Sheila Sharp

Biology and function of the reversible sulfation pathway catalysed by human sulfotransferases and sulfatases

Sulfation and sulfate conjugate hydrolysis play an important role in metabolism, and are catalysed by members of the sulfotransferase and sulfatase enzyme super-families. In general, sulfation is a deactivating, detoxication pathway, but for some chemicals the sulfate conjugates are much more reactive than the parent compound. The range of compounds which are sulfated is enormous, yet we still understand relatively little of the function of this pathway. This review summarises current knowledge of the sulfation system and the enzymes involved, and illustrates how heterologous expression of sulfotransferases (SULTs) and sulfatases is aiding our appreciation of the properties of these important proteins. The role of sulfation in the bioactivation of procarcinogens and promutagens is discussed, and new data on the inhibition of the sulfotransferase(s) involved by common dietary components such as tea and coffee are presented. The genetic and environmental factors which are known to influence the activity and expression of human SULTs and sulfatases are also reviewed.

ESTRADIOL FORMATION BY HUMAN OSTEOBLASTS VIA MULTIPLE PATHWAYS : RELATION WITH OSTEOBLAST FUNCTION

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV‐HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17β‐hydroxysteroid dehydrogenase (17β‐HSD), and steroid sulfatase. Aromatase, sulfatase, and 17β‐HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17β‐HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17β‐HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E1) and estradiol (E2), respectively. The 17β‐HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17β‐HSD activity indicated both oxidative (E2 to E1; T to A) and reductive (E1 to E2; A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone‐sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17β‐HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention. J. Cell. Biochem. 75:528–537, 1999.

Epigenetics: methylation-associated repression of heparan sulfate 3-O-sulfotransferase gene expression contributes to the invasive phenotype of H-EMC-SS chondrosarcoma cells

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell‐tissue‐organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H‐HEMC‐SS chondrosarcoma cells showed a typical hypermethylation profile of 3‐OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5‐aza‐2′‐deoxycytidine (5‐Aza‐dc), a DNA‐methyltransferase inhibitor, up‐regulated expression of 3‐OST1, 3‐OST2, and 3‐OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5‐Aza‐dc treatment or reintroduction of 3‐OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion‐exchange and gel‐filtration chromatography. Notably, 5‐Aza‐dc treatment of HEMC cells or expression of 3‐OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3‐OST genes resulting in altered HSPG sulfation and point to a defect of HS‐3‐O‐sulfation as a factor in cancer progression.—Bui, C., Ouzzine, M., Talhaoui, I., Sharp, S., Prydz, K., Coughtrie, M. W. H., Fournel‐Gigleux, S. Epigenetics: methylation‐associated repression of heparan sulfate 3‐O‐sulfo‐transferase gene expression contributes to the invasive phenotype of H‐EMC‐SS chondrosarcoma cells. FASEB J. 24, 436–450 (2010). www.fasebj.org

Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris)

The global decline in the abundance and diversity of insect pollinators could result from habitat loss, disease, and pesticide exposure. The contribution of the neonicotinoid insecticides (e.g., clothianidin and imidacloprid) to this decline is controversial, and key to understanding their risk is whether the astonishingly low levels found in the nectar and pollen of plants is sufficient to deliver neuroactive levels to their site of action: the bee brain. Here we show that bumblebees (Bombus terrestris andax) fed field levels [10 nM, 2.1 ppb (w/w)] of neonicotinoid accumulate between 4 and 10 nM in their brains within 3 days. Acute (minutes) exposure of cultured neurons to 10 nM clothianidin, but not imidacloprid, causes a nicotinic acetylcholine receptor‐dependent rapid mitochondrial depolarization. However, a chronic (2 days) exposure to 1 nM imidacloprid leads to a receptor‐dependent increased sensitivity to a normally innocuous level of acetylcholine, which now also causes rapid mitochondrial depolarization in neurons. Finally, colonies exposed to this level of imidacloprid show deficits in colony growth and nest condition compared with untreated colonies. These findings provide a mechanistic explanation for the poor navigation and foraging observed in neonicotinoid treated bumblebee colonies.—Moffat, C., Pacheco, J. G., Sharp, S., Samson, A. J., Bollan, K. A., Huang, J., Buckland, S. T., Connolly, C. N. Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris). FASEB J. 29, 2112‐2119 (2015). www.fasebj.org

Sulfation of endogenous compounds and xenobiotics : interactions and function in health and disease

Sulfation is a major detoxication mechanism for endogenous compounds and xenobiotics performed by a family of sulfotransferase isoenzymes. Understanding the normal cellular functions of these different sulfotransferases and the way in which endogenous and exogenous factors are able to influence their activity and expression will provide us with the information necessary to develop novel therapeutic strategies for conditions where sulfation may be implicated. This concept is discussed and is illustrated by examples including adverse drug reactions, fetal development and cancer.

Design, production and characterization of antibodies discriminating between the phenol-and monoamine-sulphating forms of human phenol sulphotransferase

1. Phenol sulphotransferases (PSTs) are important enzymes in xenobiotic and endobiotic detoxication, and a key component of the bodys chemical defence mechanism. 2. Human phenol-(P-PST) and monoamine-(M-PST) sulphating forms of PST share 93% amino acid sequence identity, and to date the various antibodies produced against PSTs all recognize both enzymes. 3. We have identified two peptides based on the cDNA-derived amino acid sequences of human P-PST and M-PST, which elicited for the first time antibodies capable of discriminating between these highly homologous enzymes. 4. These antibodies represent valuable tools for studying the expression, distribution and function of human phenol sulphotransferases.

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.

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.

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.

Heterogeneous expression of sulphotransferases in periportal and perivenous Hepatocytes prepared from male and female rat liver

Sulphotransferase (ST) is a family of enzymes responsible for metabolism and detoxication of endobiotics and xenobiotics. We investigated the hepatic acinar distribution of three sulphotransferases: phenol sulphotransferase (PST), oestrogen sulphotransferase (EST), and hydroxysteroid sulphotransferase (HST) in male and female rat livers by measurement of enzyme activities in isolated periportal and perivenous hepatocytes. The distribution was confirmed by immunohistochemistry. EST activity was located predominantly in the perivenous hepatocytes in male rats but not in female rats, where residual activity is catalysed by another ST. HST activity was not significantly different in periportal and perivenous hepatocytes in either male or female rats. For PST, a more widespread distribution was observed, with slight predominance in the periportal regions. The results indicate heterogeneous distribution of ST isoenzymes in the periportal and perivenous hepatocytes isolated from male and female rat livers.