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M W Coughtrie

Publications and source records attributed to M W Coughtrie.

At least 73 records · Page 4Linked to original sources
PubMed1994

Immunohistochemical localisation of hydroxysteroid sulphotransferase in human breast carcinoma tissue: a preliminary study.

Understanding the function and regulation of the metabolism of steroid hormones by breast tumours will be instrumental to the development of novel treatments for this widespread disease. We have examined the expression of hydroxysteroid sulphotransferase, an enzyme which inactivates many steroids, in particular androgens, in normal breast tissue and in six ductal-type mammary carcinomas using immunohistochemistry. The enzyme is not expressed in the epithelial cells which line the normal breast duct, but is present in significant amounts in neoplastic cells, suggesting that the gene encoding this protein is activated at some stage of the neoplastic transformation. The implications of this finding for the role of steroid metabolism in breast cancer are discussed.

Breast↗
PubMed1994

Sulfation of aromatic hydroxamic acids and hydroxylamines by multiple forms of human liver sulfotransferases.

Sulfation activity towards various heterocyclic and homocyclic aromatic hydroxamic acids and hydroxylamines was determined in adult human liver cytosol and with partially purified human liver sulfotransferases (STs). In adult human liver cytosols comparable ST activities towards N-hydroxy-2-acetyl-amino-5-phenylpyridine (N-OH-2AAPP), N-hydroxy-4-acetylaminobiphenyl (N-OH-4AABP) and N-hydroxy-4'fluoro-4-acetylaminobiphenyl (N-OH-4FAABP) were found, while the sulfation rates towards N-hydroxy-2-acetylaminofluorene (N-OH-2AAF), N-hydroxy-2-acetylaminonaphthalene (N-OH-2AAN), N-hydroxy-2-acetylaminophenanthrene (N-OH-2AAP) and N-hydroxy-4-acetylaminostilbene (N-OH-4AAS) were two- to five-fold lower. In adult liver cytosol ST activity was found towards all hydroxylamines tested. No significant differences were found for the various hydroxylamines. In general, the ST activities towards the various hydroxamic acids and hydroxylamines were comparable to phenol ST activity using adult liver cytosols. Partial purification of adult human liver STs was achieved by DEAE-Sepharose chromatography followed by anion exchange FPLC. Two separated protein peaks showing both N-OH-2AAPP and N-OH-2APP ST activities were observed and were designated human hydroxylamine/hydroxamic acid sulfotransferase (hHST) 1 and 2. Immunoblot analysis using an anti-rat estrogen ST antibody demonstrated cross reactivity with both hHSTs at a subunit mol. wt of 32 kDa corresponding to the phenol-sulfating form of phenol ST (P-PST). ST activity towards dopamine was low with both hHSTs, but hHST1 also contained significant capacity to sulfate dehydroepiandrosterone. The highest ST activity towards N-OH-2AAPP and N-OH-2APP was measured at pH 5.5 with both hHSTs. The Km values of the two hHSTs for sulfation of N-OH-2AAPP and N-OH-2APP were comparable, while the Vmax values for sulfation of N-OH-2APP were higher than for N-OH-2AAP with both hHSTs. FPLC anion exchange analysis of human platelet STs demonstrated that sulfation of N-OH-4ABP and N-OH-4AABP was associated with P-PST rather than M-PST (platelets do not possess any significant DHEA ST activity). Our results show that the various hydroxamic acids and hydroxylamines are converted by at least two hHSTs. The results presented here for the human liver hydroxamic acid and hydroxylamine ST activities are discussed in relation to those observed in the rat.

Adult↗
PubMed1994

Activation of benzylic alcohols to mutagens by human hepatic sulphotransferases.

Four primary and five secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons were tested for mutagenicity in Salmonella typhimurium TA98 in the presence of 3'-phosphoadenosine-5'-phosphosulphate, the cofactor for sulphotransferases, and varying amounts of hepatic cytosol from three or four different human subjects, a 3-year-old child, an adult female, an adult male and one unknown. All compounds except one, 4H-cyclopenta[def]phenanthren-4-ol, were activated to mutagens. The interindividual variation in the activities was at most 3-fold and the individual activities towards the different substrates were correlated with each other. The same compounds had previously been tested in the presence of hepatic cytosol from rats and all compounds activated in one species were also activated in the other species. However, there were marked quantitative differences, which were further complicated by the observation of a substantial sex difference in the rat. Male and female rat liver cytosol showed higher sulphotransferase activities towards 1-hydroxymethylpyrene, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 4H-cyclopenta[def]chrysen-4-ol than human liver cytosol. The largest difference in activity was seen with 7-hydroxymethyl-12-methylbenz[a]anthracene, reaching a factor of approximately 100 between human and female rat. However, with other benzylic alcohols, the activity in human liver cytosol was in the range of that found in the less active sex of rat (3-hydroxy-3,4-dihydrocyclopenta[cd]pyrene, 2-hydroxymethylpyrene) or the more active sex of rat [1-(1-pyrenyl)ethanol].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed.

Arylsulfotransferase↗
PubMed1993

Common food additives are potent inhibitors of human liver 17 alpha-ethinyloestradiol and dopamine sulphotransferases.

Interactions between dietary xenobiotics, drugs and biologically active endogenous compounds are a potential source of idiosyncratic adverse pathology. We have examined the inhibition of the sulphation of a number of xenobiotics and endobiotics in human liver cytosol by 15 food additives and constituents. Sulphation of dehydroepiandrosterone was resistant to inhibition by all compounds tested; however, dopamine sulphotransferase (ST) activity was inhibited strongly by (+/-)-catechin, (+)-catechin, octyl gallate, tartrazine and vanillin. Sulphation of the xenobiotic steroid 17 alpha-ethinyloestradiol (EE2) was inhibited by vanillin, erythrosin B and octyl gallate. Of these compounds, only vanillin was found to be sulphated to a significant extent by both human liver and platelets, and vanillin was determined to be a substrate for the monoamine-sulphating isoenzyme of phenolsulphotransferase. Vanillin was found to inhibit 50% of liver EE2 ST activity (IC50) at a concentration of approximately 1.3 microM and the mode of inhibition was non-competitive. The implications of these results for the adverse side effects associated with food additives and oral contraceptives are discussed.

Arylsulfotransferase↗
PubMed1993

Purification and immunochemical characterization of a male-specific rat liver oestrogen sulphotransferase.

Sulphation of oestrogens represents an important regulatory mechanism for these biologically active compounds. We have characterized and purified a form of rat liver sulphotransferase (ST), existing as a 32,500 Da monomer, which sulphates oestrogens, and have used this preparation to produce antibodies against oestrogen ST. The enzyme was active against oestrone, oestriol and beta-oestradiol, but not towards androgens. Using the antibody as a probe for immunoblotting, it was determined that the enzyme is expressed solely in male rats, and predominantly in the liver. Of the tissues examined, the only major extrahepatic tissue found to have any oestrogen ST was the brain (although the levels were very low), indicating that there might be a role for the sulphation of oestrogens in the brain. Examination of human liver and platelet cytosols by immunoblotting showed that the antibody recognized two major proteins of 32 and 34 kDa, which were presumed to correspond to the two principal phenol ST isoenzymes present in man.

Animals↗
PubMed1993

Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans.

A member of the rat liver hydroxysteroid sulfotransferase (ST) enzyme family metabolising dehydroepiandrosterone (DHEA) was purified from female rats and used to raise rabbit polyclonal antibodies. Characterisation of this antibody preparation demonstrated that it was specific for DHEA ST, and recognised a single 30-kDa protein on immunoblot analysis of rat liver cytosol which was expressed preferentially in female rat liver, and immunohistochemical localisation of the protein in female rat liver determined that DHEA ST was distributed homogeneously in the cytoplasm of hepatocytes. Examination of the extrahepatic expression of this protein showed it to be located predominantly in the liver, although a small amount of enzyme activity was found in the kidney which was not apparently subject to the same sex difference as the hepatic activity. Immunological analysis suggested that this activity was not due to the action of DHEA ST, but to another, unidentified ST isozyme. The antibody cross-reacted strongly with adult human liver DHEA ST, recognising a protein of 35 kDa on immunoblotting. Using this antibody preparation, the distribution of DHEA ST in mid-trimester human fetal tissues was examined, and it was shown that the enzyme is expressed in the adrenal and liver, but not to any significant extent in the kidney or lung. This antibody therefore provides a powerful tool for investigating the function of DHEA ST.

Adrenal Glands↗
PubMed1992

Inhibition of human liver steroid sulfotransferase activities by drugs: a novel mechanism of drug toxicity?

The inhibition of steroid and phenol sulfotransferase activities in human liver cytosol by a wide range of commonly used drugs was studied. Dehydroepiandrosterone (DHEA) and estrone sulfotransferase activities were strongly inhibited by a number of compounds, with IC50 values ranging between 440 pM and 147 microM. For DHEA sulfotransferase, clomiphene, testosterone, danazol and spironolactone were the best inhibitors, with IC50 values less than 5 microM, whereas for estrone sulfotransferase cyclizine, ibuprofen, chlorpheniramine and dimenhydrinate resulted in the strongest inhibition, again with IC50 values of less than 5 microM. The xenobiotic substrate 1-naphthol was refractory to substantial inhibition, with the exception of clomiphene. The majority of the drugs which inhibited steroid ST activities strongly were either synthetic steroids, antisteroidals or were tertiary amine drugs such as tricyclic antidepressants and antihistamines, many of which exhibit adverse side effects manifesting particularly as sexual dysfunction and disruption of hormone action in clinical use. The importance of these findings for our understanding of the molecular basis of adverse drug reactions is discussed.

Cytosol↗
PubMed1992

The distribution of UDP-glucuronosyltransferases in rat liver parenchymal and nonparenchymal cells.

Activities for the glucuronidation of 1-naphthol, morphine and bilirubin as well as for the sulfation of 2-naphthol have been determined in homogenates of parenchymal, Kupffer and endothelial cells isolated from livers of untreated and Aroclor 1254-pretreated rats. In addition, Western blot analyses using different polyclonal antibodies against UDP-glucuronosyltransferases (UDP-GTs) were performed with similar preparations. All enzymes under investigation were expressed at high levels in liver parenchymal cells. The constitutive expression and inducibility of UDP-GT isozyme(s) for 1-naphthol glucuronidation was also clearly demonstrated in Kupffer and endothelial cells. Furthermore, the presence of other UDP-GT isozymes was detected in preparations from these cells. No significant sulfation of 2-naphthol was detectable in Kupffer and endothelial cell homogenates. While the glucuronidation of 1-naphthol and morphine was significantly induced in all cell types by Aroclor 1254-pretreatment of the animals, the glucuronidation of bilirubin and the sulfation of 2-naphthol remained unchanged. Since the specific activity of conjugation reactions is much lower in liver nonparenchymal cells than in liver parenchymal cells, and nonparenchymal cells contribute only about 6% to the total liver protein, protection of the cells themselves rather than contribution to the overall metabolism of xenobiotics seems to be the significant role of these xenobiotic-metabolizing enzymes in the sinusoidal lining cells.

Animals↗
PubMed1992

Estrogen and phenol sulfotransferase activities in human fetal lung.

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.

Arylsulfotransferase↗
PubMed1992

Inhibition of human and rabbit liver steroid and xenobiotic UDP-glucuronosyltransferases by tertiary amine drugs--implications for adverse drug reactions.

1. To investigate the hypothesis that disruption of glucuronidation of endogenous compounds by drugs represents a potential mechanism for pathogenesis of adverse drug reactions, the effects of a range of tertiary amine and amide drugs (many with effects on sex hormone function) on steroid hormone and xenobiotic UDP-glucuronosyltransferase activities in human and rabbit liver microsomes were studied in vitro. 2. Chlorpromazine, amitriptyline, imipramine, promethazine and cyproheptadine were consistently the most potent inhibitors of the glucuronidation of testosterone, androsterone, oestriol and 1-naphthol, the steroid activities being more susceptible to inhibition (up to 90%). 3. Carbamazepine, diphenhydramine, sulphadimethoxine, dimenhydrinate and (+/-)-chlorpheniramine had little effect on the UDPGT activities measured. 4. The structural features within this group of compounds required for inhibitory potency were the presence of a rigid tricyclic ring (e.g. phenothiazine) and either a dimethylaminopropyl or a methylpiperidine side-chain. 5. The implications of these data for involvement of disruption of the normal cellular function of glucuronidation in the pathogenesis of frequently observed adverse side-effects associated with these compounds are discussed.

Amines↗
PubMed1991

Glucuronidation of imipramine in rabbit and human liver microsomes: assay conditions and interaction with other tertiary amine drugs.

The present work confirms that rabbit liver microsomes afford a good in vitro model for studying the glucuronidation of drugs containing tertiary amino groups. Furthermore, the availability of the assay procedure described above will allow us to understand further the basis for the inter-species differences in the metabolism of tertiary amines, and will eventually aid in our identification of the UDPGT isozyme(s) responsible for the conjugation of these compounds in man.

Amines↗
PubMed1991

Investigation of the molecular basis of the genetic deficiency of UDP-glucuronosyltransferase in Crigler-Najjar syndrome.

Liver biopsy samples were obtained from eight Crigler-Najjar patients. Bilirubin UDPGT activity, assayed by a microassay with HPLC analysis, was not detectable in type I livers, and low levels (9-26% of controls) of monoglucuronide conjugates only were observed in type II livers. 1-Naphthol UDPGT activity was normal in most patients, where membrane integrity was maintained by correct sample procurement and preparation. Our data on type II livers suggest that a defect in UDPGA transport is an unlikely cause of the hyperbilirubinaemia, but reduced affinity for UDPGA was observed in one sample. Analysis of four patient liver samples by immunoblot analysis revealed the heterogeneous nature of this inherited disease within the patient population, and one sample where 1-naphthol UDPGT activity was considerably reduced appeared to correlate with the non-detection of a phenol UDPGT protein. Progress towards a molecular genetic diagnosis of Crigler-Najjar syndromes is discussed.

Aged↗
PubMed1991

Selective induction of bilirubin UDP-glucuronosyl-transferase by perfluorodecanoic acid.

Differential effects of perfluorodecanoic acid (PFDA) on rat liver UDP-glucuronosyltransferase isoenzymes have been observed after a single i.p. administration of the compound to young male Sprague-Dawley rats. (1) Bilirubin glucuronidation was induced 2-fold. The induced state was stable for at least 3 weeks. (2) Glucuronidation of 1-naphthol, morphine and testosterone was decreased to half of the control values. These decreases were maximal after 12 days but all three activities returned to normal levels after 3 weeks. (3) Immunoblotting experiments indicated that the differential effects of PFDA on UDP-glucuronosyltransferase activities were due to modulation of enzyme protein concentrations rather than activation/inactivation mechanisms. With respect to its influence on UDP-glucuronosyltransferase isoenzymes, PFDA may be classified as a clofibrate-type inducer. The persistence of the induction after a single application however is unique among peroxisome proliferators and therefore PFDA may be a useful, elective inducer of bilirubin glucuronidation.

Animals↗
PubMed1991

Improved preparation of hepatic microsomes for in vitro diagnosis of inherited disorders of the glucose-6-phosphatase system.

Disruption of microsomal membranes after freezing liver samples can undermine the reliability of in vitro enzymatic diagnosis of the type 1 glycogen storage diseases. However, freezing of biopsy material is necessary if biopsy samples are to be safely transported to the place of assay. We have therefore examined several different methods (each of which could easily be carried out in routine hospital laboratories) of preparing and freezing liver tissue before analysis for glucose-6-phosphatase (EC 3.1.3.9) enzyme activity, and determination of microsomal intactness. Our study showed that homogenizing fresh liver, and centrifuging the homogenate at 10,000 x g for 10 min at 4 degrees C, followed by freezing the resulting supernatant material at -80 degrees C, provided the optimum source of material for subsequent preparation of microsomes for analysis of glucose-6-phosphatase activity. We also demonstrated that 1-naphthol UDP glucuronosyltransferase (EC 2.4.1.17) activity could be used to assess microsomal intactness in cases of type 1a glycogen storage disease, where mannose-6 phosphatase activity cannot be used.

Animals↗
PubMed1990

Purification and immunochemical characterization of a rat liver sulphotransferase conjugating paracetamol.

Paracetamol sulphotransferase (ST) was purified 250-fold from male rat liver, and the pure enzyme used to elicit antibodies in rabbit. The enzyme was active towards paracetamol at pH 9.0, as well as towards several commonly used drugs, and formed sulphates at both O- and N-atoms. Comparison of the substrate specificity of paracetamol ST with that of aryl sulphotransferases isolated by other workers suggested that we have purified a previously unknown isoenzyme of rat liver ST, although the difficulties of characterization of STs based on their substrate specificities is noted. The antibody preparation recognized only one polypeptide (Mr = 35,000) on immunoblot analysis of rabbit liver cytosol, corresponding to purified paracetamol ST. Analysis of the tissue distribution of this protein demonstrated that its expression was restricted to the liver, as was the enzyme activity. The observed sex difference in paracetamol ST (males greater than females) was determined by immunoblot analysis to be the result of reduced enzyme protein levels in females. In human liver cytosol, the antibody recognized two polypeptides, probably corresponding to M- and P-phenol STs, suggesting significant sequence similarity between rat and human phenol sulphotransferases.

Acetaminophen↗