsic mutagenicity of this reactive ester was significantly inhibited by glutathione and glutathione S-transferase activity in dialyzed rat liver cytosol. The metabolic formation of SMF was elucidated by enhanced mutagenicity of HMF in the presence of rat hepatic cytosol enriched with the sulfo-group donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The PAPS- and cytosol-dependent mutagenicity of HMF was markedly lessened by sulfotransferase inhibitors such as 2,6-dichloro-4-nitrophenol and dehydroepiandrosterone. These results suggest that HMF can be metabolically activated to an allylic sulfuric acid ester which may play a role as an ultimate electrophilic metabolite in toxification of the parent compound in vivo.

Topics: Animals; Biotransformation; Cytosol; Dehydroepiandrosterone; Furaldehyde; Glutathione; Glutathione Transferase; Kinetics; Liver; Mutagenicity Tests; Mutagens; Nitrophenols; Phosphoadenosine Phosphosulfate; Rats; Salmonella typhimurium; Sulfotransferases; Sulfuric Acids

Human scalp skin high speed supernatants were used to test whether minoxidil sulfotransferase (MNX-ST) and phenol sulfotransferase (PST) activities were present. Platelet homogenates from the same skin donors were used to test whether levels of sulfotransferase activities in the blood platelet would reflect levels of the enzyme activities in skin. Dopamine, p-nitrophenol and minoxidil were used as substrates for skin and platelet thermolabile (TL PST), thermostable (TS PST) and MNX-ST activities, respectively. Biochemical properties of each skin enzyme were the same as the platelet enzymes with respect to apparent Km values for substrates, pH optima, thermal stabilities and responses to inhibition by 2,6-dichloro-4-nitrophenol (DCNP). An unexpected finding was that skin and platelet MNX-ST thermal stabilities and responses to DCNP were more similar to TL PST than to TS PST, the enzyme reported to be responsible for MNX-ST activity. There were significant positive correlations of platelet sulfotransferases with the relative levels of activities of the same skin sulfotransferases. Unexpected findings were significant positive correlations of MNX-ST and TL PST activities. Partially purified platelet TS PST assayed with minoxidil as the substrate showed a response to DCNP and thermal stability that were the same as TS PST. Platelet TL PST assayed with minoxidil showed thermal stability and a response to DCNP that were essentially the same as TL PST. The results indicated that not only TS PST, but also TL PST activities in human skin and platelet contributed to MNX-ST activity. It will be feasible to test whether measures of platelet PST activities will predict physiologic responses to minoxidil.

Topics: Arylsulfotransferase; Blood Platelets; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Enzyme Stability; Humans; Minoxidil; Nitrophenols; Skin; Sodium Chloride; Substrate Specificity; Sulfotransferases; Temperature

Diclofenac sodium, famotidine and ketorolac tromethamine were determined by flow injection analysis (FIA) with spectrophotometric detection. The sample solutions (5-50 micrograms ml-1 of diclofenac sodium, 10-80 micrograms ml-1 of famotidine and 10-120 micrograms ml-1 of ketorolac tromethamine) in methanol were injected into a flow system containing 0.01% (w/v) of 2,4,dichloro-6-nitrophenol (DCNP) in methanol. The colour produced due to the formation of a charge transfer complex was measured with a spectrophotometric detector set at 450 nm. A sampling rate of 40 per hour was achieved with high reproducibility of measurements (RSD below 1.6%). The FIA method was applied to the determination of diclofenac sodium, famotidine and ketorolac tromethamine in pharmaceutical formulations.

Topics: Cyclooxygenase Inhibitors; Diclofenac; Drug Combinations; Famotidine; Flow Injection Analysis; Ketorolac Tromethamine; Methanol; Nitrophenols; Tolmetin; Tromethamine

The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.

Topics: Animals; Arylsulfotransferase; DNA, Complementary; Dopamine; Enzyme Stability; Humans; Kinetics; Nitrophenols; Phosphoadenosine Phosphosulfate; Rats; Substrate Specificity; Sulfotransferases; Temperature

2,6-Dichloro-4-nitrophenol (DCNP)-35sulfate was identified and quantified by an HPLC-radiometric assay following its biosynthesis in vitro from 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAP35S) by phenolsulfotransferase (PST) of rat liver cytosol. Acid hydrolysis of DCNP-35sulfate produced almost stoichiometric release of inorganic 35sulfate and DCNP. In two-substrate experiments of sulfation of p-nitrophenol (p-NP) or dopamine (prototype substrates for P and M human PST forms), 10 microM DCNP inhibited the reactions by about 15 and 78%, respectively. This contrasts with its action on PST of human origin where the P-PST was more sensitive to DCNP inhibition. In all mixed bi-substrate experiments, a reciprocal relationship between the two sulfated products was observed. Kinetic data showed that p-NP inhibited the sulfation of DCNP competitively. Likewise the sulfation of p-NP and dopamine was competitively inhibited by DCNP. However, non-competitive inhibition was observed in the sulfation of p-NP by DCNP, measured at varying concentrations of PAP35S. The above kinetic data suggest that DCNP is an alternate-substrate inhibitor of rat liver PST.

Topics: Animals; Arylsulfotransferase; Cytosol; Dopamine; Harmine; Kinetics; Liver; Nitrophenols; Rats; Sulfates

The aim of the present study was to examine the effects of the hepatotoxic drug acetaminophen (AA) on the synthesis rates of glutathione (GSH), activated sulphate (PAPS; adenosine 3'-phosphate 5'-phosphosulphate) and the AA metabolites AA-GSH and AA-sulphate after selective inhibition of GSH biosynthesis or sulphation in isolated rat hepatocytes. Selective inhibition of the two interdependent metabolic pathways was accomplished by buthionine sulphoximine (BSO) and 2,6-dichloro-4-nitrophenol (DCNP). The synthesis rates of GSH and PAPS were determined simultaneously by a previously described method based on trapping of radioactivity (35S) in the pre-labelled GSH and PAPS pools. Pre-incubation with 10 mM BSO for 30 min depleted GSH by 38% (P < 0.05) and PAPS by 27% (P < 0.05). The depletion resulted in increased PAPS synthesis at low, non-toxic [5-19 nmol/(10(6) cells.min)] (P < 0.05) and at high, toxic [7-30 nmol/10(6) cells.min)] (P < 0.05) AA concentrations. In both cases sulphur is diverted from GSH biosynthesis to sulphoxidation and PAPS synthesis, thereby maintaining the PAPS pool and preserving the sulphation capacity. This corresponds to the finding that AA sulphation was unaffected by BSO irrespective of AA concentration [6 vs 5 and 20 vs 17 nmol/(10(6) cells.hr), respectively]. Even though the GSH synthesis was halved after BSO pre-incubation, the GSH conjugating capacity of AA was well preserved. Incubation with 200 microM DCNP and 5 mM AA diminished PAPS synthesis from 24 to 10 nmol/(10(6) cells.min) (P < 0.02) and reduced AA-sulphate synthesis by 67% compared to experiments without DCNP incubation [4.8 vs 14.7 nmol/(10(6) cells.hr)] (P < 0.05). GSH and AA-GSH synthesis rates did not change compared to control experiments in which sulphation was not inhibited [1165 vs 1487 nmol/(10(6) cells.min), respectively] and [1.7 vs 1.7 nmol/(10(6) cells.hr), respectively]. This indicates that increased sulphur availability due to decreased PAPS synthesis is unable to raise the cysteine pool and stimulate the gamma-glutamyl cycle and GSH synthesis.

Topics: Acetaminophen; Animals; Buthionine Sulfoximine; Cells, Cultured; Female; Glutathione; Liver; Methionine Sulfoximine; Nitrophenols; Phosphoadenosine Phosphosulfate; Rats; Rats, Wistar; Sulfates; Sulfotransferases

Minoxidil sulphotransferase (MST) activity was determined in the cytosolic fraction of rat skin and liver. MST of rat skin is similar to the P (phenol)-form of phenosulphotransferase (PST) of human tissues with respect to thermostability and inhibition by 2,6-dichloro-4-nitrophenol (DCNP). p-Nitrophenol, a prototype substrate of human P-PST form, inhibits MST at micromolar concentration while millimolar concentrations of dopamine and tyramine, substrates of human M-(monoamine)-PST, are required to elicit a similar degree of inhibition. The enzymatic transfer of 35S from sodium 35sulphate to minoxidil was also demonstrated suggesting that the rat skin is potentially capable of synthesizing 3'-phosphoadenosine-5'-phosphosulphate (PAPS) from inorganic sulphate and utilizing it for the biosynthesis of minoxidil sulphate, its active metabolite. Thus, it is conceivable that the pharmacological action of minoxidil as a promoter of hair growth could be carried out by the cutaneous tissues without the contribution of hepatic or other extrahepatic organs.

Topics: Animals; Dopamine; Enzyme Stability; Hot Temperature; Liver; Minoxidil; Nitrophenols; Phosphoadenosine Phosphosulfate; Rats; Skin; Sulfates; Sulfotransferases; Tyramine

The sulphate conjugation of serotonin and N-acetylserotonin (NAS) was studied in the mosquito, Aedes togoi, using a high-pressure liquid chromatography-radioisotopic procedure. This involved the transfer of the sulphate group from 3'-phosphoadenosine-5'-phosphosulphate (PAP35S) to serotonin or NAS by phenolsulphotransferase (PST). NAS, the acetylated product, is less polar than serotonin but it is a better substrate of PST. Its removal would conceivably be facilitated by sulphate conjugation which renders it more water-soluble. This sequential two-step reaction, comprising N-acetylation of serotonin and sulphate conjugation of the acetylated product (NAS) was also demonstrated in vitro. The developmental profile of PST activity using NAS as substrate showed that peak activity occurred at pupation and increased progressively for a few days after emergence of the adult. Based on the selective inhibition of the "P" form of PST by 2,6-dichloro-4-nitrophenol (DCNP), the sulphate conjugation of NAS appeared to be catalysed by the "M" form of PST.

Topics: Aedes; Animals; Arylsulfotransferase; Hydrolysis; Nitrophenols; Phosphoadenosine Phosphosulfate; Serotonin; Sulfates

The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 times higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T3 sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 micrograms/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6-dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cricetinae; Cytosol; Dogs; Female; Growth Hormone; Guinea Pigs; Humans; Liver; Male; Mesocricetus; Mice; Nitrophenols; Pentachlorophenol; Rabbits; Rats; Rats, Inbred ACI; Rats, Inbred F344; Rats, Sprague-Dawley; Sex Characteristics; Species Specificity; Sulfates; Triiodothyronine

The sulfation of steroid hormones and xenobiotics by human fetal lung cytosol was examined. 1-Naphthol and estrone were extensively sulfated, whereas paracetamol and dehydroepiandrosterone were not good substrates for the pulmonary enzyme. Investigation of the thermostability and inhibition by 2,6-dichloro-4-nitrophenol (DCNP) of the 1-naphthol and estrone sulfotransferase (ST) activities revealed that the estrone ST activity was more thermolabile and more readily inhibited by DCNP than was the 1-naphthol ST activity. Anion exchange chromatography by FPLC resulted in the resolution of two 1-naphthol ST activities, with the estrone ST activity co-eluting with the more basic 1-naphthol ST activity. When human fetal lung cytosol was subjected to gel filtration FPLC, both the 1-naphthol and estrone ST activities had the same native molecular weight of 63,000 Da. this is the first demonstration of estrogen ST activity in human fetal lung. These results suggest that there are at least two forms of sulfotransferase in human fetal lung and that this tissue is capable of sulfating both xenobiotics and endogenous compounds.

Topics: Arylsulfotransferase; Chromatography, Liquid; Cytosol; Enzyme Stability; Estrogens; Female; Fetus; Glucuronosyltransferase; Hot Temperature; Humans; Lung; Male; Nitrophenols

N-Nitrosomethyl(2-hydroxyethyl)amine (NMHEA), when administered by gavage, is a strong liver carcinogen in F344 female rats, but a weak liver carcinogen in male rats. After repeated exposure to NMHEA, either in drinking water or by gavage, female rats accumulated higher levels of DNA-guanine adducts than did their male counterparts, suggesting a correlation with the observed disparity in carcinogenicity. NMHEA has been shown to alkylate rat liver DNA in vivo in a dose-dependent manner. Chemical investigations of NMHEA suggest that it becomes a strong electrophile when a good leaving group is substituted on the hydroxyl. We have proposed that NMHEA is activated to its ultimate carcinogenic form by conjugation with sulfate. The sulfate ester was postulated to undergo rapid cyclization to 3-methyl-1,2,3-oxadizolinium ion, which has previously been found to be a potent methylating agent in vitro. The effect of sulfotransferase inhibitors on the DNA alkylation in rats by NMHEA was studied in vivo. Dichloronitrophenol, a powerful inhibitor of phenol sulfotransferase, had little effect on the methylation and O6-hydroxyethylation of DNA guanine in female rats, while depressing the hydroxyethylation of the N-7 position of guanine. Dichloronitrophenol, however, dramatically enhanced the methylation of DNA in male rats. It also slightly inhibited the N-nitrosodimethylamine-induced methylation of DNA. On the other hand, propylene glycol, an alcohol sulfotransferase inhibitor, had a profound inhibitory effect on DNA methylation induced by NMHEA, very little effect on the formation of N7-(2-hydroxyethyl)guanine, but a very strong effect on the O6-hydroxyethylguanine lesions. NMHEA-induced alkylation was also studied in male and female brachymorphic mice, which are deficient in the ability to synthesize the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate required for sulfotransferase activity, and their heterozygous siblings. No significant differences were seen between the heterozygous and brachymorphic mice in overall levels of alkylation, except in the case of 7-hydroxyethylation. In contrast to rats, male mice showed higher levels of formation of all DNA guanine adducts than did the females. However, propylene glycol was found to depress all the levels of alkylation in the brachymorphic mice, except for N7-(2-hydroxyethyl)guanine, as was observed in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Alkylation; Animals; Biotransformation; Dimethylnitrosamine; DNA; Female; Guanine; Hydrolysis; Liver; Male; Nitrophenols; Nitroso Compounds; Rats; Rats, Inbred F344; Sex Factors; Sulfotransferases

1. The isolated perfused rat liver forms three sulphated metabolites from each of the flavonoids, quercetin and catechin: these are secreted into the bile and the perfusate. 2. Quercetin gives two double conjugates, containing sulphate and glucuronic acid, and one sulphate: catechin gives one such double conjugate and two sulphates. 3. This sulphation is not inhibited by 60 microM 2,6-dichloro-4-nitrophenol which almost completely inhibits the sulphation of harmol in this perfused liver system. 4. The sulphation of harmol by the perfused liver is not inhibited by the flavonoids. 5. Unfractionated sulphotransferases from rat liver catalyse sulphate conjugation of quercetin and catechin in vitro by a reaction inhibited by pentachlorophenol or dichloronitrophenol: the flavonoids inhibit the sulphation of 4-nitrophenol by this system. 6. The results with the two systems are discussed and shown to be compatible.

Topics: Animals; Bile; Catechin; Chromatography, Thin Layer; Glucuronidase; Harmine; Hydrolysis; In Vitro Techniques; Liver; Nitrophenols; Perfusion; Quercetin; Rats; Sulfates; Sulfur Radioisotopes

Within a few minutes of incubation with SO4(2-), cultured monolayer cells retract into round shapes with drastically reduced surface area. Concomitant elevation of phosphoinositide second messenger levels, viz, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), is observed. A causal relationship with sulphation seems to be suggested by finding (a) sulphation of an added acceptor, 4-methylumbelliferone, (b) sulphation of endogenous glycosaminoglycan (GAG) polymers, (c) inhibition by phenol sulphotransferase inhibitor, DCNP (2,6-dichloro-4-nitrophenol). DCNP also inhibits the second messenger production and cell rounding. Reduced surface area appears to be caused by massive plasma membrane internalization in a distinctive endocytosis which is also seen in cell rounding from directly imposed ionic gradients. Reducing the surface area would reduce the adhesive or attachment sites. Besides demonstrating a highly efficient cell detachment potential, huge macromolecules appear to be readily internalized. The association of sulphation, signal transduction and cell detachment is novel.

Topics: Cell Line; Cell Membrane; Cells; Diglycerides; Endocytosis; Glycosaminoglycans; Humans; Hymecromone; Inositol 1,4,5-Trisphosphate; Nitrophenols; Second Messenger Systems; Sulfates; Tumor Cells, Cultured

The activities of monoamine oxidase and phenolsulfotransferase in the hypothalamus and anterior pituitary gland of spontaneously hypertensive rats and the normotensive control (Wistar Kyoto rat) rats were investigated. The monoamine oxidase activity (determined using dopamine as substrate) in both these tissues was not significantly different between the normo- and hypertensive animals. Hypothalamic phenolsulfotransferase does not sulfate-conjugate dopamine at pH of 6.5 and pituitary phenolsulfotransferase does not sulfate-conjugate dopamine or 3,4-dihydroxyphenylacetic acid at the same pH. Hypothalamic phenolsulfotransferase activity determined using 3,4-dihydroxyphenylacetic acid as substrate was significantly higher in the spontaneously hypertensive than the Wistar Kyoto rats, while pituitary enzyme (determined using phenol as substrate) was the same in both strains of animals. We proposed that in the spontaneously hypertensive rats the higher level of hypothalamic phenolsulfotransferase could (by removing 3,4-dihydroxyphenylacetic acid as sulfated acid) increase the deamination of dopamine by monoamine oxidase. This could in turn result in the presence of high amount of sulfated 3,4-dihydroxyphenylacetic acid in the anterior pituitary gland reported in our earlier study, and be partly responsible for the reduced central dopaminergic activity found in the hypertensive rats.

Topics: Animals; Arylsulfotransferase; Chromatography, High Pressure Liquid; Hypertension; Hypothalamus; Male; Monoamine Oxidase; Nitrophenols; Pentachlorophenol; Pituitary Gland, Anterior; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Substrate Specificity

The role of sulfation in the metabolic activation of the liver carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) in male rat liver was investigated. N-OH-FAABP was a substrate for sulfotransferases in vitro and sulfation was inhibited by the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The main metabolite of N-OH-FAABP excreted in bile in vivo, and in the isolated perfused liver, was identified as the N-O-glucuronide conjugate. Inhibition of sulfation in vivo by PCP or DCNP, or in vitro by omission of inorganic sulfate, resulted in a large increase in the excretion of the N-O-glucuronide conjugate. It was estimated that at least 21% of the dose was sulfated in control animals. Inhibition of sulfation in vivo by PCP or DCNP prevented the covalent binding of N-OH-FAABP to liver (and kidney) macromolecules by 70% and 20% respectively. HPLC analysis of the fluorobiphenyl DNA and RNA adducts showed that the formation of both N-acetylated and deacetylated (deoxy)-guanosine adducts was prevented. Furthermore, omission of inorganic sulfate in the isolated perfused liver prevented the formation of all fluorobiphenyl DNA adducts by 70-80%. It is concluded that two sulfotransferase-dependent pathways exist for the metabolic activation of N-OH-FAABP in male rat liver: (i) direct sulfation of the hydroxamic acid, resulting, upon decomposition of the FAABP-N-sulfate ester, in the formation of N-acetylated DNA adducts and (ii) deacetylation followed by sulfation of the hydroxylamine to FABP-N-sulfate, leading to the formation of deacetylation DNA adducts.

Topics: Aminobiphenyl Compounds; Animals; Biotransformation; Carcinogens; Chlorophenols; Chromatography, High Pressure Liquid; DNA; Glucuronidase; Kidney; Liver; Male; Nitrophenols; Pentachlorophenol; Rats; Rats, Inbred Strains; RNA; Sulfates; Sulfurtransferases

Topics: Animals; Arylsulfotransferase; Callithrix; Callitrichinae; Female; Male; Monoamine Oxidase; Nitrophenols; Substrate Specificity

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs, neurotransmitters, and xenobiotic compounds. Human tissues contain at least two forms of PST, which differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. One form of the enzyme is thermostable (TS) and catalyzes the sulfate conjugation of micromolar concentrations of "simple" phenols. The other form of PST is thermolabile and catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other phenolic monoamines. Quantitative structure-activity relationship (QSAR) analyses of substrate kinetic data obtained with purified human liver TS PST made it possible to design a photoreactive substrate for this form of the enzyme. Because of the very high affinity of TS PST for 2-halogenated phenols, 2-iodo-4-azidophenol (IAP) was synthesized and tested for this purpose. The Km predicted for IAP on the basis of QSAR analysis was 95 nM. The apparent Km determined experimentally was 52 nM. UV irradiation of partially purified human liver TS PST in the presence of [125 I]IAP and 3'-phosphoadenosine-5'-phosphosulfate, the sulfate donor for the reaction, resulted in the radioactive labeling of two proteins, with molecular weights of 32,000 and 34,000, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibitors of the reaction catalyzed by TS PST, including 2,6-dichloro-4-nitrophenol-3'-phosphoadenosine- 5'-phosphate and NaCl, as well as 2-iodophenol, a competing substrate, inhibited the photolabeling of both of these proteins by [125I]IAP in a concentration-dependent fashion. Partially purified TS PST was then radioactively labeled with [125]IAP and was subjected to gel filtration high performance liquid chromatography to verify that the photo-affinity-labeled proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coeluted with TS PST enzyme activity. Photoaffinity labeling of TS PST will be useful in studies of the molecular characteristics of the protein and its active site, as well as in the purification of this important drug-metabolizing enzyme.

Topics: Affinity Labels; Arylsulfotransferase; Azides; Chromatography, High Pressure Liquid; Hot Temperature; Humans; Iodobenzenes; Liver; Nitrophenols; Photolysis; Trichloroacetic Acid

The metabolism of T3 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, HPLC, and RIA for T3 sulfate (T3S) and 3,3'-diiodothyronine (3,3'-T2). Type I iodothyronine deiodinase activity was inhibited with propylthiouracil (PTU), and phenol sulfotransferase activity by SO4(2-) depletion or with competitive substrates or inhibitors. Under normal conditions, labeled T3 glucuronide and I- were the main products of [3'-125I]T3 metabolism. Iodide production was decreased by inhibition (PTU) or saturation (greater than 100 nM T3) of type I deiodinase, which was accompanied by the accumulation of T3S and 3,3'-T2S. Inhibition of phenol sulfotransferase resulted in decreased iodide production, which was associated with an accumulation of 3,3'-T2 and 3,3'-T2 glucuronide, independent of PTU. Formation of 3,3'-T2 and its conjugates was only observed at T3 substrate concentrations below 10 nM. Thus, T3 is metabolized in rat liver cells by three quantitatively important pathways: glucuronidation, sulfation, and direct inner ring deiodination. Whereas T3 glucuronide is not further metabolized in the cultures, T3S is rapidly deiodinated by the type I enzyme. As confirmed by incubations with isolated rat liver microsomes, direct inner ring deiodination of T3 is largely mediated by a low Km, PTU-insensitive, type III-like iodothyronine deiodinase, and production of 3,3'-T2 is only observed if its rapid sulfation is prevented.

Topics: Animals; Chromatography; Chromatography, High Pressure Liquid; Diiodothyronines; Galactosamine; Liver; Microsomes, Liver; Nitrophenols; Osmolar Concentration; Pentachlorophenol; Radioimmunoassay; Rats; Salicylamides; Sulfates; Triiodothyronine

The effect of 2,6-dichloro-4-nitrophenol (DCNP), an inhibitor of phenol sulphotransferases (EC 2.8.2.-), on the biosynthesis of aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured pig intestinal mucosal explants. At 50 microM DCNP did not affect protein synthesis but it decreased incorporation of [35S]sulphate into aminopeptidase N and other major microvillar hydrolases by 70-85% compared with controls, indicating an inhibition of their post-translational tyrosine sulphation. In labelling experiments with [35S]methionine from 0.5 to 5 h, DCNP was tested for its possible influence on synthesis, processing and microvillar expression of aminopeptidase N, but no effect on any of these parameters could be detected. It can therefore be concluded that tyrosine sulphation is not required (for instance as a sorting signal) for the targeting of newly synthesized enzymes to the microvillar membrane.

Topics: Aminopeptidases; Animals; CD13 Antigens; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Intestinal Mucosa; Microvilli; Nitrophenols; Organ Culture Techniques; Protein Biosynthesis; Sulfur Radioisotopes; Swine; Tyrosine

The 2-hydroxyamino-3-methylimidazolo[4,5-f]quinoline (N-hydroxy-IQ), a metabolite of the food mutagen--carcinogen IQ, was mutagenic to Salmonella TA98 (nitroreductase deficient). When either rat hepatic cytosol, NADPH (1 mM) or ascorbate (0.5 mM) was added to the mutagenicity assay, mutagenicity increased up to 15-, 10- and 50-fold respectively. In light of the effects of ascorbate and NADPH, it appears likely that hepatic cytosol may contain factors that protect N-hydroxy-IQ from oxidative decomposition. In contrast, hepatic monooxygenase metabolism of N-hydroxy-IQ decreased mutagenicity. When pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, was added to the mutagenicity assay, a dose-dependent inhibition of N-hydroxy-IQ mutagenicity was observed. 2,6-Dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O- acetyltransferase, did not inhibit the mutagenicity of N-hydroxy-IQ at concentrations which appear to selectively inhibit only bacterial sulfotransferase. The data suggest that bacterial O-acetyltransferase rather than sulfotransferase mutagenically activates N-hydroxy-IQ. N-hydroxy-IQ covalently bound to calf thymus DNA in vitro under non-enzymatic conditions at pH 7.4. Rat hepatic cytosolic O-acetyltransferase and sulfotransferase enhanced the covalent binding of N-hydroxy-IQ to DNA 30- and 5-fold respectively. The data suggest that the mutagenicity of N-hydroxy-IQ is due to the reactivity of N-hydroxy-IQ with DNA and the ability of N-hydroxy-IQ to be further activated by bacterial O-acetyltransferase.

Topics: Acetyltransferases; Animals; Cytosol; DNA; Imidazoles; Liver; Male; Mutagenicity Tests; Mutagens; Nitrophenols; Pentachlorophenol; Quinolines; Rats; Rats, Inbred F344; Sulfurtransferases

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and of phenolic drugs. Human platelet PST exists in at least a thermolabile form (TL PST) and a thermostable form (TS PST). The mean basal level of platelet TS PST activity in samples from American blacks is significantly higher than the basal activity in samples from whites. We carried out the studies reported here to determine whether the higher basal TS PST activity in platelet homogenates from blacks was biochemically similar to the lower basal activity in samples from whites. We also characterized variations in TS PST thermal stability. Platelet TS PST activities in samples from the two groups were almost identical with respect to pH optima, Michaelis-Menten constant values for substrates, and susceptibilities to inhibition by 2,6-dichloro-4-nitrophenol and sodium chloride. Thermolabile and thermostable TS PST were present in samples from both blacks and whites. Thermal stabilities of TS PST in samples from 167 volunteers (104 blacks, 63 whites) were expressed as heated sample-to-control sample ratios. Bimodal frequency distribution histograms of the heated-to-control ratios revealed subgroups of samples with thermolabile TS PST activities from 13.5% of blacks (heated-to-control ratio less than 0.32) and 12.7% of whites (heated-to-control ratio less than 0.27). The mean heated-to-control ratio for thermostable TS PST from blacks was significantly higher than that from whites (0.52 +/- 0.01 vs 0.43 +/- 0.01, respectively, mean +/- SEM; p less than 0.0001). Our studies demonstrated the similarity of biochemical properties of platelet TS PST at the extremes of basal activity. They also showed equivalent subgroups of blacks and whites with thermolabile TS PST. The results are an important initial step toward testing the hypothesis that inheritance may be one factor in the regulation of basal levels of activities and thermal stabilities of platelet TS PST from American blacks.

Topics: Adult; Arylsulfotransferase; Black People; Blood Platelets; Enzyme Stability; Female; Hot Temperature; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Nitrophenols; Osmolar Concentration; White People

Recent studies using isolated rat hepatocytes have indicated that the bioactive form of thyroid hormone, T3, is metabolized in liver predominantly by conjugation with glucuronic acid or sulfate. In contrast to T3 itself and the stable glucuronide, T3 sulfate is rapidly degraded by successive deiodination of the tyrosyl and phenolic rings. In the present study we have investigated the biliary excretion of T3 metabolites in male Wistar rats under pentobarbital anesthesia. The animals were injected iv with 1) saline, 2) the deiodinase inhibitor propylthiouracil (PTU; 1 mg/100 g BW), 3) the phenol sulfotransferase inhibitor dichloronitrophenol (2.6 mumol/100 g BW), or 4) a combination of both drugs. After 15 min, 10 muCi [125I]T3 were administered iv, and bile was collected for 30-min periods until 4 h after tracer injection. Secretory products were analyzed by HPLC. In control animals, 22.4% of the dose was excreted in bile mainly in the form of T3 glucuronide. In PTU-treated rats biliary excretion was increased to 36.0% of the dose (P less than .001) due to a dramatic increase in the sulfates of T3 and 3,3'-diiodothyronine. Dichloronitrophenol by itself had no effect on the biliary clearance of T3, but greatly inhibited PTU-induced excretion of sulfates. These results strongly suggest that sulfation and subsequent deiodination is an important pathway of T3 metabolism in vivo.

Topics: Animals; Arylsulfotransferase; Bile; Iodide Peroxidase; Kinetics; Male; Nitrophenols; Propylthiouracil; Rats; Rats, Inbred Strains; Sulfurtransferases; Triiodothyronine

The accumulation and utilization of [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [35S]sulfate. [35S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [35S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [35S]PAPS formation was also indirectly followed by incubating slices in the presence of beta-naphthol and measuring the levels of [35S]beta-naphthyl sulfate ([35S]beta-NS). Whereas [35S]PAPS levels rapidly reached a plateau, [35S]beta-NS formation proceeded linearly with time for at least 1 h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [35S]PAPS formation in slices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC50 = 10 microM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [35S]PAPS accumulation and [35S]beta-NS formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N-methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.

Topics: Adenine Nucleotides; Animals; Aspartic Acid; Cerebral Cortex; Glutamates; Glutamic Acid; Kinetics; Male; Naphthols; Nitrophenols; Oxadiazoles; Phosphoadenosine Phosphosulfate; Potassium; Quisqualic Acid; Rats; Rats, Inbred Strains; Sulfates; Sulfur Radioisotopes; Veratridine; Veratrine

The tyrosylsulfotransferase activities of rat cerebral fractions transferring [35S]sulfate groups from 3'-phosphoadenosine 5'-[35S]phosphosulfate to either Boc-cholecystokinin-8 (in non-sulfated form) or the acidic amino acid polymer (Glu, Ala, Tyr)n (6:3:1) were compared. They appear similar regarding subcellular distribution (both being enriched in the microsomal fraction) and inhibition by an excess of the acidic amino acid polymer, NaCl or 2,6-dichloro 4-nitrophenol. These results obtained with artificial substrates suggest that identical (or closely similar) tyrosylsulfotransferases are responsible for sulfation of tyrosine residues of several secretory proteins from various tissues.

Topics: Amino Acids; Animals; Brain; Cholecystokinin; Male; Microsomes; Nitrophenols; Polymers; Rats; Rats, Inbred Strains; Subcellular Fractions; Sulfotransferases; Sulfurtransferases

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitters. Human platelets and brain contain at least two forms of PST. One form is relatively thermolabile (TL) and catalyzes the sulfate conjugation of monoamines such as dopamine. The other is thermostable (TS) and catalyzes the sulfation of "simple" phenols such as phenol and p-nitrophenol. We found that homogenates of human liver also contain two forms of PST that are similar to brain and platelet TL and TS PST with regard to substrate specificities, thermal stabilities and sensitivities to inhibitors. Optimal conditions were determined for the assay of these two activities in human liver homogenates. The apparent Km of liver homogenate TL PST for dopamine was 27 microM. The apparent Km of the TS form of the enzyme for p-nitrophenol was 0.94 microM. Human liver TS PST also catalyzed the sulfate conjugation of dopamine, but with an apparent Km of 5 mM, over two orders of magnitude higher than that of TL PST. Two different peaks of TS PST activity were separated from the TL activity by ion exchange chromatography of human liver preparations. Both peaks of TS PST activity were partially purified and characterized. Both had similar substrate specificities and inhibitor sensitivities. Km values of TS PST peak I for p-nitrophenol and for 3'-phosphoadenosine-5'-phosphosulfate were 0.91 and 0.86 microM, respectively, while the Km values of TS PST peak II for these two cosubstrates for the reaction were 0.43 and 0.64 microM, respectively. However, the TS PST activity in peak II was significantly more thermolabile than was the activity in peak I. These results are compatible with the conclusion that human liver homogenates contain at least two forms of PST, forms with properties similar to those of TS and TL PST in homogenates of human cerebral cortex and platelets. In addition, human liver contains two isozymes of TS PST.

Topics: Adult; Aged; Arylsulfotransferase; Cholelithiasis; Colonic Neoplasms; Dopamine; Enzyme Stability; Female; Hot Temperature; Humans; Isoenzymes; Kinetics; Liver; Liver Neoplasms; Male; Nitrophenols; Sulfurtransferases

To determine whether pheochromocytoma phenol sulfotransferase (PST) activities were similar to blood platelet PST activities, we established assay conditions and biochemical properties for the human pheochromocytoma enzymes. At least two forms of PST were present in high speed supernatant (HSS) preparations of the tumors. A thermolabile form (TL) and a thermostable form (TS) were similar to those of human platelet PST with regard to pH optima, apparent Km values, responses to 2,6-dichloro-4-nitrophenol and thermal stability. PST activities were measured in 74 tumors of neuroectodermal origin that had been stored at -80 degrees C for a mean of 37.9 months. Levels of TL and TS PST activities decreased in a nonlinear fashion with time of sample storage. TL and TS PST activities of 4 samples assayed after 1.08 +/- 1.95 (mean +/- SD) month of storage were 167 +/- 73 and 3,110 +/- 1,817 U/mg protein, respectively (mean +/- SEM). Our results indicated that the TL and TS forms of PST in pheochromocytoma HSS preparations were biochemically similar to platelet PST activities.

Topics: Adolescent; Adrenal Gland Neoplasms; Adult; Aged; Arylsulfotransferase; Child; Child, Preschool; Enzyme Stability; Female; Hot Temperature; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Middle Aged; Nitrophenols; Pheochromocytoma; Substrate Specificity; Sulfurtransferases

Reverse triiodothyronine (rT3) is metabolized predominantly by outer ring deiodination to 3,3'-diiodothyronine (3,3'-T2) in the liver. Metabolism of rT3 and 3,3'-T2 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, high performance liquid chromatography, and radioimmunoassay, with closely agreeing results. Deiodinase activity was inhibited with propylthiouracil (PTU) and sulfotransferase activity by sulfate depletion or addition of salicylamide or dichloronitrophenol. Normally, little 3,3'-T2 production from rT3 was observed, and 125I- was the main product of both 3,[3'-125I]T2 and [3',5'-125I]rT3. PTU inhibited rT3 metabolism but did not affect 3,3'-T2 clearance as explained by accumulation of 3,3'-T2 sulfate. Inhibition of sulfation did not affect rT3 clearance but 3,3'-T2 metabolism was greatly diminished. The decrease in I- formation from rT3 was compensated by an increased recovery of 3,3'-T2 up to 70% of rT3 metabolized. In conclusion, significant production of 3,3'-T2 from rT3 by rat hepatocytes is only observed if further sulfation is inhibited.

Topics: Animals; Cells, Cultured; Diiodothyronines; Iodide Peroxidase; Liver; Nitrophenols; Rats; Salicylamides; Sulfates; Triiodothyronine, Reverse

A previous study (Sund & Lauterbach 1986) in the isolated guinea pig mucosa showed a complex pattern of 1-naphthol (I) metabolism and metabolite (glucuronide = II and sulphate = III) transport in relation to tissue studied (jejunum and colon) and administration side (lumen versus blood side). In the present paper aspects of I metabolism and II and III transport have been further studied. The experiments involved: Omission of inorganic sulphate in the incubation solution at one particular side or at both sides, to see if and how intestinal sulphoconjugation depended on side of sulphate ion entry, and if II and III efflux might be linked to sulphate ion influx. Similar omission experiments with inorganic phosphate, and Incubation in presence of 2,6-dichloro-4-nitrophenol (IV), a drug claimed to be a selective inhibitor of sulphoconjugation. The experiments showed: In the jejunum, sulphate ion caused a much stronger stimulation of III formation from the lumen than from the blood side, when I was added at the luminal side. In the colon, however, the sulphate ion was more effective on the blood side than on the lumen side, regardless of side at which I was added. More experiments are needed to clarify if conjugate efflux is affected by sulphate ion omission as well. Omission of inorganic phosphate did neither affect I metabolism nor II and III efflux. IV (present at both sides at once) had complex effects, involving inhibition of II and III synthesis as well as their efflux, and, in part, a change in their normal lumen: blood distribution pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biological Transport; Colon; Guinea Pigs; In Vitro Techniques; Inactivation, Metabolic; Intestinal Absorption; Intestinal Mucosa; Jejunum; Naphthols; Nitrophenols; Organ Specificity; Phosphates; Sulfates

Topics: Animals; Biphenyl Compounds; Glucuronates; Glucuronic Acid; Liver; Male; Nitrophenols; Rats; Rats, Inbred Strains; Sulfates

A new substrate, 2,6-dichloro-4-nitrophenyl phosphate (DCNP-P), is used for the determination of acid phosphatase (EC 3.1.3.2) in serum and urine. It was hydrolyzed by acid phosphatase to 2,6-dichloro-4-nitrophenol (DCNP) and phosphoric acid. At a pH of 4.5-6.0, the absorption of DCNP liberated by acid phosphatase was much higher than that of p-nitrophenol, which is commonly used as an aglycone in the acid phosphatase assay. By using DCNP-P as a substrate for acid phosphatase activity, determinations can be made without the colour reaction which requires the addition of an alkaline solution, and can be determined by the rate assay that does not require measurement of sample blanks in serum or urine. This method using DCNP-P is highly sensitive and is the most suitable for the rate assay of acid phosphatase activity in biological fluids.

Topics: Acid Phosphatase; Humans; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Phosphoric Acids; Spectrophotometry

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitters. All human tissues that have been studied in detail contain at least two forms of PST. One form is thermolabile (TL), catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other phenolic monoamines, and is relatively resistant to inhibition by 2,6-dichloro-4-nitrophenol (DCNP). The other form is thermostable (TS), catalyzes the sulfate conjugation of micromolar concentrations of simple phenols such as p-nitrophenol, and is relatively sensitive to DCNP inhibition. These two forms of PST have been physically separated and partially purified from several human tissues, including an easily accessible tissue, the blood platelet. The biochemical properties of platelet PST are very similar to those of PST in human brain, liver, and small intestine. Individual differences in the basal activity of TS PST in the platelet are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex (r = .94, n = 15, P less than 0.001). In addition, both platelet TS and TL PST activities are correlated significantly with the extent of sulfate conjugation of orally administered drugs such as acetaminophen and methyldopa. These latter observations are compatible with the conclusions that platelet PST activity may reflect the activity of the enzyme at sites of drug metabolism, and that variation in PST activity is one factor responsible for individual differences in the sulfate conjugation of orally administered drugs.

Topics: Administration, Oral; Arylsulfotransferase; Biotransformation; Blood Platelets; Catechols; Cerebral Cortex; Genetic Variation; Hot Temperature; Humans; Intestine, Small; Isoenzymes; Liver; Neurotransmitter Agents; Nitrophenols; Pharmaceutical Preparations; Phenols; Phosphoadenosine Phosphosulfate; Sulfurtransferases

Sulfation of phenols and similar low-molecular-weight substrates in the rat in vivo is a rather complex process. Besides enzyme kinetic parameters, cosubstrate availability (indirectly measured by serum sulfate concentration) and competition with glucuronidation also play a role. For some substrates extensive extrahepatic sulfation occurs, accounting for more than 50% of the total-body sulfation capacity. However, the hepatic contribution may be under-estimated when drugs are administered into the hepatic portal vein, because saturation of hepatic metabolism may occur under those conditions. Inside the liver, sulfation is located primarily in zone 1, the periportal area. This can be shown in the single-pass perfused rat liver by perfusion in either the normal or retrograde flow direction. In the rat sulfate conjugates are eliminated preferentially in urine, whereas glucuronides are excreted to a high extent in bile. Therefore, it is important to collect both bile and urine in the characterization of pharmacokinetics of conjugation in vivo. Selective inhibition of sulfation by pentachlorophenol and 2,6-dichloro-4-nitrophenol facilitates studies of the role of sulfation in elimination of its substrates, and the competition between sulfation and glucuronidation for the same substrate.

Topics: Alkaloids; Animals; Dogs; Glucuronosyltransferase; Harmine; Hymecromone; Kinetics; Liver; Liver Circulation; Nitrophenols; Pentachlorophenol; Perfusion; Phosphoadenosine Phosphosulfate; Rats; Sulfates; Sulfobromophthalein; Sulfurtransferases

Topics: 3,4-Dihydroxyphenylacetic Acid; Arylsulfotransferase; Blood Platelets; Catecholamines; Homovanillic Acid; Humans; Nitrophenols; Phenol; Phenols; Phosphoadenosine Phosphosulfate; Sulfurtransferases; Tyramine; Vanilmandelic Acid

Human lung phenol sulfotransferase was about 1000-fold purified using the earlier described procedure. 2-Naphthol, p-nitrophenol, phenol, salicylamide, p-methylphenol, o-methoxyphenol, adrenaline, and dopamine were tested as substrates for human lung PST using the barium hydroxide procedure and the ECTEOLA-cellulose method. Km values for sulfate donor (PAPS) and for different sulfate acceptors were determined. 2,6-Dichloro-4-nitrophenol was found to be a competitive inhibitor of human lung PST with Ki = 8.87 +/- 0.08 microM. High salt concentration and Mg2+, Mn2+, and Zn2+ inhibited lung PST. The molecular weight of human lung PST was estimated as 38,000 and 35,000 by gel filtration and SDS-gel electrophoresis, respectively.

Topics: Adult; Arylsulfotransferase; Chromatography; Electrophoresis, Polyacrylamide Gel; Fractional Precipitation; Humans; Hydrogen-Ion Concentration; Lung; Middle Aged; Molecular Weight; Nitrophenols; Substrate Specificity; Sulfurtransferases

Brain phenolsulfotransferase (PST) is involved in the sulfation of simple phenols like dopamine and of precursors of biologically active peptides like cholecystokinin octapeptide (CCK-8). Therefore, inhibition of brain PST would provide a new approach to studying the sulfation of CCK-8 and other sulfated compounds. Since 2,6-dichloro-4-nitrophenol (DCNP) produces a prolonged and selective inhibition of the sulfoconjugation of exogenous phenols by the liver, we decided to examine the applicability of DCNP to studies of sulfation of CCK-8 and other compounds by brain. DCNP was capable of completely inhibiting PST activity in rat brain homogenates incubated with p-nitrophenol, phenol or dopamine as substrates. The IC50 values for p-nitrophenol and dopamine were 12 and 14 microM respectively. The concentrations of DCNP in brain cortex and plasma were measured by high pressure liquid chromatography (HPLC) after a dose of 100 mumoles/kg, i.p. Peak concentrations of 380 microM in plasma and 25 mumoles/kg in brain were achieved 30 min after injection. Subsequently, DCNP concentrations decreased with half-lives of 8 and 6 hr in plasma and brain cortex, respectively. To establish if DCNP can inhibit CCK sulfation in vivo, rats were injected with 100 micromoles/kg, i.p., of the drug 30 min before injection of 35SO4(2-) into the cerebral cortex and were killed 4.5 hr later. DCNP caused a 55% inhibition of [35S]CCK-8-SO4 formation as measured by HPLC. No change in the content of endogenous CCK-8-SO4 was detectable, however, in the brain cortex of rats treated with DCNP for up to 4 days, indicating that the PST which remained active was capable of maintaining CCK-8 content at steady state.

Topics: Animals; Arylsulfotransferase; Brain; In Vitro Techniques; Kinetics; Male; Nitrophenols; Peptides; Phenols; Rats; Rats, Inbred Strains; Sincalide; Sulfurtransferases

A possible route of metabolism of beta-adrenoceptor stimulating drugs used in obstetrics consists of conjugation by the enzyme phenol sulphotransferase. The substrate specificities of the platelet and the placental enzymes towards these drugs were tested. The specific activities of the beta-adrenoceptor agonists were found to be relatively similar to those obtained with the catecholamine dopamine but their Km values were considerably higher.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Blood Platelets; Female; Humans; Nitrophenols; Placenta; Pregnancy; Substrate Specificity; Sulfurtransferases

Topics: Animals; Biotransformation; Glucuronosyltransferase; Kinetics; Liver; Nitrophenols; Pentachlorophenol; Pharmaceutical Preparations; Rats; Sulfurtransferases

The sulfotransferase inhibitors 2,6-dichloro-4-nitrophenol and pentachlorophenol were used to investigate the role of sulfate ester formation during the in vivo bioactivation of 2,4- and 2,6-dinitrotoluene (DNT). Male F-344 rats were administered one of the sulfotransferase inhibitors (40 mu mol/kg i.p.) 45 min prior to oral administration of 28 mg/kg [ring-14C]-2,4-DNT or [3-3H]-2,6-DNT and killed 12 h later. Pentachlorophenol had no significant effect on the urinary excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. The sulfotransferase inhibitors decreased the total hepatic macromolecular covalent binding of 2,4-DNT by 33%, and of 2,6-DNT by 69%. Purification of hepatic DNA by hydroxylapatite chromatography indicated covalent binding of 2,4- and 2,6-DNT at levels of 45 and 94 pmol equivalents/mg DNA, respectively. The sulfotransferase inhibitors decreased the binding of the hepatocarcinogen 2,6-DNT to hepatic DNA by greater than 95%. 2,6-Dichloro-4-nitrophenol decreased the binding of 2,4-DNT to DNA by greater than 84% while the decrease due to pentachlorophenol was 33%. These results suggest that sulfation is important in the biotransformation of 2,4- and 2,6-DNT to reactive metabolites which covalently bind to DNA. 3H2O was detected in the urine of rats administered [3-3H]-2,6-DNT. Pentachlorophenol decreased 3H2O formation to the same extent as it decreased the total hepatic macromolecular covalent binding of 2,6-DNT, suggesting that 3H exchange at the 3 position of 2,6-DNT occurs following sulfate ester formation. These results are consistent with a nitrenium-carbonium ion resonance of the sulfate ester-derived reactive intermediate of 2,6-DNT.

Topics: Animals; Biotransformation; Carcinogens; Chlorophenols; Dinitrobenzenes; DNA; Liver; Liver Neoplasms; Macromolecular Substances; Male; Nitrobenzenes; Nitrophenols; Pentachlorophenol; Rats; Rats, Inbred F344; Sulfurtransferases; Tritium

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Topics: Animals; Carbon Radioisotopes; Kinetics; Liver; Male; Nitrophenols; Pentachlorophenol; Rats; Rats, Inbred F344; Structure-Activity Relationship; Sulfurtransferases; Toluene

Human platelet phenolsulphotransferase exists in two functional forms. M and P. In this study the substrate specificity of the two forms has been further delineated by correlating activities in different individuals with various substrates. m-Tyramine, noradrenaline, adrenaline, 5-hydroxytryptamine, p-hydroxyamphetamine, isoprenaline, salbutamol and l-naphthol were all specific substrates for the M form of the enzyme. Paracetamol, a mixed substrate, was predominantly metabolized by the M form. Salicylamide at 5 microM was a substrate for the P form but became and M substrate at higher concentration. Phenol itself, a specific substrate for phenolsulphotransferase P at 10 microM, also became an M substrate at 1 mM concentration. These substrate specificities were confirmed with the selective inhibitor, dichloronitrophenol. In this study, we measured phenolsulphotransferase activity in platelets from 13 individuals selected on the basis of their wide variation in ability to sulphoconjugate paracetamol and salicylamide in vivo. There was no significant relationship between the in vivo pattern with either drug and the activity of platelet phenolsulphotransferase assayed with paracetamol or salicylamide respectively.

Topics: Acetaminophen; Arylsulfotransferase; Blood Platelets; Humans; Isoenzymes; Kinetics; Nitrophenols; Salicylamides; Substrate Specificity; Sulfurtransferases

Topics: Acetaminophen; Arylsulfotransferase; Female; Humans; Nitrophenols; Placenta; Pregnancy; Salicylamides; Sulfurtransferases

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.

Topics: Arylsulfotransferase; Blood Platelets; Dopamine; Humans; Isoenzymes; Methoxyhydroxyphenylglycol; Molecular Weight; Nitrophenols; Phenol; Phenols; Phosphoadenosine Phosphosulfate; Serotonin; Sulfurtransferases

The pharmacokinetics of 2,6-dichloro-4-nitrophenol (DCNP) have been studied in the rat. Upon i.v. injection the plasma decay curve of DCNP showed a rapid distribution phase. After 30 min the plasma concentration reached a value that was constant for at least 90 min, indicating very slow elimination of DCNP. The volume of distribution was 88 ml/kg and a high degree of binding (over, 99%) of DCNP in vitro to bovine serum albumin was found. The concentration of DCNP in the liver was between 30 and 50% of the plasma values. While in vivo the effect of DCNP persisted for a long time, its action was readily reversible in the single-pass perfused rat liver. In vivo, the effect of the dose of DCNP on the inhibition of sulfation of the phenolic compound harmol was investigated. Upon the i.v. injection of 26 mumole DCNP/kg an instantaneous and complete inhibition of sulfation of harmol was found. Using this property of DCNP, the rate of sulfation of harmol in vivo was evaluated in relation to the dose and the time after injection of the substrate. Saturation of sulfation apparently occurred because the consumption of inorganic sulfate was extremely small.

Topics: Animals; Harmine; In Vitro Techniques; Kinetics; Liver; Male; Nitrophenols; Perfusion; Protein Binding; Rats; Sulfates

Harmol is conjugated by glucuronidation and sulfation when it is given to the rat in vivo. In the once-through perfused rat liver preparation glucuronidation of harmol shows kinetic aberrances [Pang et al., J. Pharmac. exp. Ther. 219, 134 (1981)]. In order to further delineate the mechanism behind this, sulfation was inhibited to about 10% of control by 2,6-dichloro-4-nitrophenol. The loss of sulfation was compensated by an increase in the rate of glucuronidation, keeping the total clearance by the liver virtually constant in spite of the loss of sulfation. The inhibition of sulfation eliminated the previously observed lag-phase in the kinetics of glucuronidation; the rate of glucuronidation was now almost linear with the input concentration of the substrate harmol. The constant clearance of harmol in spite of inhibition of sulfation, the occurrence of the lag-phase in glucuronidation in the presence of sulfation, and the disappearance of this lag-phase in the absence of sulfation can be explained by either diffusion-limited metabolism of harmol or a heterogeneous sub-lobular distribution of the sulfating and glucuronidating systems. Activation of glucuronidation by harmol at high concentration can be excluded.

Topics: Alkaloids; Animals; Cholestasis; Glucuronates; Harmine; Kinetics; Liver; Male; Metabolic Clearance Rate; Nitrophenols; Rats; Rats, Inbred Strains; Sulfates

The effect of 2,6-dichloro-4-nitrophenol, an inhibitor of the sulfation of the phenolic compound harmol in vivo, on the sulfation of other phenolic substances and on various conjugation reactions has been studied in the rat in vivo. Compounds chemically related to 2,6-dichloro-4-nitrophenol were also tested as sulfation inhibitors. 2,6-Dichloro-4-nitrophenol inhibited the sulfation of phenol while it had no effect on biliary excretion of dibromosulphthalein, glucuronidation of phenolphthalein, acetylation of procainamide ethobromide or glutathione conjugation of ethacrynic acid. It is concluded that of these conjugation reactions sulfation is inhibited selectively at the dose level used. Some phenols with chloro- or nitro-substituents effectively inhibited the sulfation of harmol but to a lesser extent than 2,6-dichloro-4-nitrophenol. Many other phenols did not affect the conjugation of harmol, which is both glucuronidated and sulfated.

Topics: Animals; Bile; Dose-Response Relationship, Drug; Ethacrynic Acid; Glucuronates; Harmine; Male; Nitrophenols; Phenolphthaleins; Phenols; Procainamide; Rats; Sulfates

Microsomal UDP-glucuronyltransferase and cytosolic sulphotransferase share many substrates, such as phenols and hydroxamic acids. In a search for a selective inhibitor of sulphation, several phenolic compounds were tested. 2,6-Dichloro-4-nitrophenol is introduced as a selective inhibitor of sulphation in vivo, having no effect on UDP-glucuronyltransferase activity. As substrate for both conjugating enzymes the phenolic drug harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) was used. In the rat in vivo 2,6-dichloro-4-nitrophenol caused almost complete inhibition of harmol sulphation after a single intraperitoneal injection (26mumol/kg) for 48h; the percentage of harmol sulphated decreased from 75% in controls to 5% in the treated rats. The percentage of harmol glucuronidated increased from 25 to 95%. Pentachlorophenol was equally effective but also highly toxic. Salicylamide had only a very-short-lasting inhibitory effect on sulphation. In vitro, 2,6-dichloro-4-nitrophenol inhibited sulphation of harmol by a rat liver postmitochondrial supernatant completely at 1mum, whereas even at 100mum it had no effect on glucuronidation of harmol. It is concluded that 2,6-dichloro-4-nitrophenol is a selective inhibitor of sulphation and, further, that its long duration of action makes it suitable for studies on the regulatory role of sulphation in some biological processes.

Topics: Animals; Bile; Glucuronosyltransferase; Harmaline; Harmine; In Vitro Techniques; Liver; Male; Nitrophenols; Pentachlorophenol; Rats; Sulfurtransferases; Time Factors