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Choughule Phd 2011 University of Dundee DOCTOR OF PHILOSOPHY Characterisation of the sulfotransferases catalysing the sulfation of xenobiotics and steroids in bovine liver Choughule, Kanika Award date: 2011 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 DOCTOR OF PHILOSOPHY Characterisation of the sulfotransferases catalysing the sulfation of xenobiotics and steroids in bovine liver Kanika Choughule 2011 University of Dundee Conditions for Use and Duplication Copyright of this work belongs to the author unless otherwise identified in the body of the thesis. It is permitted to use and duplicate this work only for personal and non-commercial research, study or criticism/review. You must obtain prior written consent from the author for any other use. Any quotation from this thesis must be acknowledged using the normal academic conventions. It is not permitted to supply the whole or part of this thesis to any other person or to post the same on any website or other online location without the prior written consent of the author. Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. Characterisation of the sulfotransferases catalysing the sulfation of xenobiotics and steroids in bovine liver. Kanika Choughule Thesis submitted for the degree of Doctor of Philosophy at the University of Dundee October 2011 Declaration I declare that this thesis is based on results gained from investigations that I have personally conducted and that this thesis is of my own composition. Any work other than my own is clearly indicated in the text with reference to any publications from relevant researchers. This thesis has not been previously presented, in whole or in part for a higher degree. Kanika Choughule I confirm that Kanika Choughule has spent the equivalent of at least 9 terms in research work in the Division of Medical Sciences, Ninewells Hospital and medical School, University of Dundee. She has fulfilled the conditions of the University of Dundee thereby qualifying her to submit this thesis in application for the degree of Doctor of Philosophy. Professor Michael W.H. Coughtrie Acknowledgements Firstly, I would like to thank my supervisor, Prof Mike Coughtrie for his support and guidance throughout my PhD. Thanks to all the past and present members of the MC lab, especially Zoe Riches, Theodora Tucker, Sheila Sharp and Alison Milne for their technical expertise. Thanks to Chris Jones and Ashley Dorning for the entertainment in the lab. Also thanks to Elizabeth Faini for help with administrative issues like organising my travel to Pfizer in Kalamazoo, MI and for organising the travel to conferences. A special thanks to the team at Pfizer in Kalamazoo, MI, USA, especially Chuck Locuson for offering me the opportunity to work in the states and for making me feel welcome and comfortable during my stay in America. Thanks to Matt Zaya whose technical guidance and expertise played a major part in the success of my work at Pfizer. Thanks to Dawn Merritt for coming up with the short project idea and Scott Grover for his help on hepatocytes. A big thank you to all my family and friends. Thanks to my parents for providing me the encouragement to do this PhD. Thanks to my flatmates Eleftheria Margariti and Hadeel Odeh for listening (or at least pretending to listen) to all my moaning about failed experiments and things not working. Thanks to Suraj Deshpande for the attempted interest in my work and for providing hints and tips for working with Microsoft Word. Finally thanks to all my friends I met during my stay in Dundee for the wonderful memories I have of this place. I shall cherish these memories forever. Abbreviations ADP Adenosine di phosphate APS Ammonium persulfate ATP Adenosine tri phosphate BSA Bovine Serum Albumin bSULT Bovine sulfotransferase cDNA Complementary DNA CYP Cytochrome P450 DHEA Dehydroepiandrosterone dH2O Distilled water DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid ER Estrogen receptor GI Gastrointestinal GST Glutathione S-transferase HEPES 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid HMM Hepatocyte maintenance media IgG Immunoglobulin G IPTG Isopropyl β-D-1-thiogalactopyranoside LB Luria Broth MAP Multiple antigenic peptide MBP Maltose binding protein mRNA Messenger RNA NADPH Nicotinamide adenine dinucleotide phosphate N-OH-pHIP 2- (Hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine PAPS 3’-phosphoadenosine 5’-phosphosulfate PBS Phosphate buffered saline PCR Polymerase chain reaction PSB Phosphosulfate binding loop SULT Sulfotransferase TBS Tris buffered saline TEMED N,N,N',N'-Tetramethylethylenediamine UGT Uridine 5'-diphospho-glucuronosyltransferase UPLC Ultra Performance Liquid Chromatography Abstract Cattle are a very important part of the human food chain. Administration of veterinary drugs and other xenobiotic compounds to cattle can often result in the accumulation of metabolic residues in edible tissues that can potentially affect humans through the food chain. An understanding of drug metabolism in this species is vital to ensure safe use of drugs in cattle and eventually the provision of safer animal derived food products to man. Sulfation catalysed by sulfotransferases (SULTs) is an important phase 2 drug metabolising reaction. It is not only involved in the detoxification of drugs and xenobiotics but also in the bioactivation of procarcinogens. It is also important in the metabolism of several drugs used routinely in cattle. However, very little work has been carried out on SULTs in cattle. A variety of in vitro tools are available to study drug metabolising enzymes (DMEs) like SULTs. These include tissue microsomes, cytosol, recombinant enzymes and isolated cells such as the hepatocytes. Recombinant SULTs are important tools that can be used for the study of isoform specific drug biotransformation, drug-drug interactions and the effect of genetic polymorphisms on the activity of specific isoforms. As the liver is the major drug metabolising organ in the body, it is essential to study expression and activity of cytosolic liver sulfotransferases. Hepatocytes contain DMEs and drug transporters along with all the necessary cofactors that represent in vivo conditions. This makes hepatocytes a better representative of in vivo conditions as compared to microsomes, cytosol or recombinant enzymes. In this study we have characterised sulfotransferases in cytosol, recombinant enzymes and hepatocytes. Antibodies previously raised against human sulfotransferase isoforms were used in the detection of cytosolic bovine sulfotransferases. Probe substrates established for activity with human SULTs were used for assessing the activity of recombinant and cytosolic bovine sulfotransferases. Cytosol was prepared from 8 male livers and 12 female livers (8 untreated and 4 treated with an exogenous progestin). SULT1B1, SULT1E1 and SULT2A1 were detected in bovine liver cytosol. Expression of SULT2A1 in the bovine liver was sex specific with males expressing almost twice as much SULT2A1 compared to the females. However, no activity was detected with dehydroepiandrosterone (DHEA) which is used as a probe substrate for SULT2A1 in humans. Pregnenolone is metabolised by SULT2A1 and SULT2B1 in humans. Activity towards this substrate was detected in the bovine liver, however no sex related differences in activity were observed. 4-nitrophenol is metabolised by several members of the SULT1 family in humans such as SULT1A1, SULT1B1 and SULT1C. 17β-estradiol is a probe substrate for human SULT1E1. Activity was detected with 4-nitrophenol in male and female bovine livers. Male liver cytosol followed Michaelis-Menten kinetics whereas the female liver cytosol displayed partial substrate inhibition. This suggests that different enzymes have been involved in the biotransformation of 4-nitrophenol in the male and female liver. Activity towards 17β-estradiol in the female liver was almost 4 times higher than in the male liver. Recombinant bovine sulfotransferases (SULT1A1, SULT1B1, SULT1E1 and SULT2A1) were expressed in E. coli. All bovine SULTs except SULT2A1 were expressed in the soluble fraction. Like human SULT1A1, bovine SULT1A1 also displayed partial substrate inhibition, however the extent of inhibition (as seen with the Ki values) was lower compared to human SULT1A1. Bovine SULT1B1 followed Michaelis-Menten kinetics with 4-nitrophenol. Substrate specificity profiling carried out with equal amounts of bovine SULT1A1 and SULT1B1 revealed that SULT1B1 was better at sulfating phenolic compounds as compared to bovine SULT1A1. SULT1A1 is highly expressed in the human liver and is the major enzyme involved in drug metabolism in the human liver. This might not be the case in cattle given that SULT1B1
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